Genetics and functions of the SARS coronavirus spike protein

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LSU Doctoral Dissertations Graduate School

2005

Genetics and functions of the SARS coronavirusspike proteinChad Michael PetitLouisiana State University and Agricultural and Mechanical College, [email protected]

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GENETICS AND FUNCTIONS OF THE SARS CORONAVIRUS SPIKE PROTEIN

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and

Agricultural and Mechanical College in partial fulfillment of the

requirements for the degree of Doctor of Philosophy

in

The Interdepartmental Program in Veterinary Medical Sciences through the

Department of Pathobiological Sciences

By Chad M. Petit

B.S. Louisiana State University, 2001 B.S. Louisiana State University, 2001 B.S. Louisiana State University, 2001

December, 2005

ii

© Copyright 2005 Chad M. Petit

All Rights Reserved

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ACKNOWLEDGEMENTS

First, and foremost, I would like to thank my loving wife Lauren for her patience,

understanding and support that she has given me throughout my graduate career. I can

only hope that I will be able to reciprocate all that she has given me. I would also like to

thank my family especially my parents (Mom &Gene, Dad & Kim, Tammy & Mike,

Randy & Josie) for their support. Also, I would like to thank my grandparents (Joyce &

Arnie, Billy & Betty) and in memory of Leander & Betty Petit and William Gassen. I

would also especially like to thank my grandmother Mona Gassen for being a second

mother to me and who, along with my mother Gwyneth Duhe, made many sacrifices in

order to make sure I had every encouragement and opportunity growing up. Also, I

would like to express my gratitude to my father, Kevin Petit, who provided his support,

advice, and company whenever needed throughout my professional career. I also would

like to thank all of my life long friends, without whom my life would not be the same.

I would like to express my gratitude to Dr. Vladimir Chouljenko and Dr. Jeff

Melancon, both of whom provided invaluable assistance, suggestions, and instruction to

aid my research presented in my dissertation research. In addition, I owe thanks to all

members of the Kousoulas laboratory who gave helpful advice whenever asked. Special

thanks to Arun Iyer, Preston Fulmer, and Rafael Luna for all their advice and friendship

that we sharded in pursuit of our professional goals. I would also like to thank the

members of Genelab, Thaya Geudry, Ryan Stiles, and Mamie Burrell, for their assistance

in DNA sequencing and primer synthesis.

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I would like thank the faculty members that served on my graduate committee,

Dr. Ding Shih, Dr. Elmer Godeny, Dr. Patrick DiMario, and Dr. Dennis Ingram, for their

advice and guidance throughout my graduate career. Finally, I would like thanks Dr.

Konstantin G. Kousoulas for providing guidance, encouragement, support, and all of the

rope that he has given me throughout my stint in his laboratory. Without his “sink or

swim” method of mentorship, I would not have developed as much professionally as I

have.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS ............................................................................................. iii

LIST OF TABLES .......................................................................................................... viii

LIST OF FIGURES .......................................................................................................... ix

ABSTRACT..................................................................................................................... xvi

CHAPTER I: INTRODUCTION .....................................................................................1 STATEMENT OF RESEARCH PROBLEM AND HYPOTHESIS..........................1 STATEMENT OF RESEARCH OBJECTIVES .........................................................2 LITERATURE REVIEW ..............................................................................................3

Taxonomy of Coronaviridae ...........................................................................3 Coronavirus Architecture ..............................................................................5

Virion Classification and Morphology .........................................................5 Structural Proteins.........................................................................................8

Nucleocapsid Protein ................................................................................8 Membrane Protein...................................................................................10 Envelope Protein.....................................................................................11 Hemagglutinin-Esterase Glycoprotein....................................................13 Spike Glycoprotein .................................................................................15

Nonstructural Proteins ................................................................................17 The Replicase Protein .............................................................................17

Novel SARS-CoV Specific Proteins...........................................................19 U274........................................................................................................19 U122........................................................................................................20

Protein Antigenicity ....................................................................................20 Organization of the Viral Genome ..............................................................21 The Coronavirus Lifecycle ...........................................................................25

Virus Attachment and Entry .......................................................................25 Viral Receptors ...........................................................................................28

Angiotensin Converting Enzyme 2.........................................................29 CD209L (L-SIGN)..................................................................................30

Genome Expression ....................................................................................31 Replication of Genomic RNA.....................................................................38 Translation of Viral Proteins.......................................................................40 Assembly and Release of Virions ...............................................................41 Cellular Proteins Involved in Coronavirus Replication ..............................44

HNRNP A1 .............................................................................................44 PTB .........................................................................................................45 PABP ......................................................................................................46 Mitochondrial Aconitase.........................................................................47

Pathology and Disease ..................................................................................47

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REFERENCES..............................................................................................................50

CHAPTER II: GENETIC ANALYSIS OF THE SARS-CORONAVIRUS SPIKE GLYCOPROTEIN FUNCTIONAL DOMAINS INVOLVED IN CELL-SURFACE EXPRESSION AND CELL-TO-CELL FUSION .................................................................................................................79

INTRODUCTION ........................................................................................................79 RESULTS ......................................................................................................................82

Genetic Analysis of S Glycoprotein Functional Domains .....................82 Effect of Mutations on S Synthesis ..........................................................86 Ability of Mutant S Glycoproteins to be Expressed on the Cell

Surface .................................................................................................89 Effect of Mutations on S-mediated Cell-to-Cell Fusion.........................92 Detection of the Intracellular Distribution of S Mutant

Glycoproteins Via Confocal Microscopy ..........................................98 Time-dependent Endocytotic Profiles of Wild-type and Mutant

S Proteins. ..........................................................................................101 DISCUSSION ..............................................................................................................104

Functional Domains of the S Ectodomain ............................................105 Functional Domains of the Endodomain of S.......................................107

MATERIALS AND METHODS ...............................................................................114 Cells ..........................................................................................................114 Plasmids ...................................................................................................114 Production of SARS-CoV S Monoclonal Antibodies ...........................115 Western Blot and S Oligomerization Analysis .....................................115 Cell Surface Immunohistochemistry.....................................................116 Determination of Cell-surface to Total Cell S Glycoprotein

Expression..........................................................................................117 Confocal Microscopy ..............................................................................117 S Glycoprotein Endocytosis Assay ........................................................118 Quantitation of the Extent of S-mediated Cell Fusion. .......................119

REFERENCES............................................................................................................120

CHAPTER III: GENETIC ANALYSIS OF THE CYSTEINE RICH DOMAINS IN THE CARBOXYL TERMINUS OF THE SARS-COV SPIKE GLYCOPROTEIN.................................................................................127

INTRODUCTION ......................................................................................................127 RESULTS ....................................................................................................................132

Genetic Analysis of S Glycoprotein Cysteine Rich Domain................132 Effects of Mutations on S Synthesis ......................................................133 Ability of Mutant S Glycoproteins to Be Expressed on the Cell

Surface. ..............................................................................................133 Effect of Mutations of S-mediated Cell-to-Cell Fusion. ......................138

DISSCUSSION............................................................................................................145 MATERIALS AND METHODS ...............................................................................147

Cells ..........................................................................................................147 Plasmids ...................................................................................................147

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Production of SARS-CoV S Monoclonal Antibodies ...........................148 Western Blot Analysis.............................................................................148 Cell Surface Immunohistochemistry.....................................................149 Determination of Cell-surface to Total Cell S Glycoprotein

Expression..........................................................................................150 Quantitation of the Extent of S-mediated Cell Fusion ........................150

REFERENCES............................................................................................................151

CHAPTER IV: GENETIC COMPARISON OF THE SARS-COV AND BCOV S GLYCOPROTEINS ...........................................................................158

INTRODUCTION ......................................................................................................158 RESULTS ....................................................................................................................166

Comparison of the Ectodomain of the SARS-CoV and BCoV Spike Glycoproteins ..........................................................................166

Comparison of the Endodomain of the SARS-CoV and BCoV Spike Glycoproteins ..........................................................................169

Effect of Mutations of S-mediated Cell-to-Cell Fusion .......................172 DISCUSSION ..............................................................................................................179

Comparison Between the SARS-CoV and BCoV Spike Proteins.......179 S-mediated Cell-to-Cell Fusion ..............................................................181

MATERIALS AND METHODS ...............................................................................185 Cells ..........................................................................................................185 Plasmids ...................................................................................................185 Total Protein Immunohistochemistry ...................................................186 Quantitation of the Extent of S-mediated Cell Fusion ........................186

REFERENCES............................................................................................................187

CHAPTER V: CONCLUDING REMARKS...............................................................195 SUMMARY .................................................................................................................195 CURRENT AND FUTURE RESEARCH CHALLENGES....................................198 REFERENCES............................................................................................................199

APPENDIX A: ADDITIONAL WORK ......................................................................203 VIRUS LIKE PARTICLE AND PACKAGING SYSTEM ....................................203 BOVINE CORONAVIRUS INFECTIOUS CLONE ..............................................207 REFERENCES............................................................................................................210

APPENDIX B: PERMISSION TO INCLUDE PUBLISHED WORK .....................215

VITA.................................................................................................................................218

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LIST OF TABLES

Table 1.1: Coronaviruses listed by their respective antigenic group with their host organisms and diseases they cause indicated..............................................4

Table 2.1: Grouping of S mutants by their cell-surface expression and their cell-fusion properties...........................................................................................97

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LIST OF FIGURES

Figure 1.1: A schematic diagram of a typical coronavirion structure. S, spike glycoprotein; HE hemagglutinin-esterase glycoprotein (found only in a subset of coronaviruses); M, membrane glycoprotein; E, small envelope protein; N nucleocapsid phosphoprotein .................................................6

Figure 1.2: Genomic Organization of several serotypes of coronavirus. The genome structure for five previously sequenced coronavirus RNAs are shown. All genes, except for gene 1 (pp1ab) are drawn approximately to scale. The severe acute respiratory syndrome (SARS-CoV), mouse hepatitis virus (MHV), avian infectious bronchitis virus (IBV), porcine transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus (HCoV-229E) are composed of positive stranded RNA genomes of approximately 29.7, 31.2, 27.6, 28.5, and 27.2 kb respectively. The 5' end of RNA genome is capped and contains a 65- to 98-base long leader sequence (L). Structural proteins represented by shaded boxes while nonstructural proteins are shown as unshaded boxes. Vertical lines shown between open reading frames (ORFs) represent intergenic sequences. The area found bracketed by two intergenic sequences represents a single gene. Each separate ORF within that single gene is translated from a single mRNA species. Several variations exist between the serotypes of coronaviruses in the number, location, and sequence of ORFs encoding nonstructural proteins. The positive stranded RNAs encoding the coronavirus genomes are polyadenylated at their 3' end .................22

Figure 1.3: Schematic diagram of a typical coronavirus replication cycle. Interactions between the spike protein on the virion particle and specific receptor glycoproteins or glycans found on the surface of the target host cell facilitate virion binding to the plasma membrane of the host cell. Spike protein mediated fusion between plasma membrane or endosomal membrane with the viral envelop allows penetration of the cell by the virus. Once inside the cell, gene one encoded by the positive sensed RNA genome is translated into a large polyprotein. Cotranslational or posttranslational processing of this large polyprotein produces an RNA-dependent RNA polymerase as well as several other proteins that are involved in viral RNA synthesis. The genomic RNA is then used as a template by the RNA-dependent RNA polymerase and the other polyprotein products to synthesize negative stranded RNAs. These negative stranded RNAs are subsequently used to produce genomic and subgenomic mRNAs. These mRNAs have an overlapping nested set of 3' coterminal RNAs that possess a common leader sequence at the 5' end. Each of the subgenomic mRNAs only have their 5' most ORF translated into viral proteins effectively making them monocistronic. Proteins produced include the S, spike glycoprotein; M, membrane glycoprotein; E, small envelope protein; and N nucleocapsid phosphoprotein as well as

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several nonstructural proteins. Also the HE (hemagglutinin-esterase glycoprotein) is also produced in a small subset of coronaviruses. Once translated, the N protein interacts in the cytoplasm with newly synthesized genomic RNA in order to form helical nucleocapsids. The M and E proteins are inserted in the endoplasmic reticulum and anchored in the Golgi apparatus. The helical nucleocapsid, produced through N protein and RNA interactions, most likely first joins with M at the budding compartment which is located between the rough endoplasmic reticulum and the Golgi apparatus. Interactions between the M and E proteins elicit the budding of virions which encloses the helical nucleocapsid. Also associated in the Golgi, the S and HE structural proteins are translated on membrane-bound polysomes, inserted into the rough endoplasmic reticulum and then transported to the Golgi complex. During protein transport, some of the S and HE proteins interact with the M protein and are incorporated into maturing virion particles. S and HE proteins not incorporated into virions are transported to the cellular surface where they may play a role in mediating cell-cell fusion or hemaadsorption, respectively. Virions seem to be released by exocytosis-like fusion of smooth-walled vesicles that contain the virion particles with the plasma membrane. Virions also may remain attached to the plasma membranes of infected cells. The entire coronavirus replication cycle takes place solely in the cytoplasm of the host cell............................................................................26

Figure 1.4: Schematic diagram of the three proposed models of coronavirus mRNA transcription. Positive stranded RNA is represented by solid black lines while negative stranded RNA is represented by dashed grey lines. Leader RNA and antileader RNA is represented by black and grey boxes respectively. Arrows are used to indicate direction of transcription. ........33

Figure 2.1: Schematic diagram of the SARS-CoV S glycoprotein. (A) Graphical representation of the S glycoprotein showing the approximate location of the cluster to lysine mutations CL1-CL5 relative to known and indicated functional domains. (B) Shown on the top of the diagram is a graphical representation of the SARS-CoV S glycoprotein. The predicted fusion peptide and the HR1 region are enlarged below to show the sets of amino acids replaced by lysines in the cluster mutations. The heptad repeat a and d positions are labeled above the corresponding amino acid. Amino acids changed to lysine are demarcated by arrows with the name of that particular mutation shown in brackets. (C) Amino acid sequences of the carboxyl termini of the truncation and acidic cluster associated mutations. Cysteine clusters (CRM1 and CRM2) are denoted by underlined italicized text as well as a bracket encompassing their respective regions. The charged cluster is bracketed over the region. Amino acids mutated to alanines for the CL6 and CL7 cluster mutations are in bold.................................83

Figure 2.2: Western blot analysis of the expressed mutant SARS-CoV S mutant glycoproteins. (A,B) Immunoblots of wild-type (3xFLAG So

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(WT)), cluster to lysine, cluster to alanine, and carboxyl truncation mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. "Cells only" represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to SARS S glycoprotein. (B) In order to detect trimer formation more efficiently, the protein extracts of the mutants were neither boiled nor subject to treatment with beta mercaptoethanol ....................................................................87

Figure 2.3: Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (3xFLAG So (WT)) (F1, F2), CL1 (A1, A2), CL2 (B1, B2), CL3 (C1, C2), CL4 (D1, D2), CL5 (E1, E2), CL6 (L1, L2), CL7 (M1, M2), T1214 (K1, K2), T1229 (J1, J2), T1238 (I1, I2), T1247(H1, H2) and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (G1, G2), which served as a negative control. At 48 hours post-transfection, cells were immunhistochemically processed either under live conditions to show surface expression. (A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, and M2) or fixed and permeabilized conditions to show total expression(A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, and M1) with anti-FLAG antibody ..............................................................................................90

Figure 2.4: Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see Materials and Methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio ..........................................................93

Figure 2.5: Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviations calculated through comparison of the data from each of three experiments. ..........................................................................................................95

Figure 2.6: Confocal microscopic visualization of endocytosed and intracellular distribution of SARS-CoV S glycoprotein mutants. Vero cells expressing wild-type SARS-CoV S glycoprotein (3xFLAG So (WT))

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(A and B), CL6 (C and D), CL7 (E and F), T1229 (G and H), T1238 (I and J) and T1247 (K and L) were processed for confocal microscopy using two different methods in order to assess different properties of the mutants. Endocytosis patterns (A,C,E,G,I, and K) were visualized by adding anti-FLAG (green) antibody into the media 12 h prior to processing, enabling detection of the mutant protein after endocytosis from cellular surfaces. Early endosomes were also detected for these panels using a polyclonal anti-early endosomal antigen I antibody (red). For total glycoprotein detection (B, D, F, H, J, and L), cells were fixed and permeabilized prior to labeling with anti-FLAG (green). .....................................99

Figure 2.7: Analysis of the endocytotic kinetic profile of the truncation mutants and the acidic cluster mutants using confocal microscopy. After transfection, SARS-CoV S glycoprotein wild-type (3xFLAG So (WT)) (A1-A4), T1229 (B1-B4), T1238 (C1-C4), T1247 (D1-D4), CL6 (E1-E4), and CL7 (F1-F4) expressing cells were incubated with an anti-FLAG monoclonal antibody (green) for 1 hr and then returned to 37ºC for different times. Cell nuclei were labeled with To-Pro-3 Iodide (blue). Panels A1-A4, B1-B4, C1-C4, D1-D4, E1-E4, and F1-F4 correspond to 0-, 5-, 15-, and 60-min incubation times at 37ºC, respectively. ...............................102

Figure 2.8: Cluster alignment of the carboxyl terminus of the SARS-CoV S and the MHV S glycoproteins. The shaded residues indicate the position of the cysteine rich motif in their respective protein. The cysteine residues are bolded. The charged clusters are indicated by a bracket over the corresponding regions. ........................................................................................111

Figure 3.1: Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV’, human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are

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indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks. ......................................130

Figure 3.2: Schematic diagram of the SARS-CoV S glycoprotein endodomain and the cysteine cluster to alanine mutations. Amino acid sequences of the carboxyl termini and the cysteine cluster to alanine mutations are shown for the wild-type as well as the mutant proteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The transmembrane portion of the endodomain is italicized and underlined. Amino acids mutated to alanines for the C1217A, C1223A, C1230A, and C1235A cluster mutations are in bold. ..............................................................................134

Figure 3.3: Western blot analysis of the expressed mutant SARS-CoV mutant glycoproteins. Immunoblots of wild-type [So-3xF(WT)] and cysteine to alanine mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. “Cells only” represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to the SARS-CoV S glycoprotein.....................................136

Figure 3.4: Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (SARS So 3xF) (E1, E2), C1217A (A1, A2), C1223A (B1,B2), C1230A (C1,C2), C1235A (D1,D2), and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (F1, F2), which served as a negative control. At 48 hours post-transfection, cells were immunohistochemically processed wither under live conditions to show surface expression (A1, B1, C1, D1, E1, and F1) and permeablilized conditions to show total expression (A2, B2, C2, D2, E2, and F2) with anti-FLAG antibody. ...........................................139

Figure 3.5: Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see materials and methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild-type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio as a percentage of the wild-type. .....................................................................................................................141

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Figure 3.6: Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments. ........................................................................................................143

Figure 4.1: Schematic diagram of the BCoV S glycoprotein denoting the differences between RBCoV and EBCoV. (Chouljenko et al., 2001) A schematic diagram of the BCoV S protein is shown on top from amino acid 1 to amino acid 1363. The transmembrane region of the protein is noted with a grey box labeled “Tm”. The two heptad repeat regions are indicated with grey boxes labeled HR1 and HR2 respectively. A vertical line demarcates the location of the division between the S1 and S2 subunits of the protein with the amino acid number between S1 and S2 shown. Amino acid differences are denoted with the amino acid number shown and the corresponding residues listed below. ..........................................160

Figure 4.2: Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks ...................................................164

Figure 4.3. Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV’, human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are

xv

indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks .......................................167

Figure 4.4. Alignment heptad repeat regions of the S1 subunit of the spike glycoprotein from SARS-CoV and BCoV (Abraham et al., 1990; Marra et al., 2003)A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. The transmembrane region of the protein is noted with a black box labeled “Tm”. The two heptad repeat regions are indicated with striped boxes and labels HR1 and HR2 respectively. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The two regions containing the heptad repeats (amino acids 879 to 980) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from BCoV. Residues conserced between the two viruses represented are overlayed by shaded boxes .....................................170

Figure 4.5. Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins as well as truncated proteins. Each SARS-CoV S truncation is coupled with its corresponding BCoV S truncation. The cysteine cluster and the charged rich regions of the S wild-type proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks.......................................................................................173

Figure 4.6. Immunohistochemical detection of total expression of the BCoV S wild-type and truncated proteins. Vero cells were transfected with T1359 (Panel A), T1355 (Panel B), T1346 (Panel C), and T1333 (Panel D), and wild-type respiratory (BCoV-Lun So 3xF) (Panel E) and enteric (BCoV-End So 3xF) (Panel F) strains of the BCoV optimized S) labeled with an amino terminus 3xFLAG tag. At 48 hours post-transfection, cells were immunohistochemically processed wither under permeabilized conditions to show total expression with anti-FLAG antibody...........................175

Figure 4.7. Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). The fusion levels quantitated for the VERO-E6 cell line are represented by black bars while the levels for VERO cells are represented by grey bars. Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments .........................................................................................................177

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ABSTRACT

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute

respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates

membrane fusion events during virus entry and virus-induced cell-to-cell fusion.

Investigations, described herein, have focused on the genetic manipulation of the SARS-

CoV S glycoprotein in order to delineate functional domains within the protein. This was

accomplished by incorporating single point mutations, cluster-to-lysine and cluster-to-

alanine mutations, as well as carboxyl terminal truncations into the protein and

investigating these mutants in transient expression experiments. Mutagenesis of either the

coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted

fusion peptide, or adjacent but distinct regions, severely compromised S-mediated cell-to-

cell fusion, while intracellular transport and cell-surface expression were not adversely

affected. Surprisingly, a carboxyl terminal truncation of 17 amino acids substantially

increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17

amino acids regulate the S fusogenic properties. In contrast, truncation of 26 or 39 amino

acids eliminating either one or both of the two endodomain cysteine-rich motifs,

respectively, inhibited cell fusion in comparison to the wild-type S. The cysteine rich

domains were further studied by constructing cysteine cluster to alanine mutants in order

to ascertain their importance in the function of the protein. Results showed that the two

cysteine clusters proximal to the transmembrane region were vital in the functioning of

the spike protein in mediating cell-to-cell fusion. Mutagenesis of the acidic amino acid

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cluster in the carboxyl terminus of the S glycoprotein as well as modification of a

predicted phosphorylation site within the acidic cluster revealed that this amino acid

motif may play a functional role in the retention of S at cell-surfaces. A panel of

truncations for Bovine Coronavirus (BCoV) S was also constructed and compared to

truncations made for the SARS-CoV S glycoprotein. It was found that the two sets of

truncations had very little comparable effects on protein function when compared to one

another. This genetic analysis reveals that the SARS-CoV S glycoprotein contains

extracellular domains that regulate cell fusion as well as distinct endodomains that

function in intracellular transport, cell-surface expression and cell fusion.

1

CHAPTER I

INTRODUCTION

STATEMENT OF RESEARCH PROBLEM AND HYPOTHESIS

The severe acute respiratory syndrome coronavirus (SARS-CoV) is the most

recently discovered coronavirus. It first appeared in the Guangdong Province of southern

China in November of 2002. Unlike other human coronaviruses whose infections are

usually very mild, the SARS-CoV produced mortality rates as high as 15% in some age

groups (Anand et al., 2003). Due to this high mortality rate, intense research interest has

been generated to elucidate certain aspects of the virus life cycle as well as characteristics

of viral proteins that could possibly allude to why this virus is so lethal when compared to

the other serotypes of human coronavirus.

The SARS-CoV encodes for the typical genes found in all coronaviruses which

include the nonstructural replicase gene as well as the structural proteins nucleocapsid

(N), membrane protein (M), envelope protein (E), and spike glycoprotein (S), which are

assembled into virus particles. The S glycoprotein is primarily responsible for entry of

all coronaviruses into susceptible cells through binding to specific receptors on cells and

mediating subsequent virus-cell fusion (Cavanagh, 1995). Because of the roles it plays in

virus attachment, virus entry, and cell-cell fusion, the S glycoprotein seems to potentially

be the protein most likely responsible for the increase in virulence of the virus.

Recently the SARS-CoV S glycoprotein has been codon optimized for

mammalian cells in order to allow for more efficient expression of the protein (Li et al.,

2003). This optimization of the protein allows the protein to be studied using a transient

transfection system. The overall experimental approach was to introduce site specific

2

mutations and truncations into the codon optimized SARS-CoV S glycoprotein in order

to ascertain the functional impact of certain motifs found in the S glycoprotein. The

delineation of functional domains in the endodomain as well as the role of hydrophobic

amino acids found in and around the heptad repeat regions were the focus of Chapter II.

A second aspect of the work focused on cysteine rich clusters in the endodomain of the S

protein that are conserved throughout all coronaviruses. Cysteine cluster to alanine

mutations were introduced into the SARS-CoV S glycoprotein in order to determine the

importance of several conserved cysteines. These mutants were then tested for their

ability to mediate cell-cell fusion as compared to the wild-type proteins as well as their

ability to be properly transported to the cellular surface; this work is described in Chapter

III. A codon optimized version of the bovine coronavirus (BCoV) S glycoprotein was

constructed in order to do a comparison of the motifs found in the endodomain of the

BCoV S protein versus the SARS-CoV S protein. Several truncations of the BCoV S

protein were made in order to compare them with homologous truncations made in the

SARS-CoV S (described in Chapter IV).

STATEMENT OF RESEARCH OBJECTIVES

The overall goal of this research was to gain a more thorough understanding of

the functional domains found in the SARS-CoV S glycoprotein. The specific objectives

were as follows: 1) To delineate functional domains in the SARS-CoV S glycoprotein; 2)

To ascertain the importance of the conserved cysteine residues found in the endodomain

of the SARS-CoV S glycoprotein; 3) To compare the impact on protein function of

homologous truncations made in both the SARS-CoV and BCoV S glycoproteins.

3

LITERATURE REVIEW

Taxonomy of Coronaviridae

Nidovirales is the taxonomic order in which enveloped, positive-sense RNA

viruses that synthesize a 3' co-terminal set of subgenomic mRNAs during infection of

host cells are grouped in (Cavanagh, 1997). Members of this order include

coronaviruses, toroviruses, and arteriviruses. The toroviruses and coronaviruses are

further grouped into the family Coronaviridae that contain variable length genomes and

nucleocapsid structure.

There are three groups of coronaviruses that have little to no cross-reactivity

among antigens found on virions of the different groups (Table 1.1). Two of the groups

are composed of mammalian coronaviruses, while avian coronaviruses such as the

Infectious Bronchitis Virus (IBV) and Turkey Coronavirus (TCoV) form a third group

(Guy et al., 1997; Pedersen, Ward, and Mengeling, 1978). The model virus for the first

group of coronaviruses is the HCoV-229E. Coronaviruses of pigs (transmissible

gastroenteritis virus [TGEV] and porcine respiratory coronavirus [PRCoV]), cats (feline

coronavirus [FCoV], and dogs (canine coronavirus [CCoV]) (Pedersen, Ward, and

Mengeling, 1978) belong to group I. Murine Hepatitis Virus (MHV) is the prototype of

group II coronaviruses. Some viruses found in this group are the coronaviruses of

humans (HCoV-OC43), rats (rat coronavirus [RCoV]), cattle (bovine coronavirus

[BCoV]), pigs (hemagglutinating encephalomyelitis virus [HEV]), and other species

(Pedersen, Ward, and Mengeling, 1978). Analysis of the severe acute respiratory

4

5

syndrome associated coronavirus (SARS-CoV) viral genome has demonstrated that it is

phylogenetically divergent from the three known antigenic groups of coronaviruses

(Drosten et al., 2003; Ksiazek et al., 2003). Analysis of the polymerase gene alone,

however, indicates that the SARS-CoV may be an early off-shoot from the group II

coronaviruses (Snijder et al., 2003).

Coronavirus Architecture

Virion Classification and Morphology

The basic structure of coronavirus virions is depicted in Figure 1.1. Coronavirus

virions are spherical enveloped particles that have a diameter of 100 to 120 nm. Interior

to the virion is a single-stranded, positive-sense RNA genome that ranges from 27 to 32

kb in length, the largest of all known RNA viral genomes (Boursnell et al., 1987; Eleouet

et al., 1995; Herold et al., 1993; Lai and Cavanagh, 1997; Lee et al., 1991). The SARS-

CoV genome, specifically, has a length of 29,727 nucleotides (Rota et al., 2003). The

RNA genome is associated with the nucleocapsid (N) phosphoprotein which allows it to

form a long, flexible, helical nucleocapsid (Macneughton and Davies, 1978; Sturman,

Holmes, and Behnke, 1980). When not associated with virion particles, the

nucleocapsids appear as extended tubular strands of 14 to 16 nm in diameter (Risco et al.,

1996; Sturman, Holmes, and Behnke, 1980). It has been shown that for at least two

coronaviruses (TGEV and MHV), the helical nucleocapsid is enclosed within a 65 nm in

diameter, spherical, possibly icosahedral “internal core structure” that can be released

from the virion particle by NP-40 treatment (Risco et al., 1996). This virus core is

encapsidated by a lipoprotein envelope that is formed during virus budding from

6

Figure 1.1. A schematic diagram of a typical coronavirion structure. S, spike glycoprotein; HE hemagglutinin-esterase glycoprotein (found only in a subset of coronaviruses); M, membrane glycoprotein; E, small envelope protein; N nucleocapsid phosphoprotein.

7

8

intracellular membranes (Griffiths and Rottier, 1992; Oshiro, 1973; Tooze and Tooze,

1985). The most prominent feature of the coronavirus virion is the “halo of crowns”

found to line the outside of the virion. These long proteins (20 nm in length) consist of

the spike (S) glycoprotein and are present on all coronaviruses. Some other

coronaviruses have shorter spike proteins that also line the outside of the virion which

consist of the hemagglutinin-esterase (HE) glycoprotein. The matrix (M) glycoprotein,

because it spans the lipid bilayer three times (Machamer et al., 1993; Machamer et al.,

1990; Machamer and Rose, 1987), is thought to be a component of both the internal core

structure and the envelope. The envelope protein (E) also makes up part of the viral

envelope, although it is present in much smaller quantities than the other viral envelope

proteins (Godet et al., 1992; Vennema et al., 1996; Yu et al., 1994).

Structural Proteins

Nucleocapsid Protein

The coronavirus nucleocapsid protein, N, is a 50 to 60 kDa highly basic protein

that interacts with the viral genome in order to form the viral nucleocapsid. More

specifically, the SARS-CoV N protein is 422 amino acids that shares only a 20-30%

homology with other coronaviruses (Marra et al., 2003; Rota et al., 2003). Translation of

the protein occurs on free polysomes and is rapidly phosphorylated on serine residues in

the cytosol (Stern and Sefton, 1982); the extent and physiological relevance of

phosphorylation, however, remains unclear (Stohlman and Lai, 1979; Wilbur et al.,

1986). The synthesized protein consists of three highly conserved domains that are

separated by spacer regions of variable length (Parker and Masters, 1990).

9

The most prominent feature of the N protein is its ability to bind to RNA. Several

studies have pinpointed the RNA binding region of the N protein to the second of the

three conserved domains found in the N protein (Masters, 1992; Nelson and Stohlman,

1993; Nelson, Stohlman, and Tahara, 2000; Peng et al., 1995). The complexes formed

between the N protein and the viral RNA of coronaviruses and orthomyxoviruses are

more easily disrupted at high salt concentrations and offer little protection against RNase

when compared to the complexes formed by N proteins and viral RNA of rhabdoviruses

and paramyxoviruses (Masters and Sturman, 1990).

The second type of interaction is N protein interaction with itself. Disulfide

linked multimeric forms of the N protein have been shown to exist (Robbins et al., 1986).

It has also been shown for the SARS-CoV that the carboxyl terminal conserved domain

functions as a dimerization domain (He et al., 2004). The third type of interaction is the

N protein’s interaction with the matrix glycoprotein (Sturman, Holmes, and Behnke,

1980) which leads to the formation of virus particles.

In addition to the structural role of N, it is hypothesized that it may function in

viral RNA synthesis, transcription, translation, and virus budding (He et al., 2003; Lai

and Cavanagh, 1997; Tahara et al., 1998). Specifically, it has been shown that the N

protein participates in RNA synthesis. It was found that RNA synthesis was inhibited by

greater than 90% when antibodies to N were introduced into an in vitro synthesizing

system prepared from MHV-infected cells (Compton et al., 1987). This inhibition

implies a critical role for the N protein in transcription and replication of the viral RNA.

Although N plays a critical role in RNA synthesis, the relative amounts of free N protein

in infected cells are quite large and do not appear to be the rate limiting factor for MHV

10

RNA synthesis (Perlman et al., 1986). Also, some N protein may be complexed with

cellular membranes (An, Maeda, and Makino, 1998; Stern and Sefton, 1982) where it

functions in budding of the virus. The SARS-CoV N protein has not been extensively

studied, except that the SARS-CoV N protein may selectively activate the AP-1 signal

transduction pathway (He et al., 2003).

Membrane Protein

The membrane glycoprotein, the most abundant envelope component, is a triple-

membrane spanning protein with a short amino-terminus on the virion exterior surface,

an α-helical domain, and a large carboxyl terminal domain inside the virion envelope

(Armstrong et al., 1984; Locker et al., 1992; Machamer et al., 1993; Routledge et al.,

1991). For some coronaviruses, however such as TGEV, the carboxyl terminus of the M

protein is exposed on the surface of the virion (Risco et al., 1995). The protein is

inserted into the ER membrane through the action of a signal sequence (Rottier,

Armstrong, and Meyer, 1985) after being synthesized on membrane bound polysomes.

After being synthesized, the protein undergoes posttranslational modification in the form

of glycosylation. Interestingly, for group I and group III coronaviruses, such as TGEV

and IBV, the matrix protein undergoes N-linked glycosylation (Stern and Sefton, 1982),

whereas for group II coronaviruses, such as MHV, the matrix protein undergoes O-linked

glycosylation (Holmes, Doller, and Sturman, 1981; Niemann et al., 1982; Niemann et al.,

1984). Through the use of a virus like particle system it has been demonstrated that

neither glycosylation of the M protein nor its interaction with the S glycoprotein is

necessary for virus assembly (de Haan et al., 1998; de Haan et al., 1999). These

observations are consistent with studies using the glycosylation inhibitors tunicamycin

11

(Rossen et al., 1998; Stern and Sefton, 1982) and monensin (Niemann et al., 1982) in

infected cells. Mature matrix protein accumulates in the Golgi apparatus and is not

transported to the plasma membrane (Machamer et al., 1990; Machamer and Rose, 1987;

Swift and Machamer, 1991). The nature of the membrane-targeting sequence that causes

this accumulation in the Golgi varies for each coronavirus (Locker et al., 1992;

Machamer et al., 1993).

One of the main functions of the M protein is to direct the incorporation of the S

glycoprotein (Nguyen and Hogue, 1997; Opstelten et al., 1995) and the N protein

(Narayanan et al., 2000) into the budding virion particle. The M protein itself does not,

however, determine the actual budding site, since when expressed by itself it migrates

beyond the budding compartment and localizes in the late-Golgi complex (Klumperman

et al., 1994). Interactions with other viral proteins have been shown to be mediated

through the carboxyl terminus of the protein (Corse and Machamer, 2003; de Haan et al.,

1998; Kuo and Masters, 2002). For several coronaviruses, cells expressing the M and E

proteins alone have been shown to produce virion like particles which are exported from

the cell (Baudoux et al., 1998; Bos et al., 1996; Corse and Machamer, 2000; Corse and

Machamer, 2003; de Haan et al., 1998; Vennema et al., 1996). For the SARS-CoV,

however, this is not the case. Expression of M and E alone is not sufficient for capsid

formation, however the expression of M and N alone is able to produce virus capsids

(Huang et al., 2004).

Envelope Protein

The small envelope protein, E, is typically a 9 to 12 kDa protein that is a

component of the virion envelope (Godet et al., 1992; Liu et al., 1991; Yu et al., 1994).

12

When compared to the other structural proteins, however, the E protein is in much lower

abundance relative to the M, N, and S proteins (Godet et al., 1992; Liu and Inglis, 1991;

Yu et al., 1994). Within the three groups of coronaviruses, the E proteins are well

conserved, but between the three groups, they show limited homology. All of the

proteins do share a general structure: a short hydrophilic region on the amino terminus,

followed by a large hydrophobic region, preceding a large hydrophilic carboxyl terminus

(Liu and Inglis, 1991). Recent studies of the topology of the protein have found for

MHV and IBV that the E protein is an integral membrane protein presenting its carboxyl

terminus on the cytoplasmic face of the endoplasmic reticulum or Golgi which

corresponds to the interior of the assembled virion (Corse and Machamer, 2000;

Raamsman et al., 2000; Vennema et al., 1996). For IBV, this Golgi targeting of the

molecule has been shown to be associated with the carboxyl tail, in the absence of the

membrane-bound domain (Corse and Machamer, 2002). The orientation of the amino

terminus of the protein has not been well established; one study shows a luminal

orientation for the IBV E protein (Corse and Machamer, 2000) and another study shows

the amino terminus being buried within the membrane near the cytoplasmic face for the

MHV protein (Maeda et al., 2001). E protein is localized primarily in the perinuclear

space of infected cells, although it also has been detected on the cell surface (Godet et al.,

1992; Yu et al., 1994). The main function of the E protein is its role in the formation of

the coronavirus envelope. For several coronaviruses, expression of MHV M and E alone

has been shown to be sufficient for the formation of virion like particles which are

exported from the cell (Baudoux et al., 1998; Bos et al., 1996; Corse and Machamer,

2000; Corse and Machamer, 2003; de Haan et al., 1998; Vennema et al., 1996). For the

13

SARS-CoV, however, expression of M and E alone is not sufficient for capsid formation,

although expression of M and N is sufficient for capsid formation (Huang et al., 2004).

Another function of the E protein is its ability to induce apoptosis in the infected cell (An

et al., 1999).

The SARS-CoV envelope protein is approximately 76 amino acids long with an

approximate molecular weight of 10-15 kDa. Several studies of the SARS-CoV E

protein have shown additional properties that may be specific to the SARS-CoV. The

SARS-CoV E protein has the ability to form cation-selective ion channels (Wilson et al.,

2004) similar to those of the influenza virus protein, M2 (Duff and Ashley, 1992; Duff et

al., 1994; Pinto, Holsinger, and Lamb, 1992), the HIV-1 proteins VPu (Ewart et al., 1996)

and VPr (Piller et al., 1996), and the hepatitis C virus protein, p7 (Griffin et al., 2003;

Pavlovic et al., 2003; Premkumar et al., 2004). In addition, a segment of the SARS-CoV

E protein appears to form three disulfide bonds with another segment of corresponding

cysteines in the carboxyl-terminus of the S glycoprotein (Wu et al., 2003). The SARS-

CoV E protein appears to have a unique structural feature in that it forms a highly

unusually short, palindromic transmembrane helical hairpin around a previously

unidentified pseudo-center of symmetry (Arbely et al., 2004). It is through the action of

this hairpin, by way of deforming the lipid bilayer and increasing membrane curvature,

that there finally may be a molecular explanation of the E proteins pivotal role in virus

budding (Arbely et al., 2004).

Hemagglutinin-Esterase Glycoprotein

Although not found in the SARS-CoV, another protein found on some

coronaviruses is the hemagglutinin-esterase (HE) glycoprotein (Rota et al., 2003). The

14

protein exists as a homodimer of 65 to 70 kDa proteins that form short spikes on the

surface of some group II coronavirus virions as well as turkey coronavirus virions

(Hogue, Kienzle, and Brian, 1989; Kienzle et al., 1990; Kunkel and Herrler, 1993;

Schultze et al., 1991). Cotranslational N-linked glycosylation occurs in the rough

endoplasmic reticulum after which the dimerization of proteins occurs by disulfide

interactions to form a 165 to 170 kDa protein complex (Hogue, Kienzle, and Brian, 1989;

Kienzle et al., 1990; Yokomori et al., 1989). This protein complex is then incorporated

into the envelope of budding virus particles after being transported to the Golgi where the

N-linked glycans are converted to the complex form (Yokomori et al., 1989). HE that

does not incorporate into the virion envelope is expressed on cellular surfaces (Kienzle et

al., 1990). Some studies have hypothesized that the HE protein is dispensable for viral

replication since the presence or absence is highly inconsistent even among the group II

coronaviruses. In addition, the HE gene is frequently mutated or completely deleted

during serial virus passaging in cell culture (Yokomori, Banner, and Lai, 1991). The HE

protein of various coronaviruses binds 9-O-acetylated neuraminic acid residues which is

comparable to the binding activity of S in BCoV and HCoV-OC43 (Schultze and Herrler,

1992; Schultze et al., 1991; Vlasak et al., 1988). This activity is expected to contribute to

the hemagglutination and hemadsorption activities of coronaviruses. The HE protein also

has an acetylesterase activity that cleaves acetyl groups from 9-O-acetylated neuraminic

acid (Vlasak et al., 1988; Yokomori et al., 1989) which reverses or prevents the

hemagglutination and hemadsorption activity induced by S and HE. This acetylesterase

activity, along with the inhibition of BCoV infectivity through neutralization of HE with

15

monoclonal antibodies (Deregt et al., 1989), points to a role for HE in virus entry or virus

release from infected cells.

Spike Glycoprotein

The SARS spike glycoprotein, a 1,255-amino-acid type I membrane glycoprotein

(Rota et al., 2003), is the major protein present in the viral membrane forming the typical

spike structure found on all coronavirions. Mature proteins form oligomers in the form

of homotrimers (Delmas and Laude, 1990; Xiao et al., 2004) and are known to be

glycosylated. Posttranslational glycosylation occurs on at least four different sites of the

spike protein (Krokhin et al., 2003; Ying et al., 2004). Once fully processed the protein

exists as a 180 kDa single protein species with the homotrimer species of approximately

500 kDa. Although some S oligomers can be found on the infected cell surface where it

may mediate cell-cell fusion, most newly synthesized S accumulates in the Golgi of

infected cells where it participates in virus particle assembly (Griffiths and Rottier, 1992;

Vennema et al., 1990). The S glycoprotein of mouse hepatitis virus (MHV) is cleaved

into (Cavanagh, 1995) S1 and S2 subunits, although cleavage is not necessarily required

for virus-cell fusion (Bos, Luytjes, and Spaan, 1997; Gombold, Hingley, and Weiss,

1993; Stauber, Pfleiderera, and Siddell, 1993). Similarly, the SARS-CoV S glycoprotein

seems to be cleaved into S1 and S2 subunits in Vero-E6 infected cells (Wu et al., 2004),

but it is not known whether this cleavage affects S-mediated cell fusion. This cleavage of

the SARS-CoV S protein, however, may be attributed to overexpression of the protein

and may not occur in the context of the virus (personal communication). The S protein in

group I coronaviruses appears not to be cleaved although some of the viruses can induce

cell-cell fusion (De Groot et al., 1989). The S1 subunit contains the receptor binding

16

domain which causes it to be the main determinate of viral tropism (Hingley et al., 1994;

Sanchez et al., 1999; Suzuki and Taguchi, 1996). Angiotensin converting enzyme 2

(ACE2) (Li et al., 2003) and CD209L (L-SIGN) (Jeffers et al., 2004) have been identified

to be two receptors for the SARS-CoV. The S2 subunit contains internal hydrophobic

sequences which are thought to be responsible for the membrane fusion activity of the

protein (Luo and Weiss, 1998). It is worth noting, however, that sequences in multiple

sites of S2 as well as in S1 can affect fusion activity (Gallagher, Escarmis, and

Buchmeier, 1991; Routledge et al., 1991). Expression of the S protein alone has been

shown to induce membrane fusion on cells that are susceptible to coronavirus infection

(De Groot et al., 1989; Taguchi, 1993; Yoo, Parker, and Babiuk, 1991).

The internal hydrophobic sequences that are found in the S2 portion of the SARS

S glycoprotein are known as heptad repeats (HR). These regions contain a sequence motif

characteristic of coiled-coils which appear to be a common motif in many viral and

cellular fusion proteins (Skehel and Wiley, 1998). These coiled-coil regions allow the

protein to fold back upon itself as a prerequisite step to initiating the membrane fusion

event. There are usually two HR regions: an N terminal HR region adjacent to the fusion

peptide and a C-terminal HR region close to the transmembrane region of the protein.

Within the HR segments, the first amino acid (a) and fourth amino acid (d) are typically

hydrophobic amino acids that play a vital role in maintaining coiled-coil interactions.

Based on structural similarities, two classes of viral fusion proteins have been

established. Class I viral fusion proteins contain two heptad repeat regions and an N-

terminal or N-proximal fusion peptide. Class II viral fusion proteins lack heptad repeat

regions and contain an internal fusion peptide (Lescar et al., 2001). The MHV S

17

glycoprotein, which is similar to other coronavirus S glycoproteins, is a class I membrane

protein that is transported to the plasma membrane after being synthesized in the

endoplasmic reticulum (Bosch et al., 2003). Typically, the ectodomains of the S2

subunits of coronaviruses contain two regions with a 4, 3 hydrophobic (heptad) repeat the

first being adjacent to the fusion peptide and the other being in close proximity to the

transmembrane region (de Groot et al., 1987).

The S glycoprotein is primarily responsible for entry of all coronaviruses into

susceptible cells through binding to specific receptors on cells and mediating subsequent

virus-cell fusion (Cavanagh, 1995). Although the exact mechanism by which the SARS-

CoV enters the host cell has not been elucidated, it is most likely similar to other

coronaviruses. Upon receptor binding at the cell membrane, the S glycoprotein is thought

to undergo a dramatic conformational change causing exposure of a hydrophobic fusion

peptide, which is subsequently inserted into cellular membranes. This conformational

change of the S glycoprotein causes close apposition followed by fusion of the viral and

cellular membranes resulting in entry of the virion nucleocapsids into cells (Eckert and

Kim, 2001; Tsai et al., 2003; Zelus et al., 2003). This series of S-mediated virus entry

events is similar to other class I virus fusion proteins (Baker et al., 1999; Melikyan et al.,

2000; Russell, Jardetzky, and Lamb, 2001).

Nonstructural Proteins

The Replicase Protein

Two replicase polyproteins, termed pp1a and pp1ab, are produced from two open

reading frames (ORF1a and ORF1b) contained within the first 21 kb of the

approximately 29.7 kb SARS-CoV genome (Marra et al., 2003; Rota et al., 2003; Thiel et

18

al., 2003). Pp1a is an approximately 486 kDa polyprotein that has been predicted to

include a papain-like protease (PLpro), two putative membrane proteins, MP1 (nsp4) and

MP2 (nsp6), a picornavirus 3C-like protease (3CLpro), and several other products of

unknown function. Pp1ab is approximately 790 kDa. It is generated by ribosomal

frameshifting so that both ORF1a and ORF1b are included in the translation. It is

predicted that ORF1b contains a helicase domain (nsp13) (Ivanov et al., 2004) as well as

the predicted core RNA polymerase (nsp12), exonuclease (nsp14), endoribonuclease

(nsp15), and methyltransferase (nsp16) activities (Schmidt-Mende et al., 2001; Snijder et

al., 2003). A total of 16 protein products (nonstructural proteins nsp1 to nsp16),

produced from the processed pp1a and pp1ab polyproteins, are predicted to assemble into

a membrane-associated viral replication complex (Snijder et al., 2003; Stadler et al.,

2003; Thiel et al., 2003). This processing of the polyproteins has been hypothesized to be

coordinated by two virus-encoded proteinases, the picornavirus 3C-like proteinase

(3CLpro) and a papain-like proteinase (PLP) (Denison et al., 1992; Snijder et al., 2003;

Thiel et al., 2003). The precursor proteins and mature processed replicase proteins likely

mediate the progression from replication complex formation, followed by subgenomic

mRNA transcription, and finally genome replication. Essential functions carried out by

the replication complex are as follows: 1) transcription of genome-length negative and

positive stranded RNAs, 2) transcription of a 3´-coterminal nested set of subgenomic

mRNAs that have a common 5´ “leader” sequence derived from the 5´ end of the

genome, and 3) transcription of the subgenomic derived negative-stranded RNAs with

common 5´ ends and complementary leader sequences at their 3´ ends (Lai and Holmes,

2001; Thiel et al., 2003). Replicase complex activity has been shown to take place at

19

double-membrane vesicles in the host cell cytoplasm (Gosert et al., 2002; Pedersen et al.,

1999; Prentice et al., 2004).

Novel SARS-CoV Specific Proteins

In addition to the four main structural proteins (S, E, M, and N) and the viral

encoded RNA dependent RNA polymerase, there are nine other potential ORFs found in

the SARS-CoV genome that vary from 39 to 274 amino acids in length (Marra et al.,

2003; Rota et al., 2003; Thiel et al., 2003). The functions of these additional ORFs are

largely unknown and further characterization of these proteins is necessary to be able to

assess any impact on the virus life cycle.

U274

The largest of the additional ORFs (ORF 3a) and the second largest subgenomic

mRNA encode a protein that has been termed U274 because of its length of 274 amino

acids (Tan et al., 2004c). The subgenomic mRNA encoding U274 has been found in high

quantities in infected cells and also has a strong match to the transcription regulating

consensus sequence close to and upstream of its first ORF (Marra et al., 2003; Rota et al.,

2003; Thiel et al., 2003). The exact function of the protein is unknown but a region in its

carboxyl terminus is similar to calcium-transporting adenosine triphosphatases (Marra et

al., 2003; Rota et al., 2003; Snijder et al., 2003; Thiel et al., 2003). Analysis of sera taken

from patients infected with the SARS-CoV has shown that the sera contains antibodies

against the U274 protein, which indicates that the protein may play a role in the

biogenesis of SARS-CoV (Tan et al., 2004b). In cells infected with SARS-CoV and cells

transiently transfected with U274, the protein localized to the plasma membrane as well

as to the perinuclear region (Tan et al., 2004c). This surface expression may explain the

20

presence of U274 antibodies found in sera taken from infected patients mentioned above

(Tan et al., 2004b). Surfaced expressed U274 is able to undergo endocytosis and its

cytoplasmic domains contain sorting signals that allow proper transport of the protein

(Tan et al., 2004c). Interactions were observed between the U274 protein and other

structural proteins (M, E, and S) as well as the uncharacterized SARS-CoV protein U122.

This suggests that the U274 protein may play a role in virus assembly (Tan et al., 2004c).

U122

Another group specific gene product encoded by ORF7a (also known as ORFX4

or ORF8) (Marra et al., 2003; Rota et al., 2003; Snijder et al., 2003) has been partially

characterized (Fielding et al., 2004). Like the U274 protein, U122 derives its name from

its amino acid length. The U122 appears to localize to the perinuclear region and is

associated with the endoplasmic reticulum in both cells infected with the SARS-CoV and

in cells transiently transfected with the U122 protein (Fielding et al., 2004). From

sequence analysis, it appears that the protein is a type I membrane protein because of its

probable cleaved signal peptide located on its amino terminal and a carboxyl terminal

transmembrane helix (Fielding et al., 2004). Another study has indicated that over-

expression of U122 is able to induce apoptosis via a caspase-dependent pathway (Tan et

al., 2004a).

Protein Antigenicity

The induction of the host immunological response against coronavirus infection is

largely due to the spike glycoprotein (Collins et al., 1982; Koolen et al., 1990; Spaan,

Cavanagh, and Horzinek, 1988). Analysis of its antigenic structure has been useful

particularly in vaccine design (Posthumus et al., 1990). Particular emphasis has been

21

placed on locating potential neutralization epitopes on the spike glycoprotein which

include linear and conformational epitopes (Daniel et al., 1993; Kubo, Yamada, and

Taguchi, 1994; Moore, Jackwood, and Hilt, 1997; Takase-Yoden et al., 1991; Talbot and

Buchmeier, 1985; Vautherot, Laporte, and Boireau, 1992; Vautherot et al., 1992).

Induction of a protective immune response was able to be accomplished through

immunization of animals with a synthetic peptide from a particular immunodominant

region of the MHV spike protein (Daniel, Lacroix, and Talbot, 1994; Koo et al., 1999;

Koolen et al., 1990; Yu et al., 2000). This immunodominant region of the MHV spike

was also shown to be recognized by viral neutralizing antibodies (Daniel et al., 1993;

Luytjes et al., 1989; Talbot and Buchmeier, 1985; Talbot et al., 1984). As with other

coronaviruses, there have been regions of the SARS-CoV spike that have been found that

are able to elicit a neutralizing antibody response from animals injected with the synthetic

peptides from these regions (Zhang et al., 2004). Expression of the other structural

proteins (M, E, or N), in the absence of SARS-CoV spike expression, is unable to provide

protective immunity to SARS-CoV infection (Buchholz et al., 2004).

Organization of the Viral Genome

The genomes of several coronaviruses are compared and shown in Figure 1.2.

Coronaviral genomes are capped, polyadenylated, positive stranded RNAs of 27 to 32 kb

in length. Since they are positive strand genomes, they are able to serve as mRNAs and it

has been shown that the purified genomic RNA is infectious (Lomniczi, 1977;

Schochetman, Stevens, and Simpson, 1977). The leader RNA is a sequence of 65 to 98

nucleotides that is present at the 5' end of the genome as well as at the 5' ends of all

22

Figure 1.2. Genomic Organization of several serotypes of coronavirus. The genome structure for five previously sequenced coronavirus RNAs are shown. All genes, except for gene 1 (pp1ab) are drawn approximately to scale. The severe acute respiratory syndrome (SARS-CoV), mouse hepatitis virus (MHV), avian infectious bronchitis virus (IBV), porcine transmissible gastroenteritis virus (TGEV), and human respiratory coronavirus (HCoV-229E) are composed of positive stranded RNA genomes of approximately 29.7, 31.2, 27.6, 28.5, and 27.2 kb respectively. The 5' end of RNA genome is capped and contains a 65- to 98-base long leader sequence (L). Structural proteins represented by shaded boxes while nonstructural proteins are shown as unshaded boxes. Vertical lines shown between open reading frames (ORFs) represent intergenic sequences. The area found bracketed by two intergenic sequences represents a single gene. Each separate ORF within that single gene is translated from a single mRNA species. Several variations exist between the serotypes of coronaviruses in the number, location, and sequence of ORFs encoding nonstructural proteins. The positive stranded RNAs encoding the coronavirus genomes are polyadenylated at their 3' end.

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subgenomic mRNAs (Lai et al., 1983; Shieh et al., 1987; Spaan et al., 1983). This leader

sequence is followed by an untranslated region of approximately 200 to 400 nucleotides.

There is another untranslated region of approximately 200 to 500 nucleotides at the 3' end

of the genome which is followed by a poly(A) tail that varies in length. The sequences

that make up both the 5' and 3' untranslated region are essential for RNA transcription

and replication. The remaining genome is made up of 7 to 10 open reading frames that

encode the genes necessary for virus replication. Specifically for SARS, the genome

contains 10 open reading frames encoding the replicase gene, 4 structural proteins, and 5

potential nonstructural genes that are more than 50 amino acids in length (Rota et al.,

2003). The first open reading frame makes up two thirds of the viral genome

(approximately 20 to 22 kb in length) and encodes a polyprotein that is the precursor of

the viral polymerase. This gene actually consists of two over lapping open reading

frames that is effectively combined into a single open reading frame through ribosomal

frame shifting. The order in which the polymerase polyprotein and the four structural

proteins found in all coronaviruses is 5'-Pol-S-E-M-N-3'. Several other open reading

frames that encode a variety of nonstructural proteins can be found interspersed between

the known structural proteins. Some coronaviruses also contain a gene that encodes for a

hemagglutinin-esterase protein. The number of nonstructural genes present as well as

their order in the viral genome, sequence, and method of expression vary widely among

coronaviruses (Lai and Cavanagh, 1997). The functions of most of these nonstructural

proteins are widely unknown; some are even absent in genomes of some coronaviruses

(Schwarz, Routledge, and Siddell, 1990; Yokomori and Lai, 1991).

25

The Coronavirus Lifecycle

A general schematic of the major occurrences in the coronavirus lifecycle are

depicted in Figure 1.3.

Viral Attachment and Entry

The initial step in any virus life cycle is the binding of virions to the plasma

membrane of the host cell. The viral protein that is primarily responsible for the binding

of the virion to the plasma membrane of the host cell is the spike glycoprotein. It is the

spike glycoprotein that drives infection of the host cell by facilitating viral attachment to

the target cell and promoting fusion between the host cell plasma membrane and the viral

membrane. This fusion of the two membranes allows the insertion of the viral genome

into the cellular cytoplasm. Viral attachment is accomplished through the interaction of

the spike protein and specific-receptor glycoproteins on the cell surface. Given its role in

viral attachment, the spike protein is the main determinant of viral tropism.

After binding to the specific cellular receptor, the virus enters the cell through the

fusion event between the viral envelope and the plasma membrane or the endosomal

membrane of the host cell. The pH of the environment has been shown to affect the

efficiency of membrane fusion for some coronaviruses such as BCoV, MHV, and IBV

whose optimum pH for cell-cell fusion is either neutral or slightly alkaline (Li and

Cavanagh, 1992; Payne and Storz, 1988; Sturman, Ricard, and Holmes, 1990; Weismiller

et al., 1990). Given that these viruses may cause fusion of the viral envelop with the

plasma membrane at the typical physiological pH of the extracellular environment, it is

likely that they probably enter cells by virus-cell fusion at the plasma membrane. For

26

Fig. 1.3. Schematic diagram of a typical coronavirus replication cycle. Interactions between the spike protein on the virion particle and specific receptor glycoproteins or glycans found on the surface of the target host cell facilitate virion binding to the plasma membrane of the host cell. Spike protein mediated fusion between plasma membrane or endosomal membrane with the viral envelop allows penetration of the cell by the virus. Once inside the cell, gene one encoded by the positive sensed RNA genome is translated into a large polyprotein. Cotranslational or posttranslational processing of this large polyprotein produces an RNA-dependent RNA polymerase as well as several other proteins that are involved in viral RNA synthesis. The genomic RNA is then used as a template by the RNA-dependent RNA polymerase and the other polyprotein products to synthesize negative stranded RNAs. These negative stranded RNAs are subsequently used to produce genomic and subgenomic mRNAs. These mRNAs have an overlapping nested set of 3' coterminal RNAs that possess a common leader sequence at the 5' end. Each of the subgenomic mRNAs, with few exceptions, only have their 5' most ORF translated into viral proteins effectively making them monocistronic. Proteins produced include the S, spike glycoprotein; M, membrane glycoprotein; E, small envelope protein; and N nucleocapsid phosphoprotein as well as several nonstructural proteins. Also the HE (hemagglutinin-esterase glycoprotein) is produced in a small subset of coronaviruses. Once translated, the N protein interacts in the cytoplasm with newly synthesized genomic RNA in order to form helical nucleocapsids. The M and E proteins are inserted in the endoplasmic reticulum and anchored in the Golgi apparatus. The helical nucleocapsid, produced through N protein and RNA interactions, most likely first joins with M at the budding compartment which is located between the rough endoplasmic reticulum and the Golgi apparatus. Interactions between the M and E proteins elicit the budding of virions which encloses the helical nucleocapsid. Also associated in the Golgi, the S and HE structural proteins are translated on membrane-bound polysomes, inserted into the rough endoplasmic reticulum and then transported to the Golgi complex. During protein transport, some of the S and HE proteins interact with the M protein and are incorporated into maturing virion particles. S and HE proteins not incorporated into virions are transported to the cellular surface where they may play a role in mediating cell-cell fusion or hemaadsorption, respectively. Virions seem to be released by exocytosis-like fusion of smooth-walled vesicles that contain the virion particles with the plasma membrane. Virions also may remain attached to the plasma membranes of infected cells. The entire coronavirus replication cycle takes place solely in the cytoplasm of the host cell.

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MHV, however, the efficiency of virus infectivity has been shown to be reduced through

the use of lysosomotropic drugs which suggests that the virus utilizes the endosomal

pathway for entry (Gallagher, Escarmis, and Buchmeier, 1991; Krzystyniak and Dupuy,

1984; Mizzen et al., 1985). Further experimental evidence indicates that different

coronaviruses can enter cells either by the acidic pH-dependent endocytosis or by pH

independent fusion at the plasma membrane (Kooi, Cervin, and Anderson, 1991). Once

the virus has entered the host cell, the next step in life cycle is the uncoating of the virus

and the release of the genomic RNA into the cytoplasm. The mechanism for this

uncoating and release is not currently clear and may require specific cellular proteins in

addition to the incorporated viral elements. Some murine cells, despite having the

presence of a functional MHV receptor, are known to block MHV infections at

penetration, uncoating, or other steps in entry (Asanaka and Lai, 1993; Flintoff, 1984;

Yokomori et al., 1993). These cell types can by grouped into three complementation

groups which suggests that at least three cellular genes are involved in the entry process

(Asanaka and Lai, 1993)

Viral Receptors

Receptors have been elucidated for several coronaviruses. For mouse hepatitis

virus, the receptor is a murine biliary glycoprotein which belongs to the

carcinoembryonic antigen (CEA) family in the Ig superfamily (Dveksler et al., 1991;

Williams, Jiang, and Holmes, 1991). The spike glycoprotein binds to the N-terminal Ig-

like domain of the mouse hepatitis virus receptor in order to facilitate the fusion of the

two membranes (Dveksler et al., 1993b). MHV attachment to host cells can be blocked

by monoclonal antibodies to the viral receptor (Parker, Gallagher, and Buchmeier, 1989;

29

Shieh et al., 1987). Further studies have shown that the MHV is able to use other

members of the CEA family and several human CEA-related glycoproteins as receptors

for viral entry (Chen et al., 1997; Chen et al., 1995; Dveksler et al., 1993a; Nedellec et

al., 1994; Yokomori and Lai, 1992a; Yokomori and Lai, 1992b). Other coronaviruses

such as HCoV-229E, TGEV, FIPV, and most likely canine coronavirus, use the cell

membrane-bound metalloprotease, aminopeptidase N (APN) that is specific for their host

species (Benbacer et al., 1997; Delmas et al., 1992; Yeager et al., 1992) as their entry

receptor. Unlike the CEA molecules which are normally expressed in cells of the liver,

gastrointestinal tract, macrophages, and B cells, but not thymic T cells (Coutelier et al.,

1994; Godfraind et al., 1995), APN is widely dispersed on many cell types which include

respiratory and enteric epithelial cells as well as neuronal and glial cells (Kusters et al.,

1989). Some monoclonals against APN have been shown to block binding of TGEV or

HCoV-229E virions to the host cell receptor (Delmas et al., 1992; Yeager et al., 1992);

however APN protease activity is not a requirement for viral infection (Delmas et al.,

1992; Williams, Jiang, and Holmes, 1991). The cellular receptor for SARS-CoV was

originally found to be the metallopeptidase angiotensin-converting enzyme 2 (ACE2) (Li

et al., 2003; Wang et al., 2004). Recently, however, an additional receptor, CD209L (L-

SIGN), has been shown to function as a receptor for the SARS-CoV (Jeffers et al., 2004).

Angiotensin Converting Enzyme 2

Angiotensin-converting enzyme 2 is a type I transmembrane protein that is made

up of 805 amino acids. It contains a single metalloprotease active site with an HEXXH

zinc binding domain (Kuhn et al., 2004). The ACE2 protein is synthesized in the human

heart muscle, kidneys, testis (Donoghue et al., 2000), gastrointestinal tract, and the lungs

30

(Hamming et al., 2004). In particular, high levels of ACE2 expression have been found

by immunohistochemical examinations in the endothelium of intramyocardial and

intrarenal vessels and in the renal tubular epithelium (Donoghue et al., 2000). The

enzyme appears be the physiological counterweight of the related angiotensin-converting

enzyme (ACE) which is known to cleave the inactive peptide angiotensin I in order to

produce the highly potent vasoconstrictor angiotensin II (Kuhn et al., 2004). Specifically,

ACE2 has been shown to cleave angiotensin I to the metabolite angiotensin (1-9), which

in turn is cleaved to angiotensin (1-7) (Donoghue et al., 2000; Harmer et al., 2002). It

also cleaves des-arg-bradykinin, neurotensin, and kinetensin (Donoghue et al., 2000).

There is significant overlap between the tissues that express the ACE2 protein and those

tissues that are most correlated with SARS-CoV replication and symptomatic

manifestation (Hamming et al., 2004). An obvious example of this overlap would be the

lungs. The lungs are the primary site of SARS-CoV infection (Kuiken et al., 2003) ,and

the lungs express the ACE2 protein (Donoghue et al., 2000; Hamming et al., 2004;

Harmer et al., 2002; Li et al., 2003). A less obvious example is the gastrointestinal tract

and kidneys. These areas have high levels of ACE2 expression and have also been

shown to be an active site of SARS-CoV infection (Donoghue et al., 2000; Hamming et

al., 2004; Harmer et al., 2002). SARS-CoV has not, however, been found to replicate in

the human heart which is a site a high expression of ACE2 (Donoghue et al., 2000).

CD209L (L-SIGN)

CD209L, a homologue of CD209, is a type II transmembrane glycoprotein in the

C-type lectin family that serve as adhesion receptors for ICAM-2 and ICAM-3

(Bashirova et al., 2001; Geijtenbeek et al., 2000; Mummidi et al., 2001). The isoform

31

that has been identified as the receptor for the SARS-CoV is composed of 376 amino

acids. Structurally, the protein has a short cytoplasmic tail, a transmembrane domain, an

extracellular stalk that contains seven repeats of a 23 amino acid sequence

(KAAVGELXEKSKXQEIYQELTXL), and a large carboxyl terminal carbohydrate

recognition domain (Jeffers et al., 2004). This carbohydrate recognition domain has been

shown to bind specifically to high mannose glycans on glycoproteins (Geijtenbeek,

Engering, and Van Kooyk, 2002). It has not yet been determined if it is this carbohydrate

recognition domain that participates in viral interactions with the SARS-CoV spike

glycoprotein, or if the SARS-CoV spike glycoprotein recognizes the protein’s specific

amino acid sequence.

Genome Expression

Once released into the cytoplasm, the viral RNA serves as a template for the

synthesis of the viral RNA dependent RNA polymerase. This RNA dependent RNA

polymerase first synthesizes negative stranded RNAs which are used as templates for the

synthesis of multiple subgenomic RNAs as well as full length copies of the genome. It

has been shown that there are comparable levels of negative polarity genomic and

subgenomic RNA to levels of corresponding positive sense genomic and subgenomic

mRNA in infected cells (Sawicki and Sawicki, 1990; Sethna, Hofmann, and Brian, 1991;

Sethna, Hung, and Brian, 1989). Although levels were comparable, there was no single-

stranded negative sense RNA found in the infected cell, only double-stranded (Perlman et

al., 1986; Sawicki and Sawicki, 1986; Sawicki and Sawicki, 1990). These subgenomic

RNAs account for all of the viral proteins except for the ORF 1ab polyprotein.

Depending on the strain of the virus, coronaviruses produce five to seven subgenomic

32

mRNAs. All of the mRNAs form a nested set that have common 3′ ends followed by a

poly(A) tail. Each of these mRNAs, except for the smallest, contain two or more ORFs

in which only the 5′ most ORF, with few exceptions, is translated. This effectively makes

each subgenomic mRNA functionally monocistronic. The coronavirus subgenomic

RNAs and the genomic RNA share an identical 5′ end leader sequence that is 65 to 98

bases long (Lai et al., 1983; Shieh et al., 1987; Spaan et al., 1983). The leader sequence

is a unique part of the viral genome and only shares partial homology with sequences

found between each gene that have been termed both the transcription-associated

sequence (Hiscox et al., 1995) and the intergenic sequence (Baric et al., 1987). The latter

term will be used for the purposes of this literature review. The core intergenic sequence

shares a common homology with the seven to eighteen nucleotides found at the 3′ end of

the leader (Shieh et al., 1987). Depending on which cell type is used, coronavirus RNA

synthesis occurs at membranous structures associated with the endoplasmic reticulum,

late endosomes, or Golgi complex (Denison et al., 1999; Shi et al., 1999; van der Meer et

al., 1999). Studies have shown that in addition to the RNA dependent RNA polymerase,

the N protein as well as other host components may be involved in viral RNA synthesis

(Compton et al., 1987; Li et al., 1999; Li et al., 1997).

Several models have been proposed to clarify how the subgenomic mRNAs are

synthesized so that the leader sequence RNA is fused to the subgenomic mRNAs are

produced (Figure 1.4). Proposed models of transcription include a discontinuous

transcription process by which the leader and mRNA sequences come from two different

RNA molecules (Jeong and Makino, 1994; Zhang, Liao, and Lai, 1994).

33

Figure 1.4 Schematic diagram of the three proposed models of coronavirus mRNA transcription. Positive stranded RNA is represented by solid black lines while negative stranded RNA is represented by dashed grey lines. Leader RNA and antileader RNA is represented by black and grey boxes respectively. Arrows are used to indicate direction of transcription.

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35

One model uses a leader-primed transcription (Figure 1.4A) in which a discontinous

transcription occurs throughout positive-stranded RNA synthesis (Lai, 1986). The first

step in this model is the genomic RNA being transcribed into a full-length negative-

stranded copy of the RNA genome. This is followed by the transcription of the leader

RNA beginning at the 3’ end of the negative-stranded RNA. Termination of transcription

occurs at the end of the leader sequence which allows the leader to dissociate from the

template. The dissociated leader, possibly with the polymerase still bound to it, then

reanneals to any of the intergenic sequences on the negative-stranded RNA allowing the

priming of mRNA transcription. In this model, the intergenic sequences function as

promoters for mRNA transcription. These intergenic sequences, at least in the case of

MHV, have a minimal core promoter sequence of seven nucleotides (UCUAAAC) (Joo

and Makino, 1992; Makino and Joo, 1993). A novel subgenomic RNA is produced from

this seven nucleotide sequence when it is incorporated into a defective interfering (DI)

RNA which is allowed to be expressed in MHV-infected cells (Makino, Joo, and Makino,

1991). This shows that the seven nucleotide sequence is capable of priming the synthesis

of a subgenomic RNA that is normally not synthesized. The mRNAs produced in this

model include a leader sequence from the 5’ end of the same RNA molecule or a

different RNA molecule (Zhang, Liao, and Lai, 1994). Fusion of the leader RNA occurs

within the core promoter sequence (UCUAAAC), although adjacent sequences may

possibly contribute to the joining of the leader RNA and the subsequent subgenomic

RNA (van der Most, de Groot, and Spaan, 1994). Mutagenesis or deletion of the leader

RNA eradicates or severely compromises mRNA transcription (Liao and Lai, 1994;

Zhang, Liao, and Lai, 1994). Several studies have been concluded whose data support

36

this model of transcription. Cells infected with MHV contain dissociated free leader

RNA ranging in size from 50 to 90 nucleotides (Baric et al., 1987). Although in the case

of bovine coronavirus, such free leader RNAs were not found in infected cells (Chang,

Krishnan, and Brian, 1996). Another experiment in which two distinct strains of MHV

are used to infect the same cell shows that leader RNAs transcribed can be

indiscriminately joined to the subgenomic RNAs from either strain (Makino, Stohlman,

and Lai, 1986). Leader RNAs can also be incorporated into subgenomic mRNAs when

exogenously added to an in vitro transcription system using lysates obtained from MHV-

infected cells (Baker and Lai, 1990).

A second model is based on discontinous transcription during the negative-

stranded RNA synthesis (Figure 1.4B) from the full-length genomic RNA template

(Sawicki and Sawicki, 1990). In this model, the polymerase pauses transcription at one

of the intergenic sequences and then “jumps” to the 3’ end of the leader sequence in the

genomic RNA template. This jumping mechanism generates a negative-stranded

subgenomic RNA with an antisensed leader sequence at its 3’ end which serves as a

template for the synthesis of mRNAs. Intergenic sequences on the positive-stranded

strand may serve as transcriptional termination sites. It is also possible that these

sequences interact with the leader RNA to promote polymerase jumping during negative-

strand RNA synthesis. It is during this phase of negative-strand synthesis in which the

intergenic sequences facilitate leader-mRNA fusion. As with the first model proposed,

there have been several studies that support this model of transcription. One such study

shows that there are equal amounts of subgenomic RNAs and their negative-stranded

counterparts found in infected cells (Sethna, Hung, and Brian, 1989). It has also been

37

shown that the negative-stranded RNAs are exact complementary copies of the viral

subgenomic RNAs complete with 5’ poly(U) sequence and an antisensed leader sequence

at the 3’ end (Hofmann and Brain, 1991; Sethna, Hofmann, and Brian, 1991). Also, it

appears that each subgenomic mRNA is possibly transcribed from a corresponding

subgenomic-sized, negative-stranded template (Sawicki and Sawicki, 1990). These

negative-stranded RNAs have also been found in membrane associated replication

complexes (Sethna and Brian, 1997).

The third and final model proposed for the synthesis of subgenomic mRNAs are

based on the findings of incorporated subgenomic mRNAs (Figure 1.4C), along with the

full length viral genome, within the virions of some coronaviruses such as BCoV, TGEV,

and IBV (Hofmann, Sethna, and Brian, 1990; Sethna, Hung, and Brian, 1989; Zhao,

Shaw, and Cavanagh, 1993). It is postulated by this model that subgenomic RNAs

brought in with the infecting virion are used directly as templates for the synthesis of

negative-sensed subgenomic RNAs. These negative-sensed RNAs then serve as

templates for additional copies of subgenomic mRNAs (Schwarz, Routledge, and Siddell,

1990; Senanayake et al., 1992). This model does not correspond with the discontinous

nature of coronaviral RNA synthesis. Also, not all coronaviruses have been shown to

have subgenomic mRNAs incorporated in the virion. Studies have also tried to promote

mRNA amplification by transfecting subgenomic RNAs into infected cells but were

unsuccessful (Chang et al., 1994; Liao and Lai, 1994). Currently, it cannot be

unequivocally said which of these proposed models is the correct explanation for

coronavirus mRNA synthesis. It is very possible that various aspects of these proposed

models are active at different points in the virus lifecycle.

38

The leader RNA and the intergenic sequences are not the only regulators of

coronavirus mRNA transcription. Several other facets of the RNA have been found to

play a role in transcriptional regulation. One such regulator is the 3’ end of the viral

genome (Lin, Liao, and Lai, 1994; Lin et al., 1996). A significant portion of the 3’ end,

approximately 55 nucleotides at the 3’ end of the genomic RNA as well as the poly(A)

sequence, is required for the initiation of negative-stranded RNA synthesis (Lin, Liao,

and Lai, 1994). Another example of regulation is the need for the complete 3’-UTR to be

present in order for the transcription of the subgenomic mRNAs to occur (Lin et al.,

1996). These two findings seem to indicate that the 3’ end (or the 5’ end in the case of

the negative-stranded RNA) cis-acting sequence is involved in the synthesis of positive-

stranded RNA which in turn indicates that it probably interacts with the leader or

intergenic sequences of the genome to regulate mRNA transcription.

Replication of Genomic RNA The majority (95%) of the genome sized RNA in the infected cell is packaged into

nucleocapsids and virions while the other 5% is used to synthesize mRNA encoding the

polymerase polyprotein (Perlman et al., 1986; Spaan et al., 1981). The replication of the

full length RNA would seemingly require a different mechanism than transcription of the

subgenomic mRNAs since replication of the full length RNA relies on continuous

transcription rather than the discontinuous transcription of subgenomic mRNA synthesis.

This presumption, however, may not be the case. Some studies suggest that some

genome length replication may actually be performed by discontinuous transcription. It

has been shown that some genome sized MHV genomic RNA is in fact a fusion of the

leader sequence to an intergenic sequence that immediately follows the leader RNA

39

which creates a genome sized RNA with a small deletion (Zhang and Lai, 1996). It has

also been found that the leader sequence of MHV DI RNA can be replaced with that of

the helper virus leader sequence (Makino and Lai, 1989b). This substitution of the leader

sequence by the helper virus implies that the transcription of the full length genome

utilizes a step in which the leader sequence dissociates from the template and then

rebinds are some point downstream to begin transcription. This mechanism is also

utilized in subgenomic mRNA synthesis. The quantity of UCUAA repeats at the 3’ end

of the leader have been shown to undergo rapid evolution (Makino and Lai, 1989a) and it

is at these regions that high levels of RNA recombination occur (Keck et al., 1987).

These studies suggest that genomic RNA replication uses the leader dissociation

discontinous synthesis involved in mRNA transcription.

Defective Interfering (DI) RNAs have been used to delineate which cis-acting

sequences are required for the replication of the genomic RNA (Brian and Spaan, 1997;

Makino, Fujioka, and Fujiwara, 1985; Makino et al., 1988a; Makino et al., 1988b; van

der Most, Bredenbeek, and Spaan, 1991). For MHV, replication of the viral genome

requires a 135 nucleotide internal replication sequence along with approximately 400 to

800 nucleotides at both the 3’ and 5’ ends of the viral genome (Kim, Lai, and Makino,

1993; Lin and Lai, 1993). A 57 nucleotide portion of the replication sequence in the

MHV DI RNAs, which is required for DI replication, has been found to form a secondary

structure in the positive strand (Kim and Makino, 1995; Lin and Lai, 1993). Both the

higher order structure and the sequence itself have been shown to be vital for the function

of the replication signal (Repass and Makino, 1998). The length of required cis-acting

40

regions for viral genome replication and subgenomic mRNA synthesis differ slightly

from one another (Liao and Lai, 1994; Lin and Lai, 1993; Lin et al., 1996).

Translation of Viral Proteins As previously noted, transcription of the viral genome results in multiple

subgenomic mRNAs which, with the exception of the smallest mRNA, contain two or

more ORFs in which only the 5’ most ORF, with few exceptions, is translated. Structural

proteins are translated by a cap-dependent ribosomal scanning mechanism from separate

mRNAs. Translation in virus infected lysates is enhanced by a 5’ leader sequence found

in all of the subgenomic mRNAs (Tahara et al., 1994). This enhancement of translation

may give viral mRNAs an advantage as host cell translation is being shut off by the viral

infection. Besides the structural proteins, the virus encoded polymerase is encoded

within two large overlapping ORFs (ORF1AB) that utilize a ribosomal frameshifting

mechanism in order to synthesize the entire polyprotein (Boursnell et al., 1987; Brierley,

Jenner, and Inglis, 1992; Eleouet et al., 1995; Herold et al., 1993; Lee et al., 1991). This

translation is initiated by the conventional cap-dependent translation mechanism. For

several coronaviruses, the second or third ORF has the highest efficiency of translation.

This is due to the presence of an internal ribosomal entry site just before the ORF that

allows the ribosome to bypass the preceding ORFs. This allows the translation of the

targeted ORF through a cap-independent translation mechanism (Liu and Inglis, 1992b;

Thiel and Siddell, 1994). Other than the virus encoded polymerase, there are other

proteins in the genome that contain more than one ORF. These proteins are translated by

unknown mechanisms. Examples of this include an internal ORF within the N genes of

41

MHV and BCoV (Fischer et al., 1997; Senanayake et al., 1992) and the two ORFs found

on the fifth subgenomic mRNA of IBV (Liu and Inglis, 1992a).

Assembly and Release of Virions

The initial step in virion assembly is the formation of helical nucleocapsids by the

nucleocapsid proteins binding to the viral RNA. This binding of viral RNA to

nucleocapsid protein is facilitated by a sequence of nucleotides found in ORF 1B which

is only present in the genomic-length RNA. This sequence has been shown to bind the

nucleocapsid protein (Masters et al., 1994), and is thought to facilitate packaging of viral

genomes into virions. Small DI RNAs composed only of terminal sequences are able to

be packaged into virions, which indicate that there is some form of packaging signal

contained within these terminal sequences. This packaging has been observed in several

coronaviruses such as TGEV, BCoV, and IBV (Hofmann, Sethna, and Brian, 1990;

Sethna, Hung, and Brian, 1989; Zhao, Shaw, and Cavanagh, 1993). Although the

terminal signals allow packaging of the DI particle, it may not be responsible for

packaging the viral genome. A 69 nucleotide packaging signal has been identified

through the creation of artificial chimeric DI RNAs of MHV (Fosmire, Hwang, and

Makino, 1992; Makino, Yokomori, and Lai, 1990; van der Most, Bredenbeek, and Spaan,

1991). This putative packaging signal maps to a region within ORF 1b and has been

shown to function by maintaining secondary structure (Fosmire, Hwang, and Makino,

1992), which allows interaction between the packaging signal and the RNA being

packaged (Bos et al., 1997; Woo et al., 1997). Packaging of reporter RNAs has also been

accomplished by fusing a homologous region of the bovine coronavirus genome to the

nonviral RNAs (Cologna and Hogue, 2000). Packaging efficiency for large TGEV DI

42

RNAs is affected by the presence of other portions of the ORF 1B suggesting that the

packaging of viral RNA may use additional packaging signals found in this region (Izeta

et al., 1999).

Once the nucleocapsid forms, it interacts with the matrix protein at cellular

membranes (Sturman, Holmes, and Behnke, 1980) of the ER or the Golgi complex. The

nucleocapsid protein can only be incorporated into virions when complexed with the viral

RNA; no unbound protein is able to be packaged into virions (Bos et al., 1996; Vennema

et al., 1996). This suggests that the M protein must interact with the viral genome

directly. Alternatively, a conformational change may occur when the N protein interacts

with the viral genome promoting the interaction between the N and M proteins. This

interaction may enable the nucleocapsid to be packaged into budding virus particles

formed on the membranes of the ER and the Golgi while simultaneously allowing the

formation of the spherical internal core shell surrounding the nucleocapsid (Risco et al.,

1996).

Virus like particles are able to be formed by the expression of the M and E

proteins without the presence of any other additional viral proteins (Bos et al., 1996;

Vennema et al., 1996). This suggests, for most coronaviruses, that the interactions

between the M and E proteins facilitate the formation of virion particles. Mutations

introduced into the E and M proteins have shown altered virus morphology (Fischer et

al., 1998) and abrogated virus like particle formation (de Haan et al., 1998), respectively.

Unlike other coronaviruses, the SARS coronavirus can produce virus like particles

through expression of the M and N proteins alone but particles are not formed with

expression of the M and E proteins (Huang et al., 2004). Regardless of the coronavirus

43

type, these experiments show that the spike protein is dispensable in virus particle

formation. This conclusion is also supported by the fact that tunicamycin-treated cells

infected with MHV produce noninfectious particles that do not contain the spike protein

(Holmes, Doller, and Sturman, 1981). It should be noted, however, that when the spike

protein is expressed along with M and E (M and N for the SARS-CoV), virus like

particles are produced with the spike protein incorporated in them (Bos et al., 1996;

Huang et al., 2004; Vennema et al., 1996).

The budding compartment, located between the ER and Golgi, is the site at which

virus budding is first detected (Dubois-Dalcq et al., 1982; Klumperman et al., 1994;

Tooze, Tooze, and Warren, 1984; Tooze and Tooze, 1985). Interaction between the M

protein, which accumulates at the budding compartment (Klumperman et al., 1994) and

the E protein is thought to trigger virus budding. Because virus budding has only been

shown to occur at the budding compartment and the E protein has been detected at sites

other than that of virus budding (Godet et al., 1994; Yokomori and Lai, 1992a), it is

thought that the M protein dictates the location of virus budding. Although the E protein

is required for particle formation, it may only serve as a scaffolding protein which is not

essential part of virus maturation. This is because the E protein is present in such low

quantities compared to that of the M protein (Vennema et al., 1996). It may function in

the pinching off of the budding virions at the budding compartment because it has been

shown to induce curvature of intracellular membranes containing M (Vennema et al.,

1996).

Incorporation of the spike protein and the HE protein into virions is directed by

interactions with the M protein which occurs in the pre-Golgi complex (Nguyen and

44

Hogue, 1997; Opstelten et al., 1995). The glycoproteins S and HE are processed as the

virions pass through the Golgi where they may undergo additional morphological

changes resulting in the compact, electron dense internal core typical of the mature virus

particle (Risco et al., 1998; Salanueva, Carrascosa, and Risco, 1999). After the Golgi,

mature virions accrue in large, smooth-walled vesicles that fuse with plasma membrane

in order to release the virions into the extracellular space (Griffiths and Rottier, 1992).

Although virus release is restricted to certain areas of the cell, the exact mechanism

dictating this site restriction is poorly understood.

Cellular Proteins Involved in Coronavirus Replication Coronaviruses require cellular proteins in addition to its viral proteins in order to

replicate. These cellular proteins are hijacked from their normal functions in the cell to

assist the virus in its replication. Crosslinking experiments failed to show any

interactions, with the exception of the N protein, between the viral RNA and any of the

coronavirus proteins which suggest that viral proteins interact with the viral RNA

indirectly through cellular proteins. Several cellular proteins have been shown to bind to

MHV viral RNA. These portions of viral RNA are known to play regulatory functions in

other aspects of the virus lifecycle. Such regulatory elements include the 5’ and 3’ ends

of the genomic RNA, intergenic regions of the RNA genome, and the 3’ end of the

negative-strand RNA.

HNRNP A1

Several different cellular proteins have been identified that bind to the intergenic

regions of the template coronavirus RNA (Zhang and Lai, 1995a). Deletion analysis and

mutagenesis of the intergenic sites have correlated transcription efficiency with RNA

45

protein binding. This suggests that these cellular RNA binding proteins play an

important role in the regulation of coronavirus mRNA transcription. One of the proteins

was identified by partial peptide mapping to be hnRNP A1 (Li et al., 1997). This cellular

protein is known to be an RNA-binding protein that contains two RNA-binding domains

along with a glycine-rich domain responsible for protein-protein interactions. hnRNP A1

primarily exists as a nuclear protein, but it also has a shuttling function that cycles it

between the nucleus and the cytoplasm (Pinol-Roma and Dreyfuss, 1992). Its primary

function depends on its location. When in the cytoplasm, hnRNP A1 modulates mRNA

turnover and translation by binding to AU-rich elements on cytoplasmic mRNA

(Hamilton et al., 1997; Hamilton et al., 1993; Henics et al., 1994). While in the nucleus,

it is involved in pre-mRNA splicing and transport of cellular RNAs (Dreyfuss et al.,

1993). Specifically for MHV, hnRNP A1 binds the negative-strand leader along with

intergenic sequences (Furuya and Lai, 1993; Li et al., 1997). These sequences are known

to be critical elements for discontinuous viral RNA transcription. Mutagenesis of the

intergenic sequences disrupts hnRNP A1 binding which hinders efficiency of

transcription from the altered intergenic site (Furuya and Lai, 1993; Li et al., 1997; Zhang

and Lai, 1995b). Another function of hnRNP A1 may be in its interaction with the

nucleocapsid protein (Wang and Zhang, 1999) which is known to bind to the viral RNA

directly (Baric et al., 1988; Stohlman et al., 1988).

PTB

PTB, also known as hnRNP I, is another cellular protein found to be involved in

coronavirus replication. It is known to bind to the UC-rich RNA sequences found near

the 3’ end of introns, to play a role in the regulation of translation of viral and cellular

46

RNAs, as well as alternative splicing of pre-mRNAs, and is similar in function to hnRNP

A1 in that it also shuttles between the nucleus and the cytoplasm (Kaminski et al., 1995;

Svitkin et al., 1996; Valcarcel and Gebauer, 1997). Immunoprecipitation and

crosslinking studies have established PTB binds to the MHV positive-strand leader RNA

(Li et al., 1999). This region of the MHV RNA has been shown previously to be needed

for MHV RNA synthesis and to regulate transcription (Kim, Jeong, and Makino, 1993;

Liao and Lai, 1994; Tahara et al., 1994). Deletion of the mentioned binding sites causes

significant inhibition of RNA transcription (Li et al., 1999). This suggests that PTB may

play a role in coronavirus mRNA translation although it does not have a direct effect on

the cap-dependent MHV RNA translation (Choi and Lai, unpublished data).

PABP

Poly(A)-binding protein (PABP) binds to the 3’ UTR region of coronaviral RNA

which is necessary for the synthesis of negative-stranded viral RNA and both genomic

and subgenomic positive-strand RNA synthesis (Kim, Jeong, and Makino, 1993; Lin and

Lai, 1993; Lin, Liao, and Lai, 1994; Lin et al., 1996). These 3’ UTR regions contain

structures that are conserved among several different strains of coronaviruses (Hsue,

Hartshorne, and Masters, 2000; Hsue and Masters, 1997; Liu, Johnson, and Leibowitz,

2001). It is known that PABP is a highly abundant cytoplasmic protein that binds to the

3’ poly(A) tail on eukaryotic mRNAs (Gorlach, Burd, and Dreyfuss, 1994). Binding of

PABP to the 3’ UTR of DI RNA replicons correlates with the ability of the RNA to

replicate which suggests that the PABP interaction with the poly(A) tail may have an

effect on coronavirus RNA replication (Huang and Lai, 2001; Liu, Yu, and Leibowitz,

1997; Spagnolo and Hogue, 2000; Yu and Leibowitz, 1995a; Yu and Leibowitz, 1995b).

47

Mitochondrial Aconitase

Despite the absence of a consensus RNA-binding domain, crosslinking

experiments have indicated that mitochondrial aconitase binds to the MHV 3’ protein-

binding element (Burd and Dreyfuss, 1994). This binding increases the stability on the

viral mRNA enhancing the translation of viral proteins. In addition to binding to the

MHV genome, mitochondrial aconitase also colocalizes with the MHV nucleocapsid

protein suggesting a potential interaction with the MHV replication complex (Nanda and

Leibowitz, 2001).

Pathology and Disease

Coronaviruses are known to cause a wide array of pathologies including acute

respiratory disease, hepatitis, chronic demyelination in the central nervous system (CNS),

encephalitis, and enteritis (Holmes, 1996). Generally the virus infects the respiratory and

enteric mucosal surfaces (Navas-Martin and Weiss, 2003) and is able to cause both self-

limiting acute and chronic persistent infections. Some coronaviruses, such as MHV,

target other cells such as hepatocytes, endothelial cells, neurons, macrophages,

astrocytes, and oligodendrocytes (Haring and Perlman, 2001).

Before the discovery of the SARS-CoV only two coronaviruses were known to

infect humans, HCoV-229E and HCoV-OC43. The infections caused by theses viruses

were self-limiting upper respiratory tract infections (Myint, 1994) that account for

approximately 30% of all common colds. These viruses, however, have never been

reported to cause severe illness. Other coronaviruses such as TGEV (porcine), BCoV

(bovine), and IBV (avian), cause respiratory and enteric diseases that can result in severe

economic loss in the farming industry. The SARS-CoV originated in Guangdong

48

Province, China in late 2002 (Parry, 2003). From there it spread throughout Asia and by

the end of the epidemic caused more than 8000 cases of infection worldwide, according

to the Centers for Disease Control (CDC) and the World Health Organization (WHO)

(Navas-Martin and Weiss, 2003). Out of the more than 8000 cases reported, more than

800 of these resulted in death with mortality rates, depending on the age of the victim, as

high as 15% (Anand et al., 2003).

The SARS-CoV infection exhibits a wide clinical course characterized by fever,

dyspnea, lymphopenia and lower tract respiratory infection (Nie et al., 2003; Tsui et al.,

2003) along with gastrointestinal symptoms and diarrhea (Booth et al., 2003; Lee et al.,

2003; Leung et al., 2003; Peiris et al., 2003). A three stage disease model that has been

proposed consists of viral replication, immune hyperactivity, and pulmonary destruction

(Tsui et al., 2003). SARS pathology of the lung has been correlated with diffuse alveolar

damage, epithelial cell proliferation, and an increase of macrophages (Navas-Martin and

Weiss, 2004). Similar to many coronavirus infections, multinucleate giant-cell infiltrates

of macrophage and epithelial origin have been associated with putative syncytium-like

formation (Nicholls et al., 2003). As found in the fatal influenza subtype H5N1 disease

in 1997, lymphopenia, hemophagocytosis in the lung and white-pulp atrophy of the

spleen are also observed during the SARS-CoV infection (To et al., 2001). This

hemophagocytosis found in the lung supports a cytokine dysregulation (Fisman, 2000)

which may have a role in the pathogenesis of SARS due to the proinflammatory

cytokines being released by stimulatory macrophages in the alveoli.

Based on the known pathology of the SARS-CoV, several treatments have been

tried including the administration of steroids to try to control the exacerbated cytokine

49

response (Lai et al., 2003). However, treatments of the infection have been largely

ineffective (Koren et al., 2003; Lee et al., 2003; Tsui et al., 2003). SARS-CoV infection

is also resistant to ribavirin, a nucleoside analog that normally has a broad antiviral

activity and is presently being used for the treatment of respiratory syncytial virus (RSV)

(Everard et al., 2001) and hepatitis C (Lipman and Cotler, 2003; Martin et al., 1998).

Interferons α, β, and γ have also been tested for their antiviral properties against the

SARS-CoV in vitro with interferon β showing the most promise (Cinatl et al., 2003).

The exact mechanism that allowed the emergence of the SARS-CoV possible is

unknown. Although the coronavirus biological vector is not known, it is speculated that

the virus jumped from an animal species to humans (Holmes, 2003). Several domestic

and wild animals from the Guangdong Province have been examined in order to

determine if any of them were carrying the SARS-CoV. Viruses similar to SARS have

been isolated from Himalayan palm civets, raccoon dogs, and Chinese ferret badgers

found in a retail market in China (Guan et al., 2003). It is speculated that the SARS-CoV

does indeed have an animal reservoir (Holmes, 2003).

In addition to the recent impact that the SARS-CoV has had on public health,

other coronaviruses have an economic impact in the cattle industry. Primarily, bovine

coronaviruses were known to be enteropathogenic viruses that caused severe diarrhea in

neonatal calves. Coronavirus virions were able to be isolated from diarrhea fluid and

intestinal samples from animals infected with the disease (Doughri et al., 1976; Mebus et

al., 1973). Theses enteric viruses were also able to be isolated from the feces of adult

cattle with winter dysentery (Saif et al., 1988). It was not until recently that

coronaviruses were able to be isolated from the nasal secretions and lung tissues of cattle

50

infected with fatal cases of shipping fever pneumonia. These isolated viruses were

termed respiratory coronaviruses (Storz et al., 2000a; Storz et al., 2000b) in order to

differentiate them from the commonly found enteric strains known as enteropathogenic

bovine coronaviruses. There two related viruses are separated by phenotypic variation,

antigenic differences, and genetic divergence (Chouljenko et al., 1998; Chouljenko et al.,

2001; Lin et al., 2000; Storz et al., 2000a). A comparison study between respiratory and

enteropathogenic coronavirus isolated from the same animal with fatal shipping

pneumonia suggests that the difference in viral tropism is due to slight variations in the

S1 subunit of the Spike glycoprotein (Chouljenko et al., 1998; Gelinas et al., 2001;

Hasoksuz et al., 2002; Rekik and Dea, 1994).

It is the continuing research on all coronaviruses as well as the SARS-CoV

specifically that will allow a better understanding of the SARS virus and its implications

on public health.

REFERENCES

An, S., Chen, C. J., Yu, X., Leibowitz, J. L., and Makino, S. (1999). Induction of apoptosis in murine coronavirus-infected cultured cells and demonstration of E protein as an apoptosis inducer. J Virol 73(9), 7853-9.

An, S., Maeda, A., and Makino, S. (1998). Coronavirus transcription early in infection. J

Virol 72(11), 8517-24. Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R., and Hilgenfeld, R. (2003).

Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science 300(5626), 1763-7.

Arbely, E., Khattari, Z., Brotons, G., Akkawi, M., Salditt, T., and Arkin, I. T. (2004). A

highly unusual palindromic transmembrane helical hairpin formed by SARS coronavirus E protein. J Mol Biol 341(3), 769-79.

Armstrong, J., Niemann, H., Smeekens, S., Rottier, P., and Warren, G. (1984). Sequence

and topology of a model intracellular membrane protein, E1 glycoprotein, from a coronavirus. Nature 308(5961), 751-2.

51

Asanaka, M., and Lai, M. M. (1993). Cell fusion studies identified multiple cellular

factors involved in mouse hepatitis virus entry. Virology 197(2), 732-41. Baker, K. A., Dutch, R. E., Lamb, R. A., and Jardetzky, T. S. (1999). Structural basis for

paramyxovirus-mediated membrane fusion. Mol Cell 3(3), 309-19. Baker, S. C., and Lai, M. M. (1990). An in vitro system for the leader-primed

transcription of coronavirus mRNAs. Embo J 9(12), 4173-9. Baric, R. S., Nelson, G. W., Fleming, J. O., Deans, R. J., Keck, J. G., Casteel, N., and

Stohlman, S. A. (1988). Interactions between coronavirus nucleocapsid protein and viral RNAs: implications for viral transcription. J Virol 62(11), 4280-7.

Baric, R. S., Shieh, C. K., Stohlman, S. A., and Lai, M. M. (1987). Analysis of

intracellular small RNAs of mouse hepatitis virus: evidence for discontinuous transcription. Virology 156(2), 342-54.

Bashirova, A. A., Geijtenbeek, T. B., van Duijnhoven, G. C., van Vliet, S. J., Eilering, J.

B., Martin, M. P., Wu, L., Martin, T. D., Viebig, N., Knolle, P. A., KewalRamani, V. N., van Kooyk, Y., and Carrington, M. (2001). A dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes HIV-1 infection. J Exp Med 193(6), 671-8.

Baudoux, P., Carrat, C., Besnardeau, L., Charley, B., and Laude, H. (1998). Coronavirus

pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes. J Virol 72(11), 8636-43.

Benbacer, L., Kut, E., Besnardeau, L., Laude, H., and Delmas, B. (1997). Interspecies

aminopeptidase-N chimeras reveal species-specific receptor recognition by canine coronavirus, feline infectious peritonitis virus, and transmissible gastroenteritis virus. J Virol 71(1), 734-7.

Booth, C. M., Matukas, L. M., Tomlinson, G. A., Rachlis, A. R., Rose, D. B., Dwosh, H.

A., Walmsley, S. L., Mazzulli, T., Avendano, M., Derkach, P., Ephtimios, I. E., Kitai, I., Mederski, B. D., Shadowitz, S. B., Gold, W. L., Hawryluck, L. A., Rea, E., Chenkin, J. S., Cescon, D. W., Poutanen, S. M., and Detsky, A. S. (2003). Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area. Jama 289(21), 2801-9.

Bos, E. C., Dobbe, J. C., Luytjes, W., and Spaan, W. J. (1997). A subgenomic mRNA

transcript of the coronavirus mouse hepatitis virus strain A59 defective interfering (DI) RNA is packaged when it contains the DI packaging signal. J Virol 71(7), 5684-7.

52

Bos, E. C., Luytjes, W., and Spaan, W. J. (1997). The function of the spike protein of mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity. J Virol 71(12), 9427-33.

Bos, E. C., Luytjes, W., van der Meulen, H. V., Koerten, H. K., and Spaan, W. J. (1996).

The production of recombinant infectious DI-particles of a murine coronavirus in the absence of helper virus. Virology 218(1), 52-60.

Bosch, B. J., van der Zee, R., de Haan, C. A., and Rottier, P. J. (2003). The coronavirus

spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex. J Virol 77(16), 8801-11.

Boursnell, M. E., Brown, T. D., Foulds, I. J., Green, P. F., Tomley, F. M., and Binns, M.

M. (1987). Completion of the sequence of the genome of the coronavirus avian infectious bronchitis virus. J Gen Virol 68 ( Pt 1), 57-77.

Brian, D. A., and Spaan, W. (1997). Recombination and coronavirus defective interfering

RNAs. Seminars in virology 8, 101-111. Brierley, I., Jenner, A. J., and Inglis, S. C. (1992). Mutational analysis of the "slippery-

sequence" component of a coronavirus ribosomal frameshifting signal. J Mol Biol 227(2), 463-79.

Buchholz, U. J., Bukreyev, A., Yang, L., Lamirande, E. W., Murphy, B. R., Subbarao,

K., and Collins, P. L. (2004). Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc Natl Acad Sci U S A 101(26), 9804-9.

Burd, C. G., and Dreyfuss, G. (1994). Conserved structures and diversity of functions of

RNA-binding proteins. Science 265(5172), 615-21. Cavanagh, D. (1995). "The Coronavirus Surface Glycoprotein." Plenum Press Inc., New

York, NY. Cavanagh, D. (1997). Nidovirales: a new order comprising Coronaviridae and

Arteriviridae. Arch Virol 142(3), 629-33. Chang, R. Y., Hofmann, M. A., Sethna, P. B., and Brian, D. A. (1994). A cis-acting

function for the coronavirus leader in defective interfering RNA replication. J Virol 68(12), 8223-31.

Chang, R. Y., Krishnan, R., and Brian, D. A. (1996). The UCUAAAC promoter motif is

not required for high-frequency leader recombination in bovine coronavirus defective interfering RNA. J Virol 70(5), 2720-9.

53

Chen, D. S., Asanaka, M., Chen, F. S., Shively, J. E., and Lai, M. M. (1997). Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors. J Virol 71(2), 1688-91.

Chen, D. S., Asanaka, M., Yokomori, K., Wang, F., Hwang, S. B., Li, H. P., and Lai, M.

M. (1995). A pregnancy-specific glycoprotein is expressed in the brain and serves as a receptor for mouse hepatitis virus. Proc Natl Acad Sci U S A 92(26), 12095-9.

Chouljenko, V. N., Kousoulas, K. G., Lin, X., and Storz, J. (1998). Nucleotide and

predicted amino acid sequences of all genes encoded by the 3' genomic portion (9.5 kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses. Virus Genes 17(1), 33-42.

Chouljenko, V. N., Lin, X. Q., Storz, J., Kousoulas, K. G., and Gorbalenya, A. E. (2001).

Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia. J Gen Virol 82(Pt 12), 2927-33.

Cinatl, J., Morgenstern, B., Bauer, G., Chandra, P., Rabenau, H., and Doerr, H. W.

(2003). Treatment of SARS with human interferons. Lancet 362(9380), 293-4. Collins, A. R., Knobler, R. L., Powell, H., and Buchmeier, M. J. (1982). Monoclonal

antibodies to murine hepatitis virus-4 (strain JHM) define the viral glycoprotein responsible for attachment and cell--cell fusion. Virology 119(2), 358-71.

Cologna, R., and Hogue, B. G. (2000). Identification of a bovine coronavirus packaging

signal. J Virol 74(1), 580-3. Compton, S. R., Rogers, D. B., Holmes, K. V., Fertsch, D., Remenick, J., and McGowan,

J. J. (1987). In vitro replication of mouse hepatitis virus strain A59. J Virol 61(6), 1814-20.

Corse, E., and Machamer, C. E. (2000). Infectious bronchitis virus E protein is targeted to

the Golgi complex and directs release of virus-like particles. J Virol 74(9), 4319-26.

Corse, E., and Machamer, C. E. (2002). The cytoplasmic tail of infectious bronchitis

virus E protein directs Golgi targeting. J Virol 76(3), 1273-84. Corse, E., and Machamer, C. E. (2003). The cytoplasmic tails of infectious bronchitis

virus E and M proteins mediate their interaction. Virology 312(1), 25-34. Coutelier, J. P., Godfraind, C., Dveksler, G. S., Wysocka, M., Cardellichio, C. B., Noel,

H., and Holmes, K. V. (1994). B lymphocyte and macrophage expression of carcinoembryonic antigen-related adhesion molecules that serve as receptors for murine coronavirus. Eur J Immunol 24(6), 1383-90.

54

Daniel, C., Anderson, R., Buchmeier, M. J., Fleming, J. O., Spaan, W. J., Wege, H., and

Talbot, P. J. (1993). Identification of an immunodominant linear neutralization domain on the S2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure. J Virol 67(3), 1185-94.

Daniel, C., Lacroix, M., and Talbot, P. J. (1994). Mapping of linear antigenic sites on the

S glycoprotein of a neurotropic murine coronavirus with synthetic peptides: a combination of nine prediction algorithms fails to identify relevant epitopes and peptide immunogenicity is drastically influenced by the nature of the protein carrier. Virology 202(2), 540-9.

de Groot, R. J., Luytjes, W., Horzinek, M. C., van der Zeijst, B. A., Spaan, W. J., and

Lenstra, J. A. (1987). Evidence for a coiled-coil structure in the spike proteins of coronaviruses. J Mol Biol 196(4), 963-6.

De Groot, R. J., Van Leen, R. W., Dalderup, M. J., Vennema, H., Horzinek, M. C., and

Spaan, W. J. (1989). Stably expressed FIPV peplomer protein induces cell fusion and elicits neutralizing antibodies in mice. Virology 171(2), 493-502.

de Haan, C. A., Kuo, L., Masters, P. S., Vennema, H., and Rottier, P. J. (1998).

Coronavirus particle assembly: primary structure requirements of the membrane protein. J Virol 72(8), 6838-50.

de Haan, C. A., Smeets, M., Vernooij, F., Vennema, H., and Rottier, P. J. (1999).

Mapping of the coronavirus membrane protein domains involved in interaction with the spike protein. J Virol 73(9), 7441-52.

Delmas, B., Gelfi, J., L'Haridon, R., Vogel, L. K., Sjostrom, H., Noren, O., and Laude, H.

(1992). Aminopeptidase N is a major receptor for the entero-pathogenic coronavirus TGEV. Nature 357(6377), 417-20.

Delmas, B., and Laude, H. (1990). Assembly of coronavirus spike protein into trimers

and its role in epitope expression. J Virol 64(11), 5367-75. Denison, M. R., Spaan, W. J., van der Meer, Y., Gibson, C. A., Sims, A. C., Prentice, E.,

and Lu, X. T. (1999). The putative helicase of the coronavirus mouse hepatitis virus is processed from the replicase gene polyprotein and localizes in complexes that are active in viral RNA synthesis. J Virol 73(8), 6862-71.

Denison, M. R., Zoltick, P. W., Hughes, S. A., Giangreco, B., Olson, A. L., Perlman, S.,

Leibowitz, J. L., and Weiss, S. R. (1992). Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events. Virology 189(1), 274-84.

55

Deregt, D., Gifford, G. A., Ijaz, M. K., Watts, T. C., Gilchrist, J. E., Haines, D. M., and Babiuk, L. A. (1989). Monoclonal antibodies to bovine coronavirus glycoproteins E2 and E3: demonstration of in vivo virus-neutralizing activity. J Gen Virol 70 ( Pt 4), 993-8.

Donoghue, M., Hsieh, F., Baronas, E., Godbout, K., Gosselin, M., Stagliano, N.,

Donovan, M., Woolf, B., Robison, K., Jeyaseelan, R., Breitbart, R. E., and Acton, S. (2000). A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9. Circ Res 87(5), E1-9.

Doughri, A. M., Storz, J., Hajer, I., and Fernando, H. S. (1976). Morphology and

morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves. Exp Mol Pathol 25(3), 355-70.

Dreyfuss, G., Matunis, M. J., Pinol-Roma, S., and Burd, C. G. (1993). hnRNP proteins

and the biogenesis of mRNA. Annu Rev Biochem 62, 289-321. Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S.,

Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R. A., Berger, A., Burguiere, A. M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S., Manuguerra, J. C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D., Schmitz, H., and Doerr, H. W. (2003). Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348(20), 1967-76.

Dubois-Dalcq, M. E., Doller, E. W., Haspel, M. V., and Holmes, K. V. (1982). Cell

tropism and expression of mouse hepatitis viruses (MHV) in mouse spinal cord cultures. Virology 119(2), 317-31.

Duff, K. C., and Ashley, R. H. (1992). The transmembrane domain of influenza A M2

protein forms amantadine-sensitive proton channels in planar lipid bilayers. Virology 190(1), 485-9.

Duff, K. C., Gilchrist, P. J., Saxena, A. M., and Bradshaw, J. P. (1994). Neutron

diffraction reveals the site of amantadine blockade in the influenza A M2 ion channel. Virology 202(1), 287-93.

Dveksler, G. S., Dieffenbach, C. W., Cardellichio, C. B., McCuaig, K., Pensiero, M. N.,

Jiang, G. S., Beauchemin, N., and Holmes, K. V. (1993a). Several members of the mouse carcinoembryonic antigen-related glycoprotein family are functional receptors for the coronavirus mouse hepatitis virus-A59. J Virol 67(1), 1-8.

Dveksler, G. S., Pensiero, M. N., Cardellichio, C. B., Williams, R. K., Jiang, G. S.,

Holmes, K. V., and Dieffenbach, C. W. (1991). Cloning of the mouse hepatitis virus (MHV) receptor: expression in human and hamster cell lines confers susceptibility to MHV. J Virol 65(12), 6881-91.

56

Dveksler, G. S., Pensiero, M. N., Dieffenbach, C. W., Cardellichio, C. B., Basile, A. A.,

Elia, P. E., and Holmes, K. V. (1993b). Mouse hepatitis virus strain A59 and blocking antireceptor monoclonal antibody bind to the N-terminal domain of cellular receptor. Proc Natl Acad Sci U S A 90(5), 1716-20.

Eckert, D. M., and Kim, P. S. (2001). Mechanisms of viral membrane fusion and its

inhibition. Annu Rev Biochem 70, 777-810. Eleouet, J. F., Rasschaert, D., Lambert, P., Levy, L., Vende, P., and Laude, H. (1995).

Complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology 206(2), 817-22.

Everard, M. L., Swarbrick, A., Rigby, A. S., and Milner, A. D. (2001). The effect of

ribavirin to treat previously healthy infants admitted with acute bronchiolitis on acute and chronic respiratory morbidity. Respir Med 95(4), 275-80.

Ewart, G. D., Sutherland, T., Gage, P. W., and Cox, G. B. (1996). The Vpu protein of

human immunodeficiency virus type 1 forms cation-selective ion channels. J Virol 70(10), 7108-15.

Fielding, B. C., Tan, Y. J., Shuo, S., Tan, T. H., Ooi, E. E., Lim, S. G., Hong, W., and

Goh, P. Y. (2004). Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus. J Virol 78(14), 7311-8.

Fischer, F., Peng, D., Hingley, S. T., Weiss, S. R., and Masters, P. S. (1997). The internal

open reading frame within the nucleocapsid gene of mouse hepatitis virus encodes a structural protein that is not essential for viral replication. J Virol 71(2), 996-1003.

Fischer, F., Stegen, C. F., Masters, P. S., and Samsonoff, W. A. (1998). Analysis of

constructed E gene mutants of mouse hepatitis virus confirms a pivotal role for E protein in coronavirus assembly. J Virol 72(10), 7885-94.

Fisman, D. N. (2000). Hemophagocytic syndromes and infection. Emerg Infect Dis 6(6),

601-8. Flintoff, W. F. (1984). Replication of murine coronaviruses in somatic cell hybrids

between murine fibroblasts and rat schwannoma cells. Virology 134(2), 450-9. Fosmire, J. A., Hwang, K., and Makino, S. (1992). Identification and characterization of

a coronavirus packaging signal. J Virol 66(6), 3522-30. Furuya, T., and Lai, M. M. (1993). Three different cellular proteins bind to

complementary sites on the 5'-end-positive and 3'-end-negative strands of mouse hepatitis virus RNA. J Virol 67(12), 7215-22.

57

Gallagher, T. M., Escarmis, C., and Buchmeier, M. J. (1991). Alteration of the pH

dependence of coronavirus-induced cell fusion: effect of mutations in the spike glycoprotein. J Virol 65(4), 1916-28.

Geijtenbeek, T. B., Engering, A., and Van Kooyk, Y. (2002). DC-SIGN, a C-type lectin

on dendritic cells that unveils many aspects of dendritic cell biology. J Leukoc Biol 71(6), 921-31.

Geijtenbeek, T. B., Torensma, R., van Vliet, S. J., van Duijnhoven, G. C., Adema, G. J.,

van Kooyk, Y., and Figdor, C. G. (2000). Identification of DC-SIGN, a novel dendritic cell-specific ICAM-3 receptor that supports primary immune responses. Cell 100(5), 575-85.

Gelinas, A. M., Boutin, M., Sasseville, A. M., and Dea, S. (2001). Bovine coronaviruses

associated with enteric and respiratory diseases in Canadian dairy cattle display different reactivities to anti-HE monoclonal antibodies and distinct amino acid changes in their HE, S and ns4.9 protein. Virus Res 76(1), 43-57.

Godet, M., Grosclaude, J., Delmas, B., and Laude, H. (1994). Major receptor-binding and

neutralization determinants are located within the same domain of the transmissible gastroenteritis virus (coronavirus) spike protein. J Virol 68(12), 8008-16.

Godet, M., L'Haridon, R., Vautherot, J. F., and Laude, H. (1992). TGEV corona virus

ORF4 encodes a membrane protein that is incorporated into virions. Virology 188(2), 666-75.

Godfraind, C., Langreth, S. G., Cardellichio, C. B., Knobler, R., Coutelier, J. P., Dubois-

Dalcq, M., and Holmes, K. V. (1995). Tissue and cellular distribution of an adhesion molecule in the carcinoembryonic antigen family that serves as a receptor for mouse hepatitis virus. Lab Invest 73(5), 615-27.

Gombold, J. L., Hingley, S. T., and Weiss, S. R. (1993). Fusion-defective mutants of

mouse hepatitis virus A59 contain a mutation in the spike protein cleavage signal. J Virol 67(8), 4504-12.

Gorlach, M., Burd, C. G., and Dreyfuss, G. (1994). The mRNA poly(A)-binding protein:

localization, abundance, and RNA-binding specificity. Exp Cell Res 211(2), 400-7.

Gosert, R., Kanjanahaluethai, A., Egger, D., Bienz, K., and Baker, S. C. (2002). RNA

replication of mouse hepatitis virus takes place at double-membrane vesicles. J Virol 76(8), 3697-708.

58

Griffin, S. D., Beales, L. P., Clarke, D. S., Worsfold, O., Evans, S. D., Jaeger, J., Harris, M. P., and Rowlands, D. J. (2003). The p7 protein of hepatitis C virus forms an ion channel that is blocked by the antiviral drug, Amantadine. FEBS Lett 535(1-3), 34-8.

Griffiths, G., and Rottier, P. (1992). Cell biology of viruses that assemble along the

biosynthetic pathway. Semin Cell Biol 3(5), 367-81. Guan, Y., Zheng, B. J., He, Y. Q., Liu, X. L., Zhuang, Z. X., Cheung, C. L., Luo, S. W.,

Li, P. H., Zhang, L. J., Guan, Y. J., Butt, K. M., Wong, K. L., Chan, K. W., Lim, W., Shortridge, K. F., Yuen, K. Y., Peiris, J. S., and Poon, L. L. (2003). Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 302(5643), 276-8.

Guy, J. S., Barnes, H. J., Smith, L. G., and Breslin, J. (1997). Antigenic characterization

of a turkey coronavirus identified in poult enteritis- and mortality syndrome-affected turkeys. Avian Dis 41(3), 583-90.

Hamilton, B. J., Burns, C. M., Nichols, R. C., and Rigby, W. F. (1997). Modulation of

AUUUA response element binding by heterogeneous nuclear ribonucleoprotein A1 in human T lymphocytes. The roles of cytoplasmic location, transcription, and phosphorylation. J Biol Chem 272(45), 28732-41.

Hamilton, B. J., Nagy, E., Malter, J. S., Arrick, B. A., and Rigby, W. F. (1993).

Association of heterogeneous nuclear ribonucleoprotein A1 and C proteins with reiterated AUUUA sequences. J Biol Chem 268(12), 8881-7.

Hamming, I., Timens, W., Bulthuis, M. L., Lely, A. T., Navis, G. J., and van Goor, H.

(2004). Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 203(2), 631-7.

Haring, J., and Perlman, S. (2001). Mouse hepatitis virus. Curr Opin Microbiol 4(4), 462-

6. Harmer, D., Gilbert, M., Borman, R., and Clark, K. L. (2002). Quantitative mRNA

expression profiling of ACE 2, a novel homologue of angiotensin converting enzyme. FEBS Lett 532(1-2), 107-10.

Hasoksuz, M., Sreevatsan, S., Cho, K. O., Hoet, A. E., and Saif, L. J. (2002). Molecular

analysis of the S1 subunit of the spike glycoprotein of respiratory and enteric bovine coronavirus isolates. Virus Res 84(1-2), 101-9.

He, R., Dobie, F., Ballantine, M., Leeson, A., Li, Y., Bastien, N., Cutts, T., Andonov, A.,

Cao, J., Booth, T. F., Plummer, F. A., Tyler, S., Baker, L., and Li, X. (2004).

59

Analysis of multimerization of the SARS coronavirus nucleocapsid protein. Biochem Biophys Res Commun 316(2), 476-83.

He, R., Leeson, A., Andonov, A., Li, Y., Bastien, N., Cao, J., Osiowy, C., Dobie, F.,

Cutts, T., Ballantine, M., and Li, X. (2003). Activation of AP-1 signal transduction pathway by SARS coronavirus nucleocapsid protein. Biochem Biophys Res Commun 311(4), 870-6.

Henics, T., Sanfridson, A., Hamilton, B. J., Nagy, E., and Rigby, W. F. (1994). Enhanced

stability of interleukin-2 mRNA in MLA 144 cells. Possible role of cytoplasmic AU-rich sequence-binding proteins. J Biol Chem 269(7), 5377-83.

Herold, J., Raabe, T., Schelle-Prinz, B., and Siddell, S. G. (1993). Nucleotide sequence of

the human coronavirus 229E RNA polymerase locus. Virology 195(2), 680-91. Hingley, S. T., Gombold, J. L., Lavi, E., and Weiss, S. R. (1994). MHV-A59 fusion

mutants are attenuated and display altered hepatotropism. Virology 200(1), 1-10. Hiscox, J. A., Mawditt, K. L., Cavanagh, D., and Britton, P. (1995). Investigation of the

control of coronavirus subgenomic mRNA transcription by using T7-generated negative-sense RNA transcripts. J Virol 69(10), 6219-27.

Hofmann, M. A., and Brain, D. A. (1991). The 5' end of coronavirus minus-strand RNAs

contains a short poly(U) tract. J Virol 65(11), 6331-3. Hofmann, M. A., Sethna, P. B., and Brian, D. A. (1990). Bovine coronavirus mRNA

replication continues throughout persistent infection in cell culture. J Virol 64(9), 4108-14.

Hogue, B. G., Kienzle, T. E., and Brian, D. A. (1989). Synthesis and processing of the

bovine enteric coronavirus haemagglutinin protein. J Gen Virol 70 ( Pt 2), 345-52.

Holmes, K. V. (1996). Coronaviridae: the viruses and their replication. Fields BN ed. In

"Fields Virolgy", pp. 1075-1103. Lippincott-Raven, Philadelphia. Holmes, K. V. (2003). SARS-associated coronavirus. N Engl J Med 348(20), 1948-51. Holmes, K. V., Doller, E. W., and Sturman, L. S. (1981). Tunicamycin resistant

glycosylation of coronavirus glycoprotein: demonstration of a novel type of viral glycoprotein. Virology 115(2), 334-44.

Hsue, B., Hartshorne, T., and Masters, P. S. (2000). Characterization of an essential RNA

secondary structure in the 3' untranslated region of the murine coronavirus genome. J Virol 74(15), 6911-21.

60

Hsue, B., and Masters, P. S. (1997). A bulged stem-loop structure in the 3' untranslated region of the genome of the coronavirus mouse hepatitis virus is essential for replication. J Virol 71(10), 7567-78.

Huang, P., and Lai, M. M. (2001). Heterogeneous nuclear ribonucleoprotein a1 binds to

the 3'-untranslated region and mediates potential 5'-3'-end cross talks of mouse hepatitis virus RNA. J Virol 75(11), 5009-17.

Huang, Y., Yang, Z. Y., Kong, W. P., and Nabel, G. J. (2004). Generation of synthetic

severe acute respiratory syndrome coronavirus pseudoparticles: implications for assembly and vaccine production. J Virol 78(22), 12557-65.

Ivanov, K. A., Thiel, V., Dobbe, J. C., van der Meer, Y., Snijder, E. J., and Ziebuhr, J.

(2004). Multiple enzymatic activities associated with severe acute respiratory syndrome coronavirus helicase. J Virol 78(11), 5619-32.

Izeta, A., Smerdou, C., Alonso, S., Penzes, Z., Mendez, A., Plana-Duran, J., and

Enjuanes, L. (1999). Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes. J Virol 73(2), 1535-45.

Jeffers, S. A., Tusell, S. M., Gillim-Ross, L., Hemmila, E. M., Achenbach, J. E.,

Babcock, G. J., Thomas, W. D., Jr., Thackray, L. B., Young, M. D., Mason, R. J., Ambrosino, D. M., Wentworth, D. E., Demartini, J. C., and Holmes, K. V. (2004). CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 101(44), 15748-53.

Jeong, Y. S., and Makino, S. (1994). Evidence for coronavirus discontinuous

transcription. J Virol 68(4), 2615-23. Joo, M., and Makino, S. (1992). Mutagenic analysis of the coronavirus intergenic

consensus sequence. J Virol 66(11), 6330-7. Kaminski, A., Hunt, S. L., Patton, J. G., and Jackson, R. J. (1995). Direct evidence that

polypyrimidine tract binding protein (PTB) is essential for internal initiation of translation of encephalomyocarditis virus RNA. Rna 1(9), 924-38.

Keck, J. G., Stohlman, S. A., Soe, L. H., Makino, S., and Lai, M. M. (1987). Multiple

recombination sites at the 5'-end of murine coronavirus RNA. Virology 156(2), 331-41.

Kienzle, T. E., Abraham, S., Hogue, B. G., and Brian, D. A. (1990). Structure and

orientation of expressed bovine coronavirus hemagglutinin-esterase protein. J Virol 64(4), 1834-8.

61

Kim, Y. N., Jeong, Y. S., and Makino, S. (1993). Analysis of cis-acting sequences essential for coronavirus defective interfering RNA replication. Virology 197(1), 53-63.

Kim, Y. N., Lai, M. M., and Makino, S. (1993). Generation and selection of coronavirus

defective interfering RNA with large open reading frame by RNA recombination and possible editing. Virology 194(1), 244-53.

Kim, Y. N., and Makino, S. (1995). Characterization of a murine coronavirus defective

interfering RNA internal cis-acting replication signal. J Virol 69(8), 4963-71. Klumperman, J., Locker, J. K., Meijer, A., Horzinek, M. C., Geuze, H. J., and Rottier, P.

J. (1994). Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding. J Virol 68(10), 6523-34.

Koo, M., Bendahmane, M., Lettieri, G. A., Paoletti, A. D., Lane, T. E., Fitchen, J. H.,

Buchmeier, M. J., and Beachy, R. N. (1999). Protective immunity against murine hepatitis virus (MHV) induced by intranasal or subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope. Proc Natl Acad Sci U S A 96(14), 7774-9.

Kooi, C., Cervin, M., and Anderson, R. (1991). Differentiation of acid-pH-dependent and

-nondependent entry pathways for mouse hepatitis virus. Virology 180(1), 108-19. Koolen, M. J., Borst, M. A., Horzinek, M. C., and Spaan, W. J. (1990). Immunogenic

peptide comprising a mouse hepatitis virus A59 B-cell epitope and an influenza virus T-cell epitope protects against lethal infection. J Virol 64(12), 6270-3.

Koren, G., King, S., Knowles, S., and Phillips, E. (2003). Ribavirin in the treatment of

SARS: A new trick for an old drug? Cmaj 168(10), 1289-92. Krokhin, O., Li, Y., Andonov, A., Feldmann, H., Flick, R., Jones, S., Stroeher, U.,

Bastien, N., Dasuri, K. V., Cheng, K., Simonsen, J. N., Perreault, H., Wilkins, J., Ens, W., Plummer, F., and Standing, K. G. (2003). Mass Spectrometric Characterization of Proteins from the SARS Virus: A Preliminary Report. Mol Cell Proteomics 2(5), 346-56.

Krzystyniak, K., and Dupuy, J. M. (1984). Entry of mouse hepatitis virus 3 into cells. J

Gen Virol 65 ( Pt 1), 227-31. Ksiazek, T. G., Erdman, D., Goldsmith, C. S., Zaki, S. R., Peret, T., Emery, S., Tong, S.,

Urbani, C., Comer, J. A., Lim, W., Rollin, P. E., Dowell, S. F., Ling, A. E., Humphrey, C. D., Shieh, W. J., Guarner, J., Paddock, C. D., Rota, P., Fields, B., DeRisi, J., Yang, J. Y., Cox, N., Hughes, J. M., LeDuc, J. W., Bellini, W. J., and Anderson, L. J. (2003). A novel coronavirus associated with severe acute respiratory syndrome. N Engl J Med 348(20), 1953-66.

62

Kubo, H., Yamada, Y. K., and Taguchi, F. (1994). Localization of neutralizing epitopes

and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. J Virol 68(9), 5403-10.

Kuhn, J. H., Li, W., Choe, H., and Farzan, M. (2004). Angiotensin-converting enzyme 2:

a functional receptor for SARS coronavirus. Cell Mol Life Sci 61(21), 2738-43. Kuiken, T., Fouchier, R. A., Schutten, M., Rimmelzwaan, G. F., van Amerongen, G., van

Riel, D., Laman, J. D., de Jong, T., van Doornum, G., Lim, W., Ling, A. E., Chan, P. K., Tam, J. S., Zambon, M. C., Gopal, R., Drosten, C., van der Werf, S., Escriou, N., Manuguerra, J. C., Stohr, K., Peiris, J. S., and Osterhaus, A. D. (2003). Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362(9380), 263-70.

Kunkel, F., and Herrler, G. (1993). Structural and functional analysis of the surface

protein of human coronavirus OC43. Virology 195(1), 195-202. Kuo, L., and Masters, P. S. (2002). Genetic evidence for a structural interaction between

the carboxy termini of the membrane and nucleocapsid proteins of mouse hepatitis virus. J Virol 76(10), 4987-99.

Kusters, J. G., Niesters, H. G., Lenstra, J. A., Horzinek, M. C., and van der Zeijst, B. A.

(1989). Phylogeny of antigenic variants of avian coronavirus IBV. Virology 169(1), 217-21.

Lai, K. N., Leung, J. C., Metz, C. N., Lai, F. M., Bucala, R., and Lan, H. Y. (2003). Role

for macrophage migration inhibitory factor in acute respiratory distress syndrome. J Pathol 199(4), 496-508.

Lai, M. M. (1986). Coronavirus leader-RNA-primed transcription: an alternative

mechanism to RNA splicing. Bioessays 5(6), 257-60. Lai, M. M., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Adv Virus

Res 48, 1-100. Lai, M. M., and Holmes, K. V. (2001). Coronaviridae: the viruses and their replication. In

"Fields Virology" (D. M. Knipe, and P. M. Howley, Eds.), pp. 1163-1185. Lippincott Williams and Wilkins, Philadelphia.

Lai, M. M., Patton, C. D., Baric, R. S., and Stohlman, S. A. (1983). Presence of leader

sequences in the mRNA of mouse hepatitis virus. J Virol 46(3), 1027-33. Lee, H. J., Shieh, C. K., Gorbalenya, A. E., Koonin, E. V., La Monica, N., Tuler, J.,

Bagdzhadzhyan, A., and Lai, M. M. (1991). The complete sequence (22

63

kilobases) of murine coronavirus gene 1 encoding the putative proteases and RNA polymerase. Virology 180(2), 567-82.

Lee, N., Hui, D., Wu, A., Chan, P., Cameron, P., Joynt, G. M., Ahuja, A., Yung, M. Y.,

Leung, C. B., To, K. F., Lui, S. F., Szeto, C. C., Chung, S., and Sung, J. J. (2003). A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J Med 348(20), 1986-94.

Lescar, J., Roussel, A., Wien, M. W., Navaza, J., Fuller, S. D., Wengler, G., and Rey, F.

A. (2001). The Fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105(1), 137-48.

Leung, W. K., To, K. F., Chan, P. K., Chan, H. L., Wu, A. K., Lee, N., Yuen, K. Y., and

Sung, J. J. (2003). Enteric involvement of severe acute respiratory syndrome-associated coronavirus infection. Gastroenterology 125(4), 1011-7.

Li, D., and Cavanagh, D. (1992). Coronavirus IBV-induced membrane fusion occurs at

near-neutral pH. Arch Virol 122(3-4), 307-16. Li, H. P., Huang, P., Park, S., and Lai, M. M. (1999). Polypyrimidine tract-binding

protein binds to the leader RNA of mouse hepatitis virus and serves as a regulator of viral transcription. J Virol 73(1), 772-7.

Li, H. P., Zhang, X., Duncan, R., Comai, L., and Lai, M. M. (1997). Heterogeneous

nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA. Proc Natl Acad Sci U S A 94(18), 9544-9.

Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran,

M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., and Farzan, M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426(6965), 450-4.

Liao, C. L., and Lai, M. M. (1994). Requirement of the 5'-end genomic sequence as an

upstream cis-acting element for coronavirus subgenomic mRNA transcription. J Virol 68(8), 4727-37.

Lin, X. Q., Chouljenko, V. N., Kousoulas, K. G., and Storz, J. (2000). Temperature-

sensitive acetylesterase activity of haemagglutinin-esterase specified by respiratory bovine coronaviruses. J Med Microbiol 49(12), 1119-27.

Lin, Y. J., and Lai, M. M. (1993). Deletion mapping of a mouse hepatitis virus defective

interfering RNA reveals the requirement of an internal and discontiguous sequence for replication. J Virol 67(10), 6110-8.

64

Lin, Y. J., Liao, C. L., and Lai, M. M. (1994). Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription. J Virol 68(12), 8131-40.

Lin, Y. J., Zhang, X., Wu, R. C., and Lai, M. M. (1996). The 3' untranslated region of

coronavirus RNA is required for subgenomic mRNA transcription from a defective interfering RNA. J Virol 70(10), 7236-40.

Lipman, M. M., and Cotler, S. J. (2003). Antiviral Therapy for Hepatitis C. Curr Treat

Options Gastroenterol 6(6), 445-453. Liu, D. X., Cavanagh, D., Green, P., and Inglis, S. C. (1991). A polycistronic mRNA

specified by the coronavirus infectious bronchitis virus. Virology 184(2), 531-44. Liu, D. X., and Inglis, S. C. (1991). Association of the infectious bronchitis virus 3c

protein with the virion envelope. Virology 185(2), 911-7. Liu, D. X., and Inglis, S. C. (1992a). Identification of two new polypeptides encoded by

mRNA5 of the coronavirus infectious bronchitis virus. Virology 186(1), 342-7. Liu, D. X., and Inglis, S. C. (1992b). Internal entry of ribosomes on a tricistronic mRNA

encoded by infectious bronchitis virus. J Virol 66(10), 6143-54. Liu, Q., Johnson, R. F., and Leibowitz, J. L. (2001). Secondary structural elements within

the 3' untranslated region of mouse hepatitis virus strain JHM genomic RNA. J Virol 75(24), 12105-13.

Liu, Q., Yu, W., and Leibowitz, J. L. (1997). A specific host cellular protein binding

element near the 3' end of mouse hepatitis virus genomic RNA. Virology 232(1), 74-85.

Locker, J. K., Rose, J. K., Horzinek, M. C., and Rottier, P. J. (1992). Membrane assembly

of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation. J Biol Chem 267(30), 21911-8.

Lomniczi, B. (1977). Biological properties of avian coronavirus RNA. J Gen Virol 36(3),

531-3. Luo, Z., and Weiss, S. R. (1998). Roles in cell-to-cell fusion of two conserved

hydrophobic regions in the murine coronavirus spike protein. Virology 244(2), 483-94.

Luytjes, W., Geerts, D., Posthumus, W., Meloen, R., and Spaan, W. (1989). Amino acid

sequence of a conserved neutralizing epitope of murine coronaviruses. J Virol 63(3), 1408-12.

65

Machamer, C. E., Grim, M. G., Esquela, A., Chung, S. W., Rolls, M., Ryan, K., and Swift, A. M. (1993). Retention of a cis Golgi protein requires polar residues on one face of a predicted alpha-helix in the transmembrane domain. Mol Biol Cell 4(7), 695-704.

Machamer, C. E., Mentone, S. A., Rose, J. K., and Farquhar, M. G. (1990). The E1

glycoprotein of an avian coronavirus is targeted to the cis Golgi complex. Proc Natl Acad Sci U S A 87(18), 6944-8.

Machamer, C. E., and Rose, J. K. (1987). A specific transmembrane domain of a

coronavirus E1 glycoprotein is required for its retention in the Golgi region. J Cell Biol 105(3), 1205-14.

Macneughton, M. R., and Davies, H. A. (1978). Ribonucleoprotein-like structures from

coronavirus particles. J Gen Virol 39(3), 545-9. Maeda, J., Repass, J. F., Maeda, A., and Makino, S. (2001). Membrane topology of

coronavirus E protein. Virology 281(2), 163-9. Makino, S., Fujioka, N., and Fujiwara, K. (1985). Structure of the intracellular defective

viral RNAs of defective interfering particles of mouse hepatitis virus. J Virol 54(2), 329-36.

Makino, S., and Joo, M. (1993). Effect of intergenic consensus sequence flanking

sequences on coronavirus transcription. J Virol 67(6), 3304-11. Makino, S., Joo, M., and Makino, J. K. (1991). A system for study of coronavirus mRNA

synthesis: a regulated, expressed subgenomic defective interfering RNA results from intergenic site insertion. J Virol 65(11), 6031-41.

Makino, S., and Lai, M. M. (1989a). Evolution of the 5'-end of genomic RNA of murine

coronaviruses during passages in vitro. Virology 169(1), 227-32. Makino, S., and Lai, M. M. (1989b). High-frequency leader sequence switching during

coronavirus defective interfering RNA replication. J Virol 63(12), 5285-92. Makino, S., Shieh, C. K., Keck, J. G., and Lai, M. M. (1988a). Defective-interfering

particles of murine coronavirus: mechanism of synthesis of defective viral RNAs. Virology 163(1), 104-11.

Makino, S., Shieh, C. K., Soe, L. H., Baker, S. C., and Lai, M. M. (1988b). Primary

structure and translation of a defective interfering RNA of murine coronavirus. Virology 166(2), 550-60.

66

Makino, S., Stohlman, S. A., and Lai, M. M. (1986). Leader sequences of murine coronavirus mRNAs can be freely reassorted: evidence for the role of free leader RNA in transcription. Proc Natl Acad Sci U S A 83(12), 4204-8.

Makino, S., Yokomori, K., and Lai, M. M. (1990). Analysis of efficiently packaged

defective interfering RNAs of murine coronavirus: localization of a possible RNA-packaging signal. J Virol 64(12), 6045-53.

Marra, M. A., Jones, S. J., Astell, C. R., Holt, R. A., Brooks-Wilson, A., Butterfield, Y.

S., Khattra, J., Asano, J. K., Barber, S. A., Chan, S. Y., Cloutier, A., Coughlin, S. M., Freeman, D., Girn, N., Griffith, O. L., Leach, S. R., Mayo, M., McDonald, H., Montgomery, S. B., Pandoh, P. K., Petrescu, A. S., Robertson, A. G., Schein, J. E., Siddiqui, A., Smailus, D. E., Stott, J. M., Yang, G. S., Plummer, F., Andonov, A., Artsob, H., Bastien, N., Bernard, K., Booth, T. F., Bowness, D., Czub, M., Drebot, M., Fernando, L., Flick, R., Garbutt, M., Gray, M., Grolla, A., Jones, S., Feldmann, H., Meyers, A., Kabani, A., Li, Y., Normand, S., Stroher, U., Tipples, G. A., Tyler, S., Vogrig, R., Ward, D., Watson, B., Brunham, R. C., Krajden, M., Petric, M., Skowronski, D. M., Upton, C., and Roper, R. L. (2003). The Genome sequence of the SARS-associated coronavirus. Science 300(5624), 1399-404.

Martin, J., Navas, S., Quiroga, J. A., Pardo, M., and Carreno, V. (1998). Effects of the

ribavirin-interferon alpha combination on cultured peripheral blood mononuclear cells from chronic hepatitis C patients. Cytokine 10(8), 635-44.

Masters, P. S. (1992). Localization of an RNA-binding domain in the nucleocapsid

protein of the coronavirus mouse hepatitis virus. Arch Virol 125(1-4), 141-60. Masters, P. S., Koetzner, C. A., Kerr, C. A., and Heo, Y. (1994). Optimization of targeted

RNA recombination and mapping of a novel nucleocapsid gene mutation in the coronavirus mouse hepatitis virus. J Virol 68(1), 328-37.

Masters, P. S., and Sturman, L. S. (1990). Background paper. Functions of the

coronavirus nucleocapsid protein. Adv Exp Med Biol 276, 235-8. Mebus, C. A., Stair, E. L., Rhodes, M. B., and Twiehaus, M. J. (1973). Neonatal calf

diarrhea: propagation, attenuation, and characteristics of a coronavirus-like agent. Am J Vet Res 34(2), 145-50.

Melikyan, G. B., Markosyan, R. M., Hemmati, H., Delmedico, M. K., Lambert, D. M.,

and Cohen, F. S. (2000). Evidence that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, induces membrane fusion. J Cell Biol 151(2), 413-23.

Mizzen, L., Hilton, A., Cheley, S., and Anderson, R. (1985). Attenuation of murine

coronavirus infection by ammonium chloride. Virology 142(2), 378-88.

67

Moore, K. M., Jackwood, M. W., and Hilt, D. A. (1997). Identification of amino acids involved in a serotype and neutralization specific epitope within the s1 subunit of avian infectious bronchitis virus. Arch Virol 142(11), 2249-56.

Mummidi, S., Catano, G., Lam, L., Hoefle, A., Telles, V., Begum, K., Jimenez, F.,

Ahuja, S. S., and Ahuja, S. K. (2001). Extensive repertoire of membrane-bound and soluble dendritic cell-specific ICAM-3-grabbing nonintegrin 1 (DC-SIGN1) and DC-SIGN2 isoforms. Inter-individual variation in expression of DC-SIGN transcripts. J Biol Chem 276(35), 33196-212.

Myint, S. H. (1994). Human coronavirus - a brief review. Rev Med Virol 4, 35-46. Nanda, S. K., and Leibowitz, J. L. (2001). Mitochondrial aconitase binds to the 3'

untranslated region of the mouse hepatitis virus genome. J Virol 75(7), 3352-62. Narayanan, K., Maeda, A., Maeda, J., and Makino, S. (2000). Characterization of the

coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74(17), 8127-34.

Navas-Martin, S., and Weiss, S. R. (2003). SARS: lessons learned from other

coronaviruses. Viral Immunol 16(4), 461-74. Navas-Martin, S. R., and Weiss, S. (2004). Coronavirus replication and pathogenesis:

Implications for the recent outbreak of severe acute respiratory syndrome (SARS), and the challenge for vaccine development. J Neurovirol 10(2), 75-85.

Nedellec, P., Dveksler, G. S., Daniels, E., Turbide, C., Chow, B., Basile, A. A., Holmes,

K. V., and Beauchemin, N. (1994). Bgp2, a new member of the carcinoembryonic antigen-related gene family, encodes an alternative receptor for mouse hepatitis viruses. J Virol 68(7), 4525-37.

Nelson, G. W., and Stohlman, S. A. (1993). Localization of the RNA-binding domain of

mouse hepatitis virus nucleocapsid protein. J Gen Virol 74 ( Pt 9), 1975-9. Nelson, G. W., Stohlman, S. A., and Tahara, S. M. (2000). High affinity interaction

between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA. J Gen Virol 81(Pt 1), 181-8.

Nguyen, V. P., and Hogue, B. G. (1997). Protein interactions during coronavirus

assembly. J Virol 71(12), 9278-84. Nicholls, J. M., Poon, L. L., Lee, K. C., Ng, W. F., Lai, S. T., Leung, C. Y., Chu, C. M.,

Hui, P. K., Mak, K. L., Lim, W., Yan, K. W., Chan, K. H., Tsang, N. C., Guan, Y., Yuen, K. Y., and Peiris, J. S. (2003). Lung pathology of fatal severe acute respiratory syndrome. Lancet 361(9371), 1773-8.

68

Nie, Q. H., Luo, X. D., Zhang, J. Z., and Su, Q. (2003). Current status of severe acute respiratory syndrome in China. World J Gastroenterol 9(8), 1635-45.

Niemann, H., Boschek, B., Evans, D., Rosing, M., Tamura, T., and Klenk, H. D. (1982).

Post-translational glycosylation of coronavirus glycoprotein E1: inhibition by monensin. Embo J 1(12), 1499-504.

Niemann, H., Geyer, R., Klenk, H. D., Linder, D., Stirm, S., and Wirth, M. (1984). The

carbohydrates of mouse hepatitis virus (MHV) A59: structures of the O-glycosidically linked oligosaccharides of glycoprotein E1. Embo J 3(3), 665-70.

Opstelten, D. J., Raamsman, M. J., Wolfs, K., Horzinek, M. C., and Rottier, P. J. (1995).

Envelope glycoprotein interactions in coronavirus assembly. J Cell Biol 131(2), 339-49.

Oshiro, L. S. (1973). Coronaviruses. In "Ultrastructure of Animal Viruses and

Bacteriophages: An Atlas" (A. J. Dalton, and F. Haguenau, Eds.), pp. 331-343. Academic Press, New York.

Parker, M. M., and Masters, P. S. (1990). Sequence comparison of the N genes of five

strains of the coronavirus mouse hepatitis virus suggests a three domain structure for the nucleocapsid protein. Virology 179(1), 463-8.

Parker, S. E., Gallagher, T. M., and Buchmeier, M. J. (1989). Sequence analysis reveals

extensive polymorphism and evidence of deletions within the E2 glycoprotein gene of several strains of murine hepatitis virus. Virology 173(2), 664-73.

Parry, J. (2003). WHO investigates China's fall in SARS cases. Bmj 326(7402), 1285. Pavlovic, D., Neville, D. C., Argaud, O., Blumberg, B., Dwek, R. A., Fischer, W. B., and

Zitzmann, N. (2003). The hepatitis C virus p7 protein forms an ion channel that is inhibited by long-alkyl-chain iminosugar derivatives. Proc Natl Acad Sci U S A 100(10), 6104-8.

Payne, H. R., and Storz, J. (1988). Analysis of cell fusion induced by bovine coronavirus

infection. Arch Virol 103(1-2), 27-33. Pedersen, K. W., van der Meer, Y., Roos, N., and Snijder, E. J. (1999). Open reading

frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J Virol 73(3), 2016-26.

Pedersen, N. C., Ward, J., and Mengeling, W. L. (1978). Antigenic relationship of the

feline infections peritonitis virus to coronaviruses of other species. Arch Virol 58(1), 45-53.

69

Peiris, J. S., Chu, C. M., Cheng, V. C., Chan, K. S., Hung, I. F., Poon, L. L., Law, K. I., Tang, B. S., Hon, T. Y., Chan, C. S., Chan, K. H., Ng, J. S., Zheng, B. J., Ng, W. L., Lai, R. W., Guan, Y., and Yuen, K. Y. (2003). Clinical progression and viral load in a community outbreak of coronavirus-associated SARS pneumonia: a prospective study. Lancet 361(9371), 1767-72.

Peng, D., Koetzner, C. A., McMahon, T., Zhu, Y., and Masters, P. S. (1995).

Construction of murine coronavirus mutants containing interspecies chimeric nucleocapsid proteins. J Virol 69(9), 5475-84.

Perlman, S., Ries, D., Bolger, E., Chang, L. J., and Stoltzfus, C. M. (1986). MHV

nucleocapsid synthesis in the presence of cycloheximide and accumulation of negative strand MHV RNA. Virus Res 6(3), 261-72.

Piller, S. C., Ewart, G. D., Premkumar, A., Cox, G. B., and Gage, P. W. (1996). Vpr

protein of human immunodeficiency virus type 1 forms cation-selective channels in planar lipid bilayers. Proc Natl Acad Sci U S A 93(1), 111-5.

Pinol-Roma, S., and Dreyfuss, G. (1992). Shuttling of pre-mRNA binding proteins

between nucleus and cytoplasm. Nature 355(6362), 730-2. Pinto, L. H., Holsinger, L. J., and Lamb, R. A. (1992). Influenza virus M2 protein has ion

channel activity. Cell 69(3), 517-28. Posthumus, W. P., Lenstra, J. A., Schaaper, W. M., van Nieuwstadt, A. P., Enjuanes, L.,

and Meloen, R. H. (1990). Analysis and simulation of a neutralizing epitope of transmissible gastroenteritis virus. J Virol 64(7), 3304-9.

Premkumar, A., Wilson, L., Ewart, G. D., and Gage, P. W. (2004). Cation-selective ion

channels formed by p7 of hepatitis C virus are blocked by hexamethylene amiloride. FEBS Lett 557(1-3), 99-103.

Prentice, E., McAuliffe, J., Lu, X., Subbarao, K., and Denison, M. R. (2004).

Identification and characterization of severe acute respiratory syndrome coronavirus replicase proteins. J Virol 78(18), 9977-86.

Raamsman, M. J., Locker, J. K., de Hooge, A., de Vries, A. A., Griffiths, G., Vennema,

H., and Rottier, P. J. (2000). Characterization of the coronavirus mouse hepatitis virus strain A59 small membrane protein E. J Virol 74(5), 2333-42.

Rekik, M. R., and Dea, S. (1994). Comparative sequence analysis of a polymorphic

region of the spike glycoprotein S1 subunit of enteric bovine coronavirus isolates. Arch Virol 135(3-4), 319-31.

70

Repass, J. F., and Makino, S. (1998). Importance of the positive-strand RNA secondary structure of a murine coronavirus defective interfering RNA internal replication signal in positive-strand RNA synthesis. J Virol 72(10), 7926-33.

Risco, C., Anton, I. M., Enjuanes, L., and Carrascosa, J. L. (1996). The transmissible

gastroenteritis coronavirus contains a spherical core shell consisting of M and N proteins. J Virol 70(7), 4773-7.

Risco, C., Anton, I. M., Sune, C., Pedregosa, A. M., Martin-Alonso, J. M., Parra, F.,

Carrascosa, J. L., and Enjuanes, L. (1995). Membrane protein molecules of transmissible gastroenteritis coronavirus also expose the carboxy-terminal region on the external surface of the virion. J Virol 69(9), 5269-77.

Risco, C., Muntion, M., Enjuanes, L., and Carrascosa, J. L. (1998). Two types of virus-

related particles are found during transmissible gastroenteritis virus morphogenesis. J Virol 72(5), 4022-31.

Robbins, S. G., Frana, M. F., McGowan, J. J., Boyle, J. F., and Holmes, K. V. (1986).

RNA-binding proteins of coronavirus MHV: detection of monomeric and multimeric N protein with an RNA overlay-protein blot assay. Virology 150(2), 402-10.

Rossen, J. W., de Beer, R., Godeke, G. J., Raamsman, M. J., Horzinek, M. C., Vennema,

H., and Rottier, P. J. (1998). The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells. J Virol 72(1), 497-503.

Rota, P. A., Oberste, M. S., Monroe, S. S., Nix, W. A., Campagnoli, R., Icenogle, J. P.,

Penaranda, S., Bankamp, B., Maher, K., Chen, M. H., Tong, S., Tamin, A., Lowe, L., Frace, M., DeRisi, J. L., Chen, Q., Wang, D., Erdman, D. D., Peret, T. C., Burns, C., Ksiazek, T. G., Rollin, P. E., Sanchez, A., Liffick, S., Holloway, B., Limor, J., McCaustland, K., Olsen-Rasmussen, M., Fouchier, R., Gunther, S., Osterhaus, A. D., Drosten, C., Pallansch, M. A., Anderson, L. J., and Bellini, W. J. (2003). Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300(5624), 1394-9.

Rottier, P., Armstrong, J., and Meyer, D. I. (1985). Signal recognition particle-dependent

insertion of coronavirus E1, an intracellular membrane glycoprotein. J Biol Chem 260(8), 4648-52.

Routledge, E., Stauber, R., Pfleiderer, M., and Siddell, S. G. (1991). Analysis of murine

coronavirus surface glycoprotein functions by using monoclonal antibodies. J Virol 65(1), 254-62.

Russell, C. J., Jardetzky, T. S., and Lamb, R. A. (2001). Membrane fusion machines of

paramyxoviruses: capture of intermediates of fusion. Embo J 20(15), 4024-34.

71

Saif, L. J., Redman, D. R., Brock, K. V., Kohler, E. M., and Heckert, R. A. (1988). Winter dysentery in adult dairy cattle: detection of coronavirus in the faeces. Vet Rec 123(11), 300-1.

Salanueva, I. J., Carrascosa, J. L., and Risco, C. (1999). Structural maturation of the

transmissible gastroenteritis coronavirus. J Virol 73(10), 7952-64. Sanchez, C. M., Izeta, A., Sanchez-Morgado, J. M., Alonso, S., Sola, I., Balasch, M.,

Plana-Duran, J., and Enjuanes, L. (1999). Targeted recombination demonstrates that the spike gene of transmissible gastroenteritis coronavirus is a determinant of its enteric tropism and virulence. J Virol 73(9), 7607-18.

Sawicki, S. G., and Sawicki, D. L. (1986). Coronavirus minus-strand RNA synthesis and

effect of cycloheximide on coronavirus RNA synthesis. J Virol 57(1), 328-34. Sawicki, S. G., and Sawicki, D. L. (1990). Coronavirus transcription: subgenomic mouse

hepatitis virus replicative intermediates function in RNA synthesis. J Virol 64(3), 1050-6.

Schmidt-Mende, J., Bieck, E., Hugle, T., Penin, F., Rice, C. M., Blum, H. E., and

Moradpour, D. (2001). Determinants for membrane association of the hepatitis C virus RNA-dependent RNA polymerase. J Biol Chem 276(47), 44052-63.

Schochetman, G., Stevens, R. H., and Simpson, R. W. (1977). Presence of infectious

polyadenylated RNA in coronavirus avian bronchitis virus. Virology 77(2), 772-82.

Schultze, B., and Herrler, G. (1992). Bovine coronavirus uses N-acetyl-9-O-

acetylneuraminic acid as a receptor determinant to initiate the infection of cultured cells. J Gen Virol 73 ( Pt 4), 901-6.

Schultze, B., Wahn, K., Klenk, H. D., and Herrler, G. (1991). Isolated HE-protein from

hemagglutinating encephalomyelitis virus and bovine coronavirus has receptor-destroying and receptor-binding activity. Virology 180(1), 221-8.

Schwarz, B., Routledge, E., and Siddell, S. G. (1990). Murine coronavirus nonstructural

protein ns2 is not essential for virus replication in transformed cells. J Virol 64(10), 4784-91.

Senanayake, S. D., Hofmann, M. A., Maki, J. L., and Brian, D. A. (1992). The

nucleocapsid protein gene of bovine coronavirus is bicistronic. J Virol 66(9), 5277-83.

Sethna, P. B., and Brian, D. A. (1997). Coronavirus genomic and subgenomic minus-

strand RNAs copartition in membrane-protected replication complexes. J Virol 71(10), 7744-9.

72

Sethna, P. B., Hofmann, M. A., and Brian, D. A. (1991). Minus-strand copies of

replicating coronavirus mRNAs contain antileaders. J Virol 65(1), 320-5. Sethna, P. B., Hung, S. L., and Brian, D. A. (1989). Coronavirus subgenomic minus-

strand RNAs and the potential for mRNA replicons. Proc Natl Acad Sci U S A 86(14), 5626-30.

Shi, S. T., Schiller, J. J., Kanjanahaluethai, A., Baker, S. C., Oh, J. W., and Lai, M. M.

(1999). Colocalization and membrane association of murine hepatitis virus gene 1 products and De novo-synthesized viral RNA in infected cells. J Virol 73(7), 5957-69.

Shieh, C. K., Soe, L. H., Makino, S., Chang, M. F., Stohlman, S. A., and Lai, M. M.

(1987). The 5'-end sequence of the murine coronavirus genome: implications for multiple fusion sites in leader-primed transcription. Virology 156(2), 321-30.

Skehel, J. J., and Wiley, D. C. (1998). Coiled coils in both intracellular vesicle and viral

membrane fusion. Cell 95(7), 871-4. Snijder, E. J., Bredenbeek, P. J., Dobbe, J. C., Thiel, V., Ziebuhr, J., Poon, L. L., Guan,

Y., Rozanov, M., Spaan, W. J., and Gorbalenya, A. E. (2003). Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331(5), 991-1004.

Spaan, W., Cavanagh, D., and Horzinek, M. C. (1988). Coronaviruses: structure and

genome expression. J Gen Virol 69 ( Pt 12), 2939-52. Spaan, W., Delius, H., Skinner, M., Armstrong, J., Rottier, P., Smeekens, S., van der

Zeijst, B. A., and Siddell, S. G. (1983). Coronavirus mRNA synthesis involves fusion of non-contiguous sequences. Embo J 2(10), 1839-44.

Spaan, W. J., Rottier, P. J., Horzinek, M. C., and van der Zeijst, B. A. (1981). Isolation

and identification of virus-specific mRNAs in cells infected with mouse hepatitis virus (MHV-A59). Virology 108(2), 424-34.

Spagnolo, J. F., and Hogue, B. G. (2000). Host protein interactions with the 3' end of

bovine coronavirus RNA and the requirement of the poly(A) tail for coronavirus defective genome replication. J Virol 74(11), 5053-65.

Stadler, K., Masignani, V., Eickmann, M., Becker, S., Abrignani, S., Klenk, H. D., and

Rappuoli, R. (2003). SARS--beginning to understand a new virus. Nat Rev Microbiol 1(3), 209-18.

73

Stauber, R., Pfleiderera, M., and Siddell, S. (1993). Proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity. J Gen Virol 74 ( Pt 2), 183-91.

Stern, D. F., and Sefton, B. M. (1982). Coronavirus proteins: structure and function of the

oligosaccharides of the avian infectious bronchitis virus glycoproteins. J Virol 44(3), 804-12.

Stohlman, S. A., Baric, R. S., Nelson, G. N., Soe, L. H., Welter, L. M., and Deans, R. J.

(1988). Specific interaction between coronavirus leader RNA and nucleocapsid protein. J Virol 62(11), 4288-95.

Stohlman, S. A., and Lai, M. M. (1979). Phosphoproteins of murine hepatitis viruses. J

Virol 32(2), 672-5. Storz, J., Lin, X., Purdy, C. W., Chouljenko, V. N., Kousoulas, K. G., Enright, F. M.,

Gilmore, W. C., Briggs, R. E., and Loan, R. W. (2000a). Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evans' criteria for causation. J Clin Microbiol 38(9), 3291-8.

Storz, J., Purdy, C. W., Lin, X., Burrell, M., Truax, R. E., Briggs, R. E., Frank, G. H., and

Loan, R. W. (2000b). Isolation of respiratory bovine coronavirus, other cytocidal viruses, and Pasteurella spp from cattle involved in two natural outbreaks of shipping fever. J Am Vet Med Assoc 216(10), 1599-604.

Sturman, L. S., Holmes, K. V., and Behnke, J. (1980). Isolation of coronavirus envelope

glycoproteins and interaction with the viral nucleocapsid. J Virol 33(1), 449-62. Sturman, L. S., Ricard, C. S., and Holmes, K. V. (1990). Conformational change of the

coronavirus peplomer glycoprotein at pH 8.0 and 37 degrees C correlates with virus aggregation and virus-induced cell fusion. J Virol 64(6), 3042-50.

Suzuki, H., and Taguchi, F. (1996). Analysis of the receptor-binding site of murine

coronavirus spike protein. J Virol 70(4), 2632-6. Svitkin, Y. V., Ovchinnikov, L. P., Dreyfuss, G., and Sonenberg, N. (1996). General

RNA binding proteins render translation cap dependent. Embo J 15(24), 7147-55. Swift, A. M., and Machamer, C. E. (1991). A Golgi retention signal in a membrane-

spanning domain of coronavirus E1 protein. J Cell Biol 115(1), 19-30. Taguchi, F. (1993). Fusion formation by the uncleaved spike protein of murine

coronavirus JHMV variant cl-2. J Virol 67(3), 1195-202.

74

Tahara, S. M., Dietlin, T. A., Bergmann, C. C., Nelson, G. W., Kyuwa, S., Anthony, R. P., and Stohlman, S. A. (1994). Coronavirus translational regulation: leader affects mRNA efficiency. Virology 202(2), 621-30.

Tahara, S. M., Dietlin, T. A., Nelson, G. W., Stohlman, S. A., and Manno, D. J. (1998).

Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs. Adv Exp Med Biol 440, 313-8.

Takase-Yoden, S., Kikuchi, T., Siddell, S. G., and Taguchi, F. (1991). Localization of

major neutralizing epitopes on the S1 polypeptide of the murine coronavirus peplomer glycoprotein. Virus Res 18(2-3), 99-107.

Talbot, P. J., and Buchmeier, M. J. (1985). Antigenic variation among murine

coronaviruses: evidence for polymorphism on the peplomer glycoprotein, E2. Virus Res 2(4), 317-28.

Talbot, P. J., Salmi, A. A., Knobler, R. L., and Buchmeier, M. J. (1984). Topographical

mapping of epitopes on the glycoproteins of murine hepatitis virus-4 (strain JHM): correlation with biological activities. Virology 132(2), 250-60.

Tan, Y. J., Fielding, B. C., Goh, P. Y., Shen, S., Tan, T. H., Lim, S. G., and Hong, W.

(2004a). Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway. J Virol 78(24), 14043-7.

Tan, Y. J., Goh, P. Y., Fielding, B. C., Shen, S., Chou, C. F., Fu, J. L., Leong, H. N., Leo,

Y. S., Ooi, E. E., Ling, A. E., Lim, S. G., and Hong, W. (2004b). Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers. Clin Diagn Lab Immunol 11(2), 362-71.

Tan, Y. J., Teng, E., Shen, S., Tan, T. H., Goh, P. Y., Fielding, B. C., Ooi, E. E., Tan, H.

C., Lim, S. G., and Hong, W. (2004c). A novel severe acute respiratory syndrome coronavirus protein, U274, is transported to the cell surface and undergoes endocytosis. J Virol 78(13), 6723-34.

Thiel, V., Ivanov, K. A., Putics, A., Hertzig, T., Schelle, B., Bayer, S., Weissbrich, B.,

Snijder, E. J., Rabenau, H., Doerr, H. W., Gorbalenya, A. E., and Ziebuhr, J. (2003). Mechanisms and enzymes involved in SARS coronavirus genome expression. J Gen Virol 84(Pt 9), 2305-15.

Thiel, V., and Siddell, S. G. (1994). Internal ribosome entry in the coding region of

murine hepatitis virus mRNA 5. J Gen Virol 75 ( Pt 11), 3041-6. To, K. F., Chan, P. K., Chan, K. F., Lee, W. K., Lam, W. Y., Wong, K. F., Tang, N. L.,

Tsang, D. N., Sung, R. Y., Buckley, T. A., Tam, J. S., and Cheng, A. F. (2001).

75

Pathology of fatal human infection associated with avian influenza A H5N1 virus. J Med Virol 63(3), 242-6.

Tooze, J., Tooze, S., and Warren, G. (1984). Replication of coronavirus MHV-A59 in

sac- cells: determination of the first site of budding of progeny virions. Eur J Cell Biol 33(2), 281-93.

Tooze, J., and Tooze, S. A. (1985). Infection of AtT20 murine pituitary tumour cells by

mouse hepatitis virus strain A59: virus budding is restricted to the Golgi region. Eur J Cell Biol 37, 203-12.

Tsai, J. C., Zelus, B. D., Holmes, K. V., and Weiss, S. R. (2003). The N-terminal domain

of the murine coronavirus spike glycoprotein determines the CEACAM1 receptor specificity of the virus strain. J Virol 77(2), 841-50.

Tsui, P. T., Kwok, M. L., Yuen, H., and Lai, S. T. (2003). Severe acute respiratory

syndrome: clinical outcome and prognostic correlates. Emerg Infect Dis 9(9), 1064-9.

Valcarcel, J., and Gebauer, F. (1997). Post-transcriptional regulation: the dawn of PTB.

Curr Biol 7(11), R705-8. van der Meer, Y., Snijder, E. J., Dobbe, J. C., Schleich, S., Denison, M. R., Spaan, W. J.,

and Locker, J. K. (1999). Localization of mouse hepatitis virus nonstructural proteins and RNA synthesis indicates a role for late endosomes in viral replication. J Virol 73(9), 7641-57.

van der Most, R. G., Bredenbeek, P. J., and Spaan, W. J. (1991). A domain at the 3' end

of the polymerase gene is essential for encapsidation of coronavirus defective interfering RNAs. J Virol 65(6), 3219-26.

van der Most, R. G., de Groot, R. J., and Spaan, W. J. (1994). Subgenomic RNA

synthesis directed by a synthetic defective interfering RNA of mouse hepatitis virus: a study of coronavirus transcription initiation. J Virol 68(6), 3656-66.

Vautherot, J. F., Laporte, J., and Boireau, P. (1992). Bovine coronavirus spike

glycoprotein: localization of an immunodominant region at the amino-terminal end of S2. J Gen Virol 73 ( Pt 12), 3289-94.

Vautherot, J. F., Madelaine, M. F., Boireau, P., and Laporte, J. (1992). Bovine

coronavirus peplomer glycoproteins: detailed antigenic analyses of S1, S2 and HE. J Gen Virol 73 ( Pt 7), 1725-37.

Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten,

D. J., and Rottier, P. J. (1996). Nucleocapsid-independent assembly of

76

coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15(8), 2020-8.

Vennema, H., Heijnen, L., Zijderveld, A., Horzinek, M. C., and Spaan, W. J. (1990).

Intracellular transport of recombinant coronavirus spike proteins: implications for virus assembly. J Virol 64(1), 339-46.

Vlasak, R., Luytjes, W., Leider, J., Spaan, W., and Palese, P. (1988). The E3 protein of

bovine coronavirus is a receptor-destroying enzyme with acetylesterase activity. J Virol 62(12), 4686-90.

Wang, P., Chen, J., Zheng, A., Nie, Y., Shi, X., Wang, W., Wang, G., Luo, M., Liu, H.,

Tan, L., Song, X., Wang, Z., Yin, X., Qu, X., Wang, X., Qing, T., Ding, M., and Deng, H. (2004). Expression cloning of functional receptor used by SARS coronavirus. Biochem Biophys Res Commun 315(2), 439-44.

Wang, Y., and Zhang, X. (1999). The nucleocapsid protein of coronavirus mouse

hepatitis virus interacts with the cellular heterogeneous nuclear ribonucleoprotein A1 in vitro and in vivo. Virology 265(1), 96-109.

Weismiller, D. G., Sturman, L. S., Buchmeier, M. J., Fleming, J. O., and Holmes, K. V.

(1990). Monoclonal antibodies to the peplomer glycoprotein of coronavirus mouse hepatitis virus identify two subunits and detect a conformational change in the subunit released under mild alkaline conditions. J Virol 64(6), 3051-5.

Wilbur, S. M., Nelson, G. W., Lai, M. M., McMillan, M., and Stohlman, S. A. (1986).

Phosphorylation of the mouse hepatitis virus nucleocapsid protein. Biochem Biophys Res Commun 141(1), 7-12.

Williams, R. K., Jiang, G. S., and Holmes, K. V. (1991). Receptor for mouse hepatitis

virus is a member of the carcinoembryonic antigen family of glycoproteins. Proc Natl Acad Sci U S A 88(13), 5533-6.

Wilson, L., McKinlay, C., Gage, P., and Ewart, G. (2004). SARS coronavirus E protein

forms cation-selective ion channels. Virology 330(1), 322-31. Woo, K., Joo, M., Narayanan, K., Kim, K. H., and Makino, S. (1997). Murine

coronavirus packaging signal confers packaging to nonviral RNA. J Virol 71(1), 824-7.

Wu, Q., Zhang, Y., Lu, H., Wang, J., He, X., Liu, Y., Ye, C., Lin, W., Hu, J., Ji, J., Xu, J.,

Ye, J., Hu, Y., Chen, W., Li, S., Bi, S., and Yang, H. (2003). The E protein is a multifunctional membrane protein of SARS-CoV. Genomics Proteomics Bioinformatics 1(2), 131-44.

77

Wu, X. D., Shang, B., Yang, R. F., Yu, H., Ma, Z. H., Shen, X., Ji, Y. Y., Lin, Y., Wu, Y. D., Lin, G. M., Tian, L., Gan, X. Q., Yang, S., Jiang, W. H., Dai, E. H., Wang, X. Y., Jiang, H. L., Xie, Y. H., Zhu, X. L., Pei, G., Li, L., Wu, J. R., and Sun, B. (2004). The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells. Cell Res 14(5), 400-6.

Xiao, X., Feng, Y., Chakraborti, S., and Dimitrov, D. S. (2004). Oligomerization of the

SARS-CoV S glycoprotein: dimerization of the N-terminus and trimerization of the ectodomain. Biochem Biophys Res Commun 322(1), 93-9.

Yeager, C. L., Ashmun, R. A., Williams, R. K., Cardellichio, C. B., Shapiro, L. H., Look,

A. T., and Holmes, K. V. (1992). Human aminopeptidase N is a receptor for human coronavirus 229E. Nature 357(6377), 420-2.

Ying, W., Hao, Y., Zhang, Y., Peng, W., Qin, E., Cai, Y., Wei, K., Wang, J., Chang, G.,

Sun, W., Dai, S., Li, X., Zhu, Y., Li, J., Wu, S., Guo, L., Dai, J., Wan, P., Chen, T., Du, C., Li, D., Wan, J., Kuai, X., Li, W., Shi, R., Wei, H., Cao, C., Yu, M., Liu, H., Dong, F., Wang, D., Zhang, X., Qian, X., Zhu, Q., and He, F. (2004). Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus. Proteomics 4(2), 492-504.

Yokomori, K., Asanaka, M., Stohlman, S. A., and Lai, M. M. (1993). A spike protein-

dependent cellular factor other than the viral receptor is required for mouse hepatitis virus entry. Virology 196(1), 45-56.

Yokomori, K., Banner, L. R., and Lai, M. M. (1991). Heterogeneity of gene expression of

the hemagglutinin-esterase (HE) protein of murine coronaviruses. Virology 183(2), 647-57.

Yokomori, K., La Monica, N., Makino, S., Shieh, C. K., and Lai, M. M. (1989).

Biosynthesis, structure, and biological activities of envelope protein gp65 of murine coronavirus. Virology 173(2), 683-91.

Yokomori, K., and Lai, M. M. (1991). Mouse hepatitis virus S RNA sequence reveals

that nonstructural proteins ns4 and ns5a are not essential for murine coronavirus replication. J Virol 65(10), 5605-8.

Yokomori, K., and Lai, M. M. (1992a). Mouse hepatitis virus utilizes two

carcinoembryonic antigens as alternative receptors. J Virol 66(10), 6194-9. Yokomori, K., and Lai, M. M. (1992b). The receptor for mouse hepatitis virus in the

resistant mouse strain SJL is functional: implications for the requirement of a second factor for viral infection. J Virol 66(12), 6931-8.

78

Yoo, D. W., Parker, M. D., and Babiuk, L. A. (1991). The S2 subunit of the spike glycoprotein of bovine coronavirus mediates membrane fusion in insect cells. Virology 180(1), 395-9.

Yu, M. W., Scott, J. K., Fournier, A., and Talbot, P. J. (2000). Characterization of murine

coronavirus neutralization epitopes with phage-displayed peptides. Virology 271(1), 182-96.

Yu, W., and Leibowitz, J. L. (1995a). A conserved motif at the 3' end of mouse hepatitis

virus genomic RNA required for host protein binding and viral RNA replication. Virology 214(1), 128-38.

Yu, W., and Leibowitz, J. L. (1995b). Specific binding of host cellular proteins to

multiple sites within the 3' end of mouse hepatitis virus genomic RNA. J Virol 69(4), 2016-23.

Yu, X., Bi, W., Weiss, S. R., and Leibowitz, J. L. (1994). Mouse hepatitis virus gene 5b

protein is a new virion envelope protein. Virology 202(2), 1018-23. Zelus, B. D., Schickli, J. H., Blau, D. M., Weiss, S. R., and Holmes, K. V. (2003).

Conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8. J Virol 77(2), 830-40.

Zhang, H., Wang, G., Li, J., Nie, Y., Shi, X., Lian, G., Wang, W., Yin, X., Zhao, Y., Qu,

X., Ding, M., and Deng, H. (2004). Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies. J Virol 78(13), 6938-45.

Zhang, X., and Lai, M. M. (1995a). Interactions between the cytoplasmic proteins and the

intergenic (promoter) sequence of mouse hepatitis virus RNA: correlation with the amounts of subgenomic mRNA transcribed. J Virol 69(3), 1637-44.

Zhang, X., and Lai, M. M. (1996). A 5'-proximal RNA sequence of murine coronavirus

as a potential initiation site for genomic-length mRNA transcription. J Virol 70(2), 705-11.

Zhang, X., Liao, C. L., and Lai, M. M. (1994). Coronavirus leader RNA regulates and

initiates subgenomic mRNA transcription both in trans and in cis. J Virol 68(8), 4738-46.

Zhang, X. M., and Lai, M. M. (1995b). Regulation of coronavirus RNA transcription is

likely mediated by protein-RNA interactions. Adv Exp Med Biol 380, 515-21. Zhao, X., Shaw, K., and Cavanagh, D. (1993). Presence of subgenomic mRNAs in

virions of coronavirus IBV. Virology 196(1), 172-8.

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CHAPTER II

GENETIC ANALYSIS OF THE SARS-CORONAVIRUS SPIKE GLYCOPROTEIN FUNCTIONAL DOMAINS INVOLVED IN CELL-SURFACE

EXPRESSION AND CELL-TO-CELL FUSION*

INTRODUCTION

An outbreak of atypical pneumonia, termed severe acute respiratory syndrome

(SARS), appeared in the Guangdong Province of southern China in November, 2002. The

mortality rates of the disease reached as high as 15% in some age groups (Anand et al.,

2003). The etiological agent of the disease was found to be a novel coronavirus (SARS-

CoV), which was first isolated from infected individuals by propagation of the virus on

Vero E6 cells (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003). Analysis of

the viral genome has demonstrated that the SARS-CoV is phylogenetically divergent

from the three known antigenic groups of coronaviruses (Drosten et al., 2003; Ksiazek et

al., 2003). Analysis of the polymerase gene alone, however, has indicated that the SARS-

CoV may be an early off-shoot from the group 2 coronaviruses (Snijder et al., 2003).

The coronaviruses are the largest of the enveloped RNA viruses with a positive-

stranded RNA genome of 28 to 32 kb (Holmes, 2003). Coronaviruses possess a wide host

range, capable of infecting mammalian and avian species. All identified coronaviruses

have a common group of indispensable genes that encode nonstructural proteins

including the RNA replicase gene open reading frame (ORF) 1ab and the structural

proteins nucleocapsid (N), membrane protein (M), envelope protein (E), and spike

glycoprotein (S), which are assembled into virus particles. A hemagglutinin-esterase

* Reprinted from Virology , Vol 341 (2), C. M. Petit, J. M. Melancon, V. N. Chouljenko, R. Colgrove, M. Farzan, D. M. Knipe, and K. G. Kousoulas, Genetic Analysis of the SARS-Coronavirus Spike Glycoprotein Functional Domains Involved in Cell-surface Expression and Cell-to-Cell Fusion, Pages 215-308, Copyright 2005, with permission from the Elsevier.

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(HE) protein is also encoded by some coronaviruses. Distributed among the major viral

genes are a series of ORFs that are specific to the different coronavirus groups. Functions

of the majority these ORFs have not been determined.

The SARS spike glycoprotein, a 1,255-amino-acid type I membrane glycoprotein


spike structure found on all coronavirions. The S glycoprotein is primarily responsible

for entry of all coronaviruses into susceptible cells through binding to specific receptors

on cells and mediating subsequent virus-cell fusion (Cavanagh, 1995). The S

glycoprotein specified by mouse hepatitis virus (MHV), is cleaved into S1 and S2

subunits, although cleavage is not necessarily required for virus-cell fusion (Bos, Luytjes,

and Spaan, 1997; Gombold, Hingley, and Weiss, 1993; Stauber, Pfleiderera, and Siddell,

1993). Similarly, the SARS-CoV S glycoprotein seems to be cleaved into S1 and S2

subunits in Vero-E6 infected cells (Wu et al., 2004), while it is not known whether this

cleavage affects S-mediated cell fusion. The SARS-CoV receptor has been recently

identified as the angiotensin converting enzyme 2 (ACE2) (Li et al., 2003). Although the

exact mechanism by which the SARS-CoV enters the host cell has not been elucidated, it

is most likely similar to other coronaviruses. Upon receptor binding at the cell

membrane, the S glycoprotein is thought to undergo a dramatic conformational change

causing exposure of a hydrophobic fusion peptide, which is subsequently inserted into

cellular membranes. This conformational change of the S glycoprotein causes close

apposition followed by fusion of the viral and cellular membranes resulting in entry of

the virion nucleocapsids into cells (Eckert and Kim, 2001; Tsai et al., 2003; Zelus et al.,

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2003). This series of S-mediated virus entry events is similar to other class I virus fusion

proteins (Baker et al., 1999; Melikyan et al., 2000; Russell, Jardetzky, and Lamb, 2001).

Heptad repeat (HR) regions, a sequence motif characteristic of coiled-coils,

appear to be a common motif in many viral and cellular fusion proteins (Skehel and

Wiley, 1998). These coiled-coil regions allow the protein to fold back upon itself as a

prerequisite step to initiating the membrane fusion event. There are usually two HR

regions: an N terminal HR region adjacent to the fusion peptide and a C-terminal HR

region close to the transmembrane region of the protein. Within the HR segments, the

first amino acid (a) and fourth amino acid (d) are typically hydrophobic amino acids that

play a vital role in maintaining coiled-coil interactions. Based on structural similarities,

two classes of viral fusion proteins have been established. Class I viral fusion proteins

contain two heptad repeat regions and an N-terminal or N-proximal fusion peptide. Class

II viral fusion proteins lack heptad repeat regions and contain an internal fusion peptide

(Lescar et al., 2001). The MHV S glycoprotein, which is similar to other coronavirus S

glycoproteins, is a class I membrane protein that is transported to the plasma membrane

after being synthesized in the endoplasmic reticulum (Bosch et al., 2003). Typically, the

ectodomains of the S2 subunits of coronaviruses contain two regions with a 4, 3

hydrophobic (heptad) repeat the first being adjacent to the fusion peptide and the other

being in close proximity to the transmembrane region (de Groot et al., 1987).

In the present study, we investigated the role of several predicted structural and

functional domains of the SARS spike glycoprotein by introducing specific alterations

within selected S glycoprotein regions. The results show that the SARS-CoV S

glycoprotein conforms to the general structure and function relationships that have been

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elucidated for other coronaviruses, most notably the MHV (Chang, Sheng, and Gombold,

2000; Ye, Montalto-Morrison, and Masters, 2004) . However, in contrast to the MHV

endodomain, the carboxyl terminus of the SARS-CoV S glycoprotein contains multiple

non-overlapping domains that function in intracellular transport, cell-surface expression,

and endocytosis as well as in S glycoprotein-mediated cell-to-cell fusion.

RESULTS

Genetic Analysis of S Glycoprotein Functional Domains

To delineate domains of the S glycoprotein that function in membrane fusion,

intracellular transport, and cell-surface expression, two types of mutations were

introduced within the S gene: a) mutations were introduced within and adjacent to the

predicted amino terminal heptad repeat (HR1) core and the predicted fusion peptide,

which are known to play important roles in membrane fusion (Bosch et al., 2004; Bosch

et al., 2003; Ingallinella et al., 2004; Tripet et al., 2004); b) mutations and carboxyl

terminal truncations of the S glycoprotein were engineered to delineate S cytoplasmic

domains that function in glycoprotein synthesis, intracellular transport, and membrane

fusion (Fig. 2.1). Specifically, to investigate the amino acid requirements of the HR1 of

the SARS-CoV S glycoprotein, the a and d amino acid positions L(898) and N(901) were

both replaced by lysine residues in the cluster mutation CL2, effectively collapsing the

predicted α-helical structure at the amino terminal terminus of the HR. This amino acid

sequence is thought to align with the L(1184) of HR2 in the formation of the HR1/HR2

core complex (Xu et al., 2004). In addition, cluster-to-lysine mutations CL3 and CL4

replaced the a and d positions within the HR1 region (Fig. 2.1B). The CL5 cluster

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Figure 2.1. Schematic diagram of the SARS-CoV S glycoprotein. (A) Graphical representation of the S glycoprotein showing the approximate location of the cluster to lysine mutations CL1-CL5 relative to known and indicated functional domains. (B) Shown on the top of the diagram is a graphical representation of the SARS-CoV S glycoprotein. The predicted fusion peptide and the HR1 region are enlarged below to show the sets of amino acids replaced by lysines in the cluster mutations. The heptad repeat a and d positions are labeled above the corresponding amino acid. Amino acids changed to lysine are demarcated by arrows with the name of that particular mutation shown in brackets. (C) Amino acid sequences of the carboxyl termini of the truncation and acidic cluster associated mutations. Cysteine clusters (CRM1 and CRM2) are denoted by underlined italicized text as well as a bracket encompassing their respective regions. The charged cluster is bracketed over the region. Amino acids mutated to alanines for the CL6 and CL7 cluster mutations are in bold.

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mutation was placed adjacent to the HR1 region to investigate whether regions proximal

to HR1 had any effect on S mediated cell fusion. Similarly, the role of the a and d

positions within the predicted fusion peptide, located immediately proximal to the N

terminus of HR1, was investigated by constructing the CL1 cluster mutation (Fig. 2.1B).

It has been shown for other viral class I fusion proteins that the carboxyl terminus

plays a regulatory role in membrane fusion (Bagai and Lamb, 1996; Sergel and Morrison,

1995; Seth, Vincent, and Compans, 2003; Tong et al., 2002; Yao and Compans, 1995).

Specifically for coronaviruses, the MHV S glycoprotein endodomain has been shown to

contain charged-rich and cysteine-rich regions, which are critical for fusion of infected

cells (Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and

Masters, 2004). The carboxyl terminal portion of the S glycoprotein contains a consensus

acidic amino acid cluster containing a motif, which is predicted by the NetPhos 2.0

software to be phosphorylated (Blom, Gammeltoft, and Brunak, 1999). To investigate the

potential role of the acidic amino acid cluster in synthesis, transport and cell fusion, serial

truncations of S were constructed. The acidic cluster was specifically targeted by

mutagenizing the predicted phosphorylation site embedded within the acidic cluster as

well as by replacing acidic residues of the acidic cluster with alanine residues. In

addition, carboxyl-terminal truncations of 8, 17, 26, and 41 amino acids were engineered

by insertion of stop codons within the S glycoprotein gene. The 8 aa truncation (T1247)

was designed to bring the predicted charged cluster DEDDSE proximal to the carboxyl

terminus of the mutated S glycoprotein (Fig. 2.1C). Similarly, the 17 aa truncation

(T1238) was designed to delete the DEDDSE acidic cluster. The SARS-CoV S

glycoprotein endodomain contains two cysteine residue clusters, a CCMTSCCSC

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(CRM1) cluster immediately adjacent to the membrane and a CSCGSCC (CRM2)

downstream of the first cluster. To address the role of these domains in S glycoprotein-

mediated cell-to-cell fusion, the 26 aa truncation (T1229) was designed to delete the

CRM2 domain (T1229), while the 41 aa truncation (T1214) deleted both the CRM1 and

CRM2 domains (Fig. 2.1C).

Effect of Mutations on S Synthesis

To investigate the effect of the different mutations on S synthesis, western

immunoblot analysis was used to detect and visualize all of the constructed mutant

glycoproteins as well as the wild type S (Fig. 2.2). Cellular lysates prepared from

transfected cells at 48 hours post transfection were electrophoretically separated by SDS-

PAGE and the S glycoproteins were detected via chemiluminescence using a monoclonal

antibody specific for the SARS-CoV S glycoprotein. Carbohydrate addition was shown to

occur in at least four different locations of the SARS-CoV S glycoprotein (Krokhin et al.,

2003; Ying et al., 2004). Furthermore, transiently expressed S glycoprotein in Vero E6

cells was proteolytically cleaved into S1 and S2 components (Wu et al., 2004). The anti-S

monoclonal antibody SW-111 detected a protein species in cellular extracts from

transfected cells, which migrated with an apparent molecular mass of approximately 180

kDa, as reported previously (Song et al., 2004). All mutated S glycoproteins produced

similar S-related protein species to that of the wild-type S indicating that none of the

engineered mutations adversely affect S synthesis and intracellular processing (Fig.

2.2A). The SARS S glycoprotein is known to form homotrimers in its native state (Song

et al., 2004). To investigate the effect of the mutations on S oligomerization, cellular

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Figure 2.2. Western blot analysis of the expressed mutant SARS-CoV S mutant glycoproteins. (A,B) Immunoblots of wild-type (3xFLAG So (WT)), cluster to lysine, cluster to alanine, and carboxyl truncation mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. "Cells only" represents a negative control in which Vero cells with no protein transfected into them were probed with the monoclonal antibody to SARS S glycoprotein. (B) In order to detect trimer formation more efficiently, the protein extracts of the mutants were neither boiled nor subject to treatment with beta mercaptoethanol.

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lysates from transfected cells were electrophoretically separated without prior boiling of

the samples and in the absence of reducing agents (Song et al., 2004). The different S

species were detected via chemiluminescence using the monoclonal SW-111 to the SARS

S glycoprotein. A S protein species was detected that had an approximate apparent

molecular mass of 500 kDa, which was consistent with previously published data (Song

et al., 2004) (Fig 2.2B). Although levels of oligomer expression seemed to vary slightly

between mutant forms, all mutated S glycoproteins produced similar species to the wild

type, indicating that none of the mutations blocked oligomerization from occurring.

Ability of Mutant S Glycoproteins to be Expressed on the Cell Surface

To determine if the mutant S glycoproteins were expressed on the surface of cells,

immunohistochemical analysis was used to label cell-surface expressed S under live cell

conditions that restrict antibody binding to cell surfaces. In addition,

immunohistochemistry was used to detect the total amount of S expressed in cells by

fixing and permeabilizing the cells prior to reaction with the antibody. A recombinant S

protein having the 3xFLAG added in-frame to the carboxyl terminus of S was used as a

negative control; since it would not be stained by the live cell reaction conditions (see

Materials and Methods). Both wild-type versions of S having the 3xFLAG, either at the

amino or carboxyl terminus of S, caused similar amounts of fusion (Fig. 2.3), which also

was similar to that obtained with the untagged wild-type S (not shown). The relative

amounts of cell-surface versus total cellular expression of S were obtained through the

use of an ELISA. A ratio between the cell-surface localized S and total cellular S

expression was then calculated and normalized to the corresponding ratio obtained with

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Figure 2.3. Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (3xFLAG So (WT)) (F1, F2), CL1 (A1, A2), CL2 (B1, B2), CL3 (C1, C2), CL4 (D1, D2), CL5 (E1, E2), CL6 (L1, L2), CL7 (M1, M2), T1214 (K1, K2), T1229 (J1, J2), T1238 (I1, I2), T1247(H1, H2) and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (G1, G2), which served as a negative control. At 48 hours post-transfection, cells were immunhistochemically processed either under live conditions to show surface expression. (A2, B2, C2, D2, E2, F2, G2, H2, I2, J2, K2, L2, and M2) or fixed and permeabilized conditions to show total expression(A1, B1, C1, D1, E1, F1, G1, H1, I1, J1, K1, L1, and M1) with anti-FLAG antibody.

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the wild-type S glycoprotein (see Materials and Methods) (Fig. 2.4). The CL1 (95%),

CL2 (78%), CL3 (86%), CL4 (80%), CL5 (92%), CL6 (86%) mutants as well as the

T1229 (91%), T1238 (94%), and T1247 (79%) truncations were expressed on the cellular

surface by the percentages indicated when compared to the cell surface expression of the

wild type protein. In contrast, the 1214T mutant expressed 77% less S on cell surfaces in

comparison to the wild-type S (Fig. 2.4).

Effect of Mutations on S-mediated Cell-to-Cell Fusion

Transiently expressed wild type S causes extensive cell-to-cell fusion (syncytial

formation), especially in the presence of the SARS-CoV ACE2 receptor (Li et al., 2003).

To determine the ability of each mutant S glycoprotein to cause cell-to-cell fusion and the

formation of syncytia, fused cells were labeled by immunohistochemistry using the anti-

FLAG antibody (Fig. 2.5), and the extent of cell-to-cell fusion caused by each mutant

glycoprotein was calculated by obtaining the average size of approximately 300 syncytia.

The average syncytium size for each mutant was then normalized to that found in wild

type S transfected cells (see Materials and Methods). The CL1 (73%), CL2 (75%), CL3

(68%), CL4 (71%), CL5 (76%), CL6 (51%) as well as the T1214 (86%) and T1247

(66%) mutants inhibited the formation of syncytia by the percentages indicated. The

T1229 truncation and the cluster mutant CL7 produced syncytia, which were on the

average 22% and 15% smaller, respectively, than that of the wild-type S. In contrast, the

T1238 mutant produced on the average 43% larger syncytia than that of the wild-type S

(Fig. 2.5).

Comparison of the membrane fusion and cell-surface expression results allowed the

grouping of the different mutant S phenotypes into four distinct groups (Table 2.1): 1) S

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Figure 2.4. Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see Materials and Methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio.

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Figure 2.5. Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviations calculated through comparison of the data from each of three experiments.

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mutant forms in group I (CL1-CL6 and T1247) resulted in high levels of cell-surface

expression (78-95% of the wild-type S); however, the average size of syncytial formed

by these mutated S glycoproteins was reduced substantially in comparison to the wild-

type S (23-48% of the wild-type). CL1 affects the predicted fusion peptide, CL2-CL4

affect the HR1 domain, and CL5 affects a region downstream of the HR1 domain. The

CL6 mutation is located within the S carboxyl terminal acidic cluster. The T1247

mutation truncates the S carboxyl terminus by 8 amino acids; 2) S mutant forms in group

II produced high levels of S cell-surface expression and an average size of syncytia

slightly smaller than that of the wild-type S. These mutations included CL7, which

modified the acidic cluster and the T1229 truncations that deleted the cysteine-rich motif

CRM2; 3) The single S mutant in group III, T1214, produced significantly less cell-

surface expression and concomitantly the average size of syncytia was substantially

reduced in comparison to the wild type S; 4) The T1238 truncation in group IV produced

high levels of cell surface expression equivalent to that of the wild-type (94% of the wild-

type S), while the average syncytium size was 43% larger than that the syncytial

produced by the wild-type S (Table 2.1).

Detection of the Intracellular Distribution of S Mutant Glycoproteins Via Confocal Microscopy To visualize the intracellular distribution of S mutant glycoproteins, cells were

transfected with plasmids encoding the wild type or S mutants and examined by confocal

microscopy at 48 h post transfection (Fig. 2.6). The wild type and all the S mutants were

detected throughout the cytoplasm of transfected cells and exhibited similar intracellular

distribution patterns (Fig. 2.6, panels B, D, F, H, J, L). To determine and compare the

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Figure 2.6. Confocal microscopic visualization of endocytosed and intracellular distribution of SARS-CoV S glycoprotein mutants. Vero cells expressing wild-type SARS-CoV S glycoprotein (3xFLAG So (WT)) (A and B), CL6 (C and D), CL7 (E and F), T1229 (G and H), T1238 (I and J) and T1247 (K and L) were processed for confocal microscopy using two different methods in order to assess different properties of the mutants. Endocytosis patterns (A,C,E,G,I, and K) were visualized by adding anti-FLAG (green) antibody into the media 12 h prior to processing, enabling detection of the mutant protein after endocytosis from cellular surfaces. Early endosomes were also detected for these panels using a polyclonal anti-early endosomal antigen I antibody (red). For total glycoprotein detection (B, D, F, H, J, and L), cells were fixed and permeabilized prior to labeling with anti-FLAG (green).

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endocytotic profiles of wild-type and S mutant forms, transfected cells were reacted with

the anti-FLAG antibody under live conditions for 12 hours at 37ºC and visualized by

confocal microscopy. The majority of the wild-type S detected by the anti- FLAG

antibody appeared to remain on cell surfaces (Fig. 2.6, panel A). In contrast, a significant

fraction of cell-surface expressed CL6 and CL7 as well as the T1229, T1238 and T1247 S

mutants appeared to partially endocytose to cytoplasmic compartments (Fig. 2.6, panels

C, E, G, I, K). The CL7 mutant, but not the other S mutants, appeared to colocalize with

the early endosomal marker EEA-1 (Fig. 2.6, panel E). The S mutants CL1, CL2, CL3,

CL4, and CL5 remained in plasma membranes exhibiting profiles similar to that of the S

wild-type glycoprotein (data not shown).

Time-dependent Endocytotic Profiles of Wild-type and Mutant S Proteins

A time-dependent endocytosis assay was utilized to better visualize the endocytotic

patterns of the wild-type and mutant S glycoproteins as well as to exclude the possibility

that the observed plasma membrane accumulation of the wild-type S and some of the S

mutants was due to recirculation of endocytosed S to cell-surfaces. In this assay, cell-

surface expressed S was reacted with anti-FLAG antibody at 4°C and subsequently, cells

were incubated at 37°C for different time periods before processing for confocal

microscopy (see Materials and Methods). Generally, these time-dependent endocytosis

studies were in agreement with the results shown in Figure 2.6. Specifically, in cells that

were not shifted to 37ºC, referred to as time zero cells, wild type and mutant spike were

detected exclusively at the surface of the cells (Fig. 2.7, panels A1, B1, C1, D1, E1, F1).

At five and fifteen minutes after the shift to 37ºC, the wild-type S remained exclusively at

the surface while the other S mutants were detected in numerous intracellular vesicles

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Figure 2.7. Analysis of the endocytotic kinetic profile of the truncation mutants and the acidic cluster mutants using confocal microscopy. After transfection, SARS-CoV S glycoprotein wild-type (3xFLAG So (WT)) (A1-A4), T1229 (B1-B4), T1238 (C1-C4), T1247 (D1-D4), CL6 (E1-E4), and CL7 (F1-F4) expressing cells were incubated with an anti-FLAG monoclonal antibody (green) for 1 hr and then returned to 37ºC for different times. Cell nuclei were labeled with To-Pro-3 Iodide (blue). Panels A1-A4, B1-B4, C1-C4, D1-D4, E1-E4, and F1-F4 correspond to 0-, 5-, 15-, and 60-min incubation times at 37ºC, respectively.

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dispersed inside the cell (Fig. 2.7, panels A2 and A3 compared to panels B2 and B3, C2

and C3, D2 and D3, E2 and E3, F2 and F3). By sixty minutes after the shift to 37ºC, the

wild type S was still localized exclusively to the surface of the cell (Fig. 2.7, panel A4),

while the T1229, T1238, CL6 and CL7 S mutants appeared to be present throughout the

cytoplasm of the cell (Fig. 2.7, panels B4, C4, E4, F4). The T1247 S mutant seemed to

undergo rapid and complete endocytosis during the 60 min observation and appeared to

localize into punctuate structures in the cytoplasm of cells unlike the fairly even cellular

distribution of all other S mutants (Fig. 2.7, panels D1-D4 compared A4, B4, C4, D4, E4,

F4).

DISCUSSION

The mechanism by which class I fusion proteins such as the coronavirus S

glycoprotein, the hemagglutinin protein (HA) of influenza virus, the gp41 of human

immunodeficiency virus (HIV), the Ebola virus surface glycoprotein (GP), and the fusion

protein (F) of paramyxovirus facilitate membrane fusion during viral entry into cells has

been extensively investigated (Eckert and Kim, 2001; Hernandez et al., 1996; Tsai et al.,

2003; White, 1992; Zelus et al., 2003). Currently, specific membrane fusion models have

been proposed all of which include the following general steps: a) binding of a receptor

through a receptor specific domain located within the ectodomain of the viral

glycoprotein; b) induction of a conformational change via low pH or binding to the

receptor that exposes a fusion peptide, typically a hydrophobic region in the membrane

anchored subunit, which inserts into the cellular lipid membrane; c) formation of a

trimer-of-hairpins like structure by α-helical peptides, termed heptad repeat segments, via

a transient pre-hairpin intermediate that facilitates the juxtaposition of the viral and

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cellular membranes which then leads to fusion of the viral envelope with cellular

membranes (reviewed in (Eckert and Kim, 2001; Hernandez et al., 1996)). Although the

most important domains of the class I fusion proteins are naturally located in their

ectodomains, it has been reported that intracytoplasmic endodomains play an important

role in intracellular transport and virus-induced cell fusion (Bagai and Lamb, 1996; Bos

et al., 1995; Chang, Sheng, and Gombold, 2000; Lontok, Corse, and Machamer, 2004;

Schwegmann-Wessels et al., 2004; Sergel and Morrison, 1995; Seth, Vincent, and

Compans, 2003; Tong et al., 2002; Waning et al., 2004; Yao and Compans, 1995).

In this paper, we show that mutations that alter the HR1 and predicted fusion

peptide domains of the SARS-CoV S glycoprotein as well as mutations located within α-

helical regions well separated from the HR1, HR2 and predicted fusion peptide domains

drastically affected S-mediated cell fusion. Importantly, mutagenesis of the S cytoplasmic

domains suggests that the carboxyl terminus of the S glycoprotein contains multiple but

distinct regulatory domains that may function in virus-induced cell fusion through

different mechanisms.

Functional Domains of the S Ectodomain

The CL1 cluster mutation is located within the predicted fusion peptide. The

constructed cluster mutations replaced the amino terminal a and d positions of the

predicted fusion peptide resulting in shortening the predicted α-helical portion of the

fusion peptide. The S mutant glycoprotein carrying the CL1 mutation was apparently

synthesized in comparable levels to the wild type S glycoprotein. As expected, although

this mutant S form was able to be expressed on cell surfaces (95% of wild-type S levels),

its ability to cause cell fusion was inhibited by more than 70% (Table 1; Group I

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mutants). This result confirms that the predicted fusion peptide it absolutely essential for

S mediated cell fusion, although the engineered collapse of the predicted region does not

significantly effect glycoprotein synthesis, processing, and cell-surface expression.

Recent studies have shown that interactions between HR1 and HR2 of SARS-CoV

are critical in producing the necessary conformation changes that result in exposure of the

fusion peptide and its insertion into apposed membranes (Ingallinella et al., 2004; Tripet

et al., 2004; Xu et al., 2004). Biochemical and x-ray crystallography studies have shown

that the HR1 and HR2 form a stable six-helix bundle, in which the HR1 helices form a

central coiled-coil surrounded by three HR2 helices in an oblique, antiparallel manner

termed the fusion core (Ingallinella et al., 2004; Tripet et al., 2004; Xu et al., 2004),

which is consistent with other class I fusion proteins (Baker et al., 1999; Bullough et al.,

1994; Caffrey et al., 1998; Chan et al., 1997; Lu, Blacklow, and Kim, 1995; Tan et al.,

1997; Weissenhorn et al., 1998a; Weissenhorn et al., 1998b; Weissenhorn et al., 1997).

Amino acid residues 902-947 in the SARS-CoV S HR1 domain fold into a predicted 12-

turn α-helix (entire length of the fusion core) with hydrophobic amino acids

predominantly occupying the a and d positions. The CL3 and CL4 mutations were

designed to change the a and d hydrophobic residues to hydrophilic (lysine) residues.

Both mutant glycoproteins were expressed on cell surfaces at reduced levels in

comparison to the wild-type S glycoprotein (14% and 20% reduction, respectively).

However, these mutations inhibited S-mediated fusion by 68% and 71%, respectively

(Table 1; Group I mutants). Therefore, the inability of the CL3 and CL4 mutants to cause

fusion is most likely due to ectodomain structural changes involving the HR1 domain.

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Inhibition of proper HR1 interaction with HR2 may be the primary cause of the observed

inhibition of S-mediated cell fusion.

The CL2 and CL5 cluster mutations are located upstream and downstream of HR1

within a predicted α-helical portion of the S ectodomain extending from residues 875 to

1014. Both CL2 and CL5 S mutant forms exhibited reduced S-mediated cell fusion (75%

and 76% reduction in comparison to the S wild-type, respectively), while they were

synthesized and expressed on cell-surfaces at levels similar to the S wild-type (Table 1;

Group I mutants). These data suggest that the inability of these S mutants to cause

extensive cell fusion was mostly due to structural alterations of the extracellular portion

of the S glycoprotein. Furthermore, these results suggest that α-helical portions of the S

ectodomain that are well-separated from HR1 and HR2 or the predicted fusion peptide

are important for S-mediated cell fusion. It is possible that these mutations affect HR1

interactions with HR2 by inhibiting ectodomain conformational changes required for

their optimal interactions. Specifically, the CL2 mutation is only 16 amino acids

downstream of the predicted fusion peptide. Therefore, this mutation may interfere with

fusion peptide-associated functions

Functional Domains of the Endodomain of S

The T1247 S mutation deletes 8 amino acids from the carboxyl terminus of S. This

S mutant exhibited a 65% reduction in cell fusion in comparison to the wild-type S, while

cell-surface expression was reduced by 20% in comparison to the wild type S (Table 1;

Group I mutants). Kinetic endocytosis experiments revealed that the T1247 S mutant

also endocytosed much faster than the wild-type S glycoprotein. Acidic cluster motifs are

known to serve as endocytotic signals (Brideau et al., 1999; Zhu et al., 1996). The rapid

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endocytosis of the T1247 S mutant form may be due to the relocation of the acidic cluster

KFDEDDSE proximal to the carboxyl terminus of the S glycoprotein after removal of the

terminal 8 amino acids resulting in more efficient endocytosis. Therefore, the inability of

the S mutant form to cause extensive cell fusion may be due primarily to its rapid

endocytosis from cell surfaces. It is worth noting that a similar deletion of 8 amino acids

from the carboxyl terminus of the MHV S glycoprotein resulted in enhanced S-mediated

cell fusion (Chang, Sheng, and Gombold, 2000). A comparison of the carboxyl terminal

amino acid sequences of these two glycoproteins reveals that the MHV S does not have a

well-defined acidic cluster in the SARS-COV S homologous location; however, the last

three amino acids of the MHV S are charged residues (HED). Therefore, the ability of the

truncated MHV S to cause more cell fusion may be due to increased surface retention

resulting from a reduction in endocytosis mediated by these charged residues.

Alternatively, the MHV deletion may cause structural changes that enhance the MHV S

fusogenicity by destabilizing the overall structure of the glycoprotein. Stabilization of the

carboxyl terminus has been shown to decrease the fusion activity of the vesicular

stomatitis virus G glycoprotein (Waning et al., 2004), therefore, conversely, it is possible

that destabilization of the carboxyl terminus may cause an increase in fusion.

Of particular interest is the T1238 truncation which removed the S carboxyl

terminal acidic domain. This S mutant glycoprotein exhibited a more than 40% increase

in cell fusion relative to the S wild type, while there was only a slight decrease in cell-

surface expression (Table 1; Group IV mutants). The fact that the overall levels of S

glycoprotein detected on cell surfaces as well as the endocytosis profile were not altered

suggests that this deletion may enhance fusion via a structural destabilization of the

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glycoprotein. In contrast, changing acidic amino acids of the acidic motif to alanine

residues inhibited cell fusion indicating that the acidic motif was important for S-

mediated cell fusion. Specifically, the CL6 cluster mutation changed the acidic residues

(DEDDSE) to alanine residues (AAAASA). This mutant protein, while being efficiently

expressed at cellular surfaces (87% of the wild type protein), exhibited a 51% reduction

in fusion activity in comparison to the wild type (Table 1; group I mutant). The observed

reduction in S-mediated cell fusion suggests that the acidic cluster plays an important

regulatory role in S-mediated cell fusion without appreciably affecting intracellular

transport and cell-surface expression. This result is in sharp contrast to the T1238

truncation that deleted the acidic cluster and caused enhanced S-mediated cell fusion. A

possible explanation for these seemingly disparate results is that modifications of the

carboxyl terminus produce differential effects on the structure and function of the protein

by rendering the S glycoprotein more or less prone to S-mediated fusion. Alternatively, it

is possible that the acidic cluster plays important roles only in the context of the entire S

glycoprotein by regulating binding to other viral or cellular proteins that may modify the

S fusogenic properties.

The acidic cluster located in the cytoplasmic portion of the SARS CoV S contains a

predicted phosphorylation site (DEDDSE). To address the role of this predicted

phosphorylation site within the acidic cluster, the CL7 cluster mutation was constructed.

The CL7 replaces the serine residue with an alanine residue within the acidic cluster.

This mutant S protein fused cells extensively (85% of the wild type) and was expressed

on cell-surfaces at levels similar to that of the wild type S (88% of the wild type) (Table

1; Group II mutants), suggesting that the potential phosphorylation site does not play an

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important role in S-mediated cell fusion and cell-surface expression. However, the CL7

mutant appeared to recycle to early endosomes in contrast to the wild type S, which

remained mostly on cell surfaces. Therefore, it is possible that the altered putative

phosphorylation site within the acidic cluster may play a yet unknown role in S retention

at cell surfaces. Conversely, lack of this signal may cause aberrant endocytosis to early

endosomes. The overall charge of the carboxyl terminus may also play some role in the

structure and function of S. In this regard, there are additional charged amino acids

dispersed upstream and downstream of the mutated charged cluster that may play some

role in S transport and S-mediated fusion through electrostatic interactions with other

viral and cellular proteins. Additional alanine scanning mutations would be needed to

resolve their potential contribution to S functions.

In contrast to the MHV S glycoprotein, in which the domains overlap, the charged

region of the SARS-CoV S glycoprotein and the two cysteine-rich motifs CRM1 and

CRM2 are separate distinct regions (Fig 2.8). The T1229 mutation deleted the CRM2

domain, while the T1214 truncation deleted both CRM1 and CRM2. Deletion of the

CRM2 slightly inhibited surface expression (91% of the wild type) while reducing fusion

activity by 22% (Table 1; Group II mutants). A similar truncation of the MHV S

glycoprotein produced a comparable effect on cell fusion (Chang, Sheng, and Gombold,

2000). The 1214 truncation severely inhibited cell surface expression by 77%, when

compared to the wild type, while cell-to-cell fusion activity was reduced by 84% (Table

1; Group III mutant). These results differ from previously published data on similar

truncations of the MHV S glycoprotein. Specifically, it was found that the replacement

of the entire cysteine rich domain with amino acid sequences derived from the

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Figure 2.8. Cluster alignment of the carboxyl terminus of the SARS-CoV S and the MHV S glycoproteins. The shaded residues indicate the position of the cysteine rich motif in their respective protein. The cysteine residues are bolded. The charged clusters are indicated by a bracket over the corresponding regions.

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cytoplasmic terminus of the herpes simplex virus 1 (HSV-1) glycoprotein D (gD)

severely inhibited MHV S glycoprotein function without necessarily affecting cell-

surface expression (Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and

Masters, 2004). In contrast, the SARS-CoV S T1214 mutant glycoprotein failed to be

expressed on cell surfaces explaining the inability of this glycoprotein to cause cell

fusion. These results suggest that the proximal cysteine residues of the SARS CoV S play

crucial roles in intracellular transport and cell-surface expression. The discrepancy with

the MHV S carboxyl terminal replacements may be due to additional gD sequences that

facilitated intracellular transport and cell-surface expression. It is worth noting that

depending on the algorithm used to predict the membrane spanning domain of S, a few of

the cysteine residues may be included in the membrane spanning region (Chang, Sheng,

and Gombold, 2000; Ye, Montalto-Morrison, and Masters, 2004). Therefore, it is

possible that deletion of these cysteine residues may lead to S misincorporation into

membranes, resulting in the apparent transport defects.

Overall, these results suggest that the S-mediated cell fusion is regulated by both

the ecto- and endodomains, which play important roles in cell-surface expression.

Furthermore, the data suggest that the 17 carboxyl terminal amino acid residues of S

exert a negative regulatory (repressor) effect on S-mediated cell fusion, while both the

carboxyl terminal acidic cluster and CRM2 domains exert secondary regulatory roles in

S-mediated cell fusion. Additional studies are required to elucidate the specific amino

acid requirements of the S endodomain that can affect S-mediated cell fusion potentially

via a transmembrane signal transduction process that leads to destabilization of the S

ectodomain.

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MATERIALS AND METHODS

Cells

African green monkey kidney (Vero) cells were obtained from the American Type

Culture Collection (Rockville, Md.). Cells were propagated and maintained in Dulbecco

modified Eagle medium (Sigma Chemical Co., St. Louis, Mo.) containing sodium

bicarbonate and 15 mM HEPES supplemented with 10% heat-inactivated fetal bovine

serum.

Plasmids

The parental plasmid used in the present study, SARS-S-Optimized, has been

previously described (Li et al., 2003). The Spike-3XFLAG-N gene construct was

generated by cloning the codon-optimized S gene, without the DNA sequence coding for

the signal peptide, into the p3XFLAG-CMV-9 plasmid vector (Sigma). PCR overlap

extension (Aiyar, Xiang, and Leis, 1996) was used to construct the alanine mutants,

cluster mutants, and the single point mutants. In order to construct the truncation

mutants, primers were designed that incorporated a stop codon and a BamHI restriction

site at the appropriate gene site. Restriction endonuclease sites HindIII and BamHI were

then used to clone the gene construct into the Spike-3XFLAG-N plasmid.

The constructed cluster mutants targeting the S ectodomain changed the following

sets of amino acids to lysine residues: CL:1 Y(855), L(859), G(862); CL2: L(898), N

(901); CL3: L(927), L(930), V(934); CL4: L(941), L(944); CL5: L(983), L(986). The

cluster mutants targeting the S endodomain changed the following amino acids to alanine

residues: CL6: D(1239), E(1240), D(1241), D(1242), E(1244); CL7: S(1243) (Figs. 2.1B

and 2.1C).

115

Production of SARS-CoV S Monoclonal Antibodies

The monoclonal antibodies SW-111 was raised against the Spike envelope

glycoprotein of the SARS virus. A synthetic, codon-optimized gene corresponding to

SAR-CoV S glycoprotein coding sequences (Li et al., 2003) was engineered to produce

truncated, secreted proteins containing C-terminal His-tags which encode either the entire

ectodomain or just the receptor binding domain of S1. These genes were cloned into

baculoviral vectors, and the resulting virus was used to infect insect cell lines (High Five)

(Invitrogen, Carlsbad, CA). The supernatants were then tested by western blotting using

anti-6-His antibodies. Protein was partially purified from supernatants by passage

through a nickel column and then concentrated by ultrafiltration. Additional protein

derived in an analogous fashion was provided by the laboratory of Stephen Harrison.

Standard protocols for mouse immunization were used. The animals were maintained in

the animal facility of Dana-Farber Cancer Institute, and hybridomas and monoclonal

antibodies were produced in the Dana Farber/Harvard Cancer Center Monoclonal

Antibody Core. Spleenocytes from immunogenized mice were fused with NS-1 myeloma

cells (ATCC) using standard protocols. Antibody-producing clones were identified by

Western blot analysis, using purified SARS-CoV S. The positive cells were then sub-

cloned and re-tested against the purified SARS-CoV S glycoprotein. Isotype analysis

revealed that the antibody belonged to the IgG-1 class.

Western Blot and S Oligomerization Analysis

Vero cell monolayers in six-well plates were transfected with the indicated plasmids

utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s

directions. At 48 hours (h) post transfection, cells were collected by low-speed

116

centrifugation, washed with Tris-buffered saline (TBS), and lysed at on ice for 15 min in

mammalian protein extraction reagent supplemented with a cocktail of protease inhibitors

(Invitrogen/Life Technologies). Insoluble cell debris was pelleted, samples were

electrophoretically separated by SDS-PAGE, transferred to nitrocellulose membranes,

and probed with anti-SARS CoV monoclonal antibody at a 1:10 dilution. Samples being

analyzed for transport and processing were boiled for 5 min and treated with beta

mercaptoethanol, while samples being analyzed for trimer formation were not.

Subsequently, blots were incubated for 1 h with a peroxidase-conjugated secondary

antibody at a 1:50,000 dilution and then visualized on X-ray film by chemiluminescence

(Pierce Chemicals, Rockford, Ill.). All antibody dilutions and buffer washes were

performed in TBS supplemented with 0.135 M CaCl2 and 0.11 M MgCl2 (TBS-Ca/Mg).

Cell Surface Immunohistochemistry



directions. At 48 h post transfection, the cells were washed with TBS-Ca/Mg and either

fixed with iced cold methanol or left unfixed (live). Immunohistochemistry was

performed by utilizing the Vector Laboratories Vectastain Elite ABC kit (Vector

Laboratories, Burlingame, CA) essentially as described in the manufacturer’s directions.

Briefly, cells were washed with TBS-Ca/Mg and incubated in TBS supplemented with

5% normal horse serum and 5% bovine serum albumin at room temperature for 1 h. After

blocking, cells were reacted with anti-FLAG antibody (1:500) in TBS blocking buffer for

3 h, washed four times with TBS-Ca/Mg, and incubated with biotinylated horse anti-

mouse antibody. Excess antibody was removed by four washes with TBS-Ca/Mg and

117

subsequently incubated with Vectastain Elite ABC reagent for 30 min. Finally, cells were

washed three times with TBS-Ca/Mg, and reactions were developed with NovaRed

substrate (Vector Laboratories) according to the manufacturer’s directions.

Determination of Cell-surface to Total Cell S Glycoprotein Expression


and processed for immunohistochemistry as described above with the exception that the

ABTS Substrate Kit , 2, 2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Vector

Laboratories) was used instead of the NovaRed substrate. After the substrate was allowed

to develop for 30 min, 100 μl of the developed substrate was transferred, in triplicate, to a

96 well plate. The samples were then analyzed for color change at a wavelength of 405

nm. The absorbance reading from cell-surface labeling experiments obtained from live

cells were divided by the total labeled absorbance readings obtained from fixed cells

which was then normalized to the wild type protein values. The measurements were then

converted to percentages reflecting the ratio of S present on cell-surfaces versus the total

S expressed in the transfected cells.

Confocal Microscopy

Vero cell monolayers grown on coverslips in six-well plates were transfected with

the indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to

the manufacturer’s directions. For the endocytosis analysis of the mutants, anti-FLAG

diluted 1:500 in TBS supplemented with 5% normal goat serum and 5% bovine serum

albumin (TBS blocking buffer) was added to the cell culture media for 12 hours before

processing. At 48 h post transfection, cells were washed with TBS and fixed with

electron microscopy-grade 3% paraformaldehyde (Electron Microscopy Sciences, Fort

118

Washington, Pa.) for 15 min, washed twice with TBS-Ca/Mg, and permeabilized with

0.1% Triton X-100. Monolayers were blocked for 1 hour with TBS blocking buffer

before incubation for 3 h with anti-FLAG antibody (sigma) diluted 1:500 in TBS

blocking buffer. Cells were then washed extensively and subsequently incubated for 1 h

with Alexafluor 488-conjugated anti-immunoglobulin G (IgG) (Molecular Probes)

diluted 1:750 in TBS blocking buffer. To visualize the early endosomes, cells were

stained with a 1:500 dilution of anti-early endosomal antigen I antibody (Affinity

Bioreagents Inc). Cells were examined by using a Leica TCS SP2 laser-scanning

microscope (Leica Microsystems, Exton, Pa.) fitted with a 63x Leica objective lens

(Planachromatic; 1.4 numerical aperture). Individual optical sections in the z axis,

averaged eight times, were collected simultaneously in the different channels at a 512 x

512 pixel resolution as described previously (Foster et al., 2004; Lee et al., 2000). Images

were compiled and rendered in Adobe Photoshop.

S Glycoprotein Endocytosis Assay

Vero cell monolayers grown on coverslips in six-well plates were transfected with

the indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to

the manufacturer’s directions. At 48 hours posttransfection, the cells were washed once

with room temperature TBS-Ca/Mg. The plates were then moved to a 4ºC cold room and

washed with 4ºC TBS-Ca/Mg. The cells were labeled for 1 hour at 4°C with anti-FLAG

antibody diluted 1:500 in TBS blocking buffer. The cells were washed with 4ºC TBS

three times and then brought back to 37°C and allowed to incubate for their respective

time points. Then, the cells were immediately fixed and permeabilized with ice cold

methanol. Monolayers were blocked for 2 hrs in TBS blocking buffer before incubation

119

for 1 h with Alexafluor 488-conjugated anti-immunoglobulin G (IgG) (Molecular Probes)

diluted 1:750 in TBS-Ca/Mg blocking buffer. After incubation, excess antibody was

removed by washing five times with TBS-Ca/Mg. The nuclei was counterstained for 15

min with TO-PRO-3 iodide (1:5,000 dilution) and visualized at 647 nm. Cells were

examined by using a Leica TCS SP2 laser-scanning microscope (Leica Microsystems,

Exton, Pa.) fitted with a 63x Leica objective lens (Planachromatic; 1.4 numerical

aperature). Individual optical sections in the z axis, averaged eight times, were collected

simultaneously in the different channels at 512 x 512 pixel resolution as described

previously (Foster et al., 2004; Lee et al., 2000). Images were compiled and rendered in

Adobe Photoshop.

Quantitation of the Extent of S-mediated Cell Fusion

Vero cell monolayers in six-well plates were transfected in triplicate with the indicated

plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the

manufacturer’s directions. Concurrently, Vero cell monolayers in six-well plates were

transfected with the plasmid encoding the ACE2 receptor protein utilizing the

Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s directions. At

24 h post transfection, cells containing the mutant plasmids, the ACE2 receptor, and

normal untransfected cells were washed with TBS-Ca/Mg, trypsinized, and overlayed in

a single well of a six-well plate at a ratio of 2 ml (cells transfected with the ACE2

receptor) : 0.5 ml (cells transfected with the mutant) : 1.5 ml (untransfected cells). All of

the cells transfected with ACE2 were pooled to ensure that every well had an equal

amount of cells with receptor expressed on their surface. After incubation for 24 h, the

cells were washed with TBS-Ca/Mg and fixed with ice cold methanol.

120

Immunohistochemistry was performed by utilizing the Vector Laboratories Vectastain

Elite ABC kit essentially as described in the manufacturer’s directions. Briefly, cells were

washed with TBS-Ca/Mg and incubated in TBS blocking buffer supplemented with

normal horse serum at room temperature for 1 h. After blocking, cells were reacted with

anti-FLAG antibody (1:500) in TBS blocking buffer for 3 h, washed four times with

TBS-Ca/Mg, and incubated with biotinylated horse anti-mouse antibody. Excess antibody

was removed by four washes with TBS-Ca/Mg and subsequently incubated with

Vectastain Elite ABC reagent for 30 min. Finally, cells were washed three times with

TBS-Ca/Mg, and reactions were developed with NovaRed substrate (Vector

Laboratories) according to the manufacturer’s directions. The average size of syncytia for

each mutant was determined by analyzing the area of approximately 300 syncytia, from

digital images, using the Image Pro Plus 5.0 software package. The averages were then

converted to percentages of the average syncytia size of the wild type SARS-CoV S.

Error bars shown represent the standard deviations calculated through comparison of the

data from each of three experiments.

REFERENCES

Aiyar, A., Xiang, Y., and Leis, J. (1996). Site-directed mutagenesis using overlap extension PCR. Methods Mol Biol 57, 177-91.

Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R., and Hilgenfeld, R. (2003).


Bagai, S., and Lamb, R. A. (1996). Truncation of the COOH-terminal region of the

paramyxovirus SV5 fusion protein leads to hemifusion but not complete fusion. J Cell Biol 135(1), 73-84.

Baker, K. A., Dutch, R. E., Lamb, R. A., and Jardetzky, T. S. (1999). Structural basis for

paramyxovirus-mediated membrane fusion. Mol Cell 3(3), 309-19.

121

Blom, N., Gammeltoft, S., and Brunak, S. (1999). Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294(5), 1351-62.

Bos, E. C., Heijnen, L., Luytjes, W., and Spaan, W. J. (1995). Mutational analysis of the

murine coronavirus spike protein: effect on cell-to-cell fusion. Virology 214(2), 453-63.

Bos, E. C., Luytjes, W., and Spaan, W. J. (1997). The function of the spike protein of

mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity. J Virol 71(12), 9427-33.

Bosch, B. J., Martina, B. E., Van Der Zee, R., Lepault, J., Haijema, B. J., Versluis, C.,

Heck, A. J., De Groot, R., Osterhaus, A. D., and Rottier, P. J. (2004). Severe acute respiratory syndrome coronavirus (SARS-CoV) infection inhibition using spike protein heptad repeat-derived peptides. Proc Natl Acad Sci U S A 101(22), 8455-60.



Brideau, A. D., del Rio, T., Wolffe, E. J., and Enquist, L. W. (1999). Intracellular

trafficking and localization of the pseudorabies virus Us9 type II envelope protein to host and viral membranes. J Virol 73(5), 4372-84.

Bullough, P. A., Hughson, F. M., Skehel, J. J., and Wiley, D. C. (1994). Structure of

influenza haemagglutinin at the pH of membrane fusion. Nature 371(6492), 37-43.

Caffrey, M., Cai, M., Kaufman, J., Stahl, S. J., Wingfield, P. T., Covell, D. G.,

Gronenborn, A. M., and Clore, G. M. (1998). Three-dimensional solution structure of the 44 kDa ectodomain of SIV gp41. Embo J 17(16), 4572-84.

Cavanagh, D. (1995). "The coronavirus surface glycoprotein." Plenum Press, Inc., New

York, N.Y. Chan, D. C., Fass, D., Berger, J. M., and Kim, P. S. (1997). Core structure of gp41 from

the HIV envelope glycoprotein. Cell 89(2), 263-73. Chang, K. W., Sheng, Y., and Gombold, J. L. (2000). Coronavirus-induced membrane

fusion requires the cysteine-rich domain in the spike protein. Virology 269(1), 212-24.

122

de Groot, R. J., Luytjes, W., Horzinek, M. C., van der Zeijst, B. A., Spaan, W. J., and Lenstra, J. A. (1987). Evidence for a coiled-coil structure in the spike proteins of coronaviruses. J Mol Biol 196(4), 963-6.

Drosten, C., Gunther, S., Preiser, W., van der Werf, S., Brodt, H. R., Becker, S.,



inhibition. Annu Rev Biochem 70, 777-810. Foster, T. P., Melancon, J. M., Baines, J. D., and Kousoulas, K. G. (2004). The herpes

simplex virus type 1 UL20 protein modulates membrane fusion events during cytoplasmic virion morphogenesis and virus-induced cell fusion. J Virol 78(10), 5347-57.

Gombold, J. L., Hingley, S. T., and Weiss, S. R. (1993). Fusion-defective mutants of


Hernandez, L. D., Hoffman, L. R., Wolfsberg, T. G., and White, J. M. (1996). Virus-cell

and cell-cell fusion. Annu Rev Cell Dev Biol 12, 627-61. Holmes, K. V. (2003). SARS-associated coronavirus. N Engl J Med 348(20), 1948-51. Ingallinella, P., Bianchi, E., Finotto, M., Cantoni, G., Eckert, D. M., Supekar, V. M.,

Bruckmann, C., Carfi, A., and Pessi, A. (2004). Structural characterization of the fusion-active complex of severe acute respiratory syndrome (SARS) coronavirus. Proc Natl Acad Sci U S A 101(23), 8709-14.

Krokhin, O., Li, Y., Andonov, A., Feldmann, H., Flick, R., Jones, S., Stroeher, U.,


Ksiazek, T. G., Erdman, D., Goldsmith, C. S., Zaki, S. R., Peret, T., Emery, S., Tong, S.,


123

Lee, B. S., Alvarez, X., Ishido, S., Lackner, A. A., and Jung, J. U. (2000). Inhibition of

intracellular transport of B cell antigen receptor complexes by Kaposi's sarcoma-associated herpesvirus K1. J Exp Med 192(1), 11-21.

Lescar, J., Roussel, A., Wien, M. W., Navaza, J., Fuller, S. D., Wengler, G., and Rey, F.




Lontok, E., Corse, E., and Machamer, C. E. (2004). Intracellular targeting signals

contribute to localization of coronavirus spike proteins near the virus assembly site. J Virol 78(11), 5913-22.

Lu, M., Blacklow, S. C., and Kim, P. S. (1995). A trimeric structural domain of the HIV-

1 transmembrane glycoprotein. Nat Struct Biol 2(12), 1075-82. Melikyan, G. B., Markosyan, R. M., Hemmati, H., Delmedico, M. K., Lambert, D. M.,


Peiris, J. S., Lai, S. T., Poon, L. L., Guan, Y., Yam, L. Y., Lim, W., Nicholls, J., Yee, W.

K., Yan, W. W., Cheung, M. T., Cheng, V. C., Chan, K. H., Tsang, D. N., Yung, R. W., Ng, T. K., and Yuen, K. Y. (2003). Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361(9366), 1319-25.




paramyxoviruses: capture of intermediates of fusion. Embo J 20(15), 4024-34. Schwegmann-Wessels, C., Al-Falah, M., Escors, D., Wang, Z., Zimmer, G., Deng, H.,

Enjuanes, L., Naim, H. Y., and Herrler, G. (2004). A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but

124

absent from severe acute respiratory syndrome-associated coronavirus. J Biol Chem 279(42), 43661-6.

Sergel, T., and Morrison, T. G. (1995). Mutations in the cytoplasmic domain of the

fusion glycoprotein of Newcastle disease virus depress syncytia formation. Virology 210(2), 264-72.

Seth, S., Vincent, A., and Compans, R. W. (2003). Mutations in the cytoplasmic domain

of a paramyxovirus fusion glycoprotein rescue syncytium formation and eliminate the hemagglutinin-neuraminidase protein requirement for membrane fusion. J Virol 77(1), 167-78.




Song, H. C., Seo, M. Y., Stadler, K., Yoo, B. J., Choo, Q. L., Coates, S. R., Uematsu, Y.,

Harada, T., Greer, C. E., Polo, J. M., Pileri, P., Eickmann, M., Rappuoli, R., Abrignani, S., Houghton, M., and Han, J. H. (2004). Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein. J Virol 78(19), 10328-35.

Stauber, R., Pfleiderera, M., and Siddell, S. (1993). Proteolytic cleavage of the murine

coronavirus surface glycoprotein is not required for fusion activity. J Gen Virol 74 ( Pt 2), 183-91.

Tan, K., Liu, J., Wang, J., Shen, S., and Lu, M. (1997). Atomic structure of a

thermostable subdomain of HIV-1 gp41. Proc Natl Acad Sci U S A 94(23), 12303-8.

Tong, S., Li, M., Vincent, A., Compans, R. W., Fritsch, E., Beier, R., Klenk, C., Ohuchi,

M., and Klenk, H. D. (2002). Regulation of fusion activity by the cytoplasmic domain of a paramyxovirus F protein. Virology 301(2), 322-333.

Tripet, B., Howard, M. W., Jobling, M., Holmes, R. K., Holmes, K. V., and Hodges, R.

S. (2004). Structural characterization of the SARS-coronavirus spike S fusion protein core. J Biol Chem 279(20), 20836-49.



125

Waning, D. L., Russell, C. J., Jardetzky, T. S., and Lamb, R. A. (2004). Activation of a paramyxovirus fusion protein is modulated by inside-out signaling from the cytoplasmic tail. Proc Natl Acad Sci U S A 101(25), 9217-22.

Weissenhorn, W., Calder, L. J., Wharton, S. A., Skehel, J. J., and Wiley, D. C. (1998a).

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil. Proc Natl Acad Sci U S A 95(11), 6032-6.

Weissenhorn, W., Carfi, A., Lee, K. H., Skehel, J. J., and Wiley, D. C. (1998b). Crystal

structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol Cell 2(5), 605-16.

Weissenhorn, W., Dessen, A., Harrison, S. C., Skehel, J. J., and Wiley, D. C. (1997).

Atomic structure of the ectodomain from HIV-1 gp41. Nature 387(6631), 426-30. White, J. M. (1992). Membrane fusion. Science 258(5084), 917-24. Wu, X. D., Shang, B., Yang, R. F., Yu, H., Ma, Z. H., Shen, X., Ji, Y. Y., Lin, Y., Wu, Y.

D., Lin, G. M., Tian, L., Gan, X. Q., Yang, S., Jiang, W. H., Dai, E. H., Wang, X. Y., Jiang, H. L., Xie, Y. H., Zhu, X. L., Pei, G., Li, L., Wu, J. R., and Sun, B. (2004). The spike protein of severe acute respiratory syndrome (SARS) is cleaved in virus infected Vero-E6 cells. Cell Res 14(5), 400-6.

Xu, Y., Lou, Z., Liu, Y., Pang, H., Tien, P., Gao, G. F., and Rao, Z. (2004). Crystal

structure of severe acute respiratory syndrome coronavirus spike protein fusion core. J Biol Chem 279(47), 49414-9.

Yao, Q., and Compans, R. W. (1995). Differences in the role of the cytoplasmic domain

of human parainfluenza virus fusion proteins. J Virol 69(11), 7045-53. Ye, R., Montalto-Morrison, C., and Masters, P. S. (2004). Genetic analysis of

determinants for spike glycoprotein assembly into murine coronavirus virions: distinct roles for charge-rich and cysteine-rich regions of the endodomain. J Virol 78(18), 9904-17.



Zelus, B. D., Schickli, J. H., Blau, D. M., Weiss, S. R., and Holmes, K. V. (2003).

Conformational changes in the spike glycoprotein of murine coronavirus are

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induced at 37 degrees C either by soluble murine CEACAM1 receptors or by pH 8. J Virol 77(2), 830-40.

Zhu, Z., Hao, Y., Gershon, M. D., Ambron, R. T., and Gershon, A. A. (1996). Targeting

of glycoprotein I (gE) of varicella-zoster virus to the trans-Golgi network by an AYRV sequence and an acidic amino acid-rich patch in the cytosolic domain of the molecule. J Virol 70(10), 6563-75.

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CHAPTER III

GENETIC ANALYSIS OF THE CYSTEINE RICH DOMAINS IN THE CARBOXYL TERMINUS OF THE SARS-COV SPIKE GLYCOPROTEIN

INTRODUCTION

An outbreak of atypical pneumonia, termed severe acute respiratory syndrome

(SARS), appeared in the Guangdong Province of southern China in November, 2002. The

mortality rates of the disease reached as high as 15% in some age groups (Anand et al.,

2003). The etiological agent of the disease was found to be a novel coronavirus (SARS-

CoV), which was first isolated from infected individuals by propagation of the virus on

Vero E6 cells (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003). Analysis of

the viral genome has demonstrated that the SARS-CoV is phylogenetically divergent

from the three known antigenic groups of coronaviruses (Drosten et al., 2003; Ksiazek et

al., 2003). Analysis of the polymerase gene alone, however, has indicated that the SARS-

CoV may be an early off-shoot from the group 2 coronaviruses (Snijder et al., 2003).





including the RNA replicase gene open reading frame (ORF) 1ab and the structural

proteins nucleocapsid (N), membrane protein (M), envelope protein (E), and spike


(HE) protein is also encoded by some coronaviruses. Distributed among the major viral

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genes are a series of ORFs that are specific to the different coronavirus groups. Functions

of the majority of these ORFs have not been determined.

The SARS spike glycoprotein, a 1255-amino-acid type I membrane glycoprotein


spike structure found on all coronavirions. The S glycoprotein facilitates virion entry of

all coronaviruses into susceptible cells by binding to specific receptors on cells and

mediating virus-cell fusion (Cavanagh, 1995). The S glycoprotein specified by mouse

hepatitis virus (MHV) is cleaved into S1 and S2 subunits, although cleavage is not

necessary for virus-cell fusion (Bos, Luytjes, and Spaan, 1997; Gombold, Hingley, and

Weiss, 1993; Stauber, Pfleiderera, and Siddell, 1993). Similarly, the SARS-CoV S

glycoprotein seems to be cleaved into S1 and S2 subunits in VeroE6-infected cells (Wu et

al., 2004), while it is not known whether this cleavage affects S-mediated cell fusion. The

SARS-CoV receptor has been recently identified as the angiotensin-converting enzyme 2

(ACE2) (Li et al., 2003). Although the exact mechanism by which the SARS-CoV enters

the host cell has not been elucidated, it is most likely similar to other coronaviruses.

Upon receptor binding at the cell membrane, the S glycoprotein is thought to undergo a

dramatic conformational change causing exposure of a hydrophobic fusion peptide,

which is subsequently inserted into cellular membranes. This conformational change of

the S glycoprotein causes close apposition followed by fusion of the viral and cellular

membranes resulting in entry of the virion nucleocapsids into cells (Eckert and Kim,

2001; Tsai et al., 2003; Zelus et al., 2003). This series of S-mediated virus entry events is

similar to other class I virus fusion proteins (Baker et al., 1999; Melikyan et al., 2000;

Russell, Jardetzky, and Lamb, 2001).

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Heptad repeat (HR) regions, a sequence motif characteristic of coiled-coils,

appear to be a common motif in many viral and cellular fusion proteins (Skehel and

Wiley, 1998). These coiled-coil regions allow the protein to fold back upon itself as a

prerequisite step to initiating the membrane fusion event. There are usually two HR

regions: an N terminal HR region adjacent to the fusion peptide and a C-terminal HR

region close to the transmembrane region of the protein. Within the HR segments, the

first amino acid (a) and fourth amino acid (d) are typically hydrophobic amino acids that

play a vital role in maintaining coiled-coil interactions. Based on structural similarities,

two classes of viral fusion proteins have been established. Class I viral fusion proteins

contain two heptad repeat regions and an N- terminal or N-proximal fusion peptide. Class

II viral fusion proteins lack heptad repeat regions and contain an internal fusion peptide

(Lescar et al., 2001). The MHV S glycoprotein, which is similar to other coronavirus S

glycoproteins, is a class I membrane protein that is transported to the plasma membrane

after being synthesized in the endoplasmic reticulum (Bosch et al., 2003). Typically, the

ectodomains of the S2 subunits of coronaviruses contain two regions with a 4, 3

hydrophobic (heptad) repeat the first being adjacent to the fusion peptide and the other

being in close proximity to the transmembrane region (de Groot et al., 1987).

In addition, the SARS-CoV S glycoprotein has a high (3%) cysteine content (39

residues. Nine of the cysteine resides are concentrated in a domain that spans the

transmembrane region and the cytoplasmic domain, with six of these residues extremely

well conserved throughout all coronaviruses (Fig 3.1). This unusual concentration of

cysteine residues along with the conservation of the residues among all coronaviruses

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Figure 3.1. Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV], human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks.

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suggest that these residues play an important role in S glycoprotein function although this

exact role is largely unknown. For MHV, studies have shown that the cysteine rich

domain is required for coronavirus-induced membrane fusion. Substituting the

cytoplasmic portion of this cysteine rich region and the cytoplasmic tail with the

cytoplasmic tail of the VSV-G protein abolished MHV S glycoprotein mediated cell-cell

fusion (Bos et al., 1995). Also, it was shown that while not being the sole functional

domain of the transmembrane anchor required for fusion activity, it was necessary for

fusion activity (Chang, Sheng, and Gombold, 2000).

In this study, cluster to alanine mutagenesis was used to elucidate which cysteine

clusters were dispensable for protein transport and SARS-CoV S mediated cell-cell

fusion. The results indicate that the SARS-CoV S glycoprotein conforms to the general

structure and function relationships that have been elucidated for the cysteine rich

domains of other coronaviruses, most notably the MHV (Bos et al., 1995; Chang, Sheng,

and Gombold, 2000). Specifically, the results show that the cysteine residues proximal to

the membrane are required for S-mediated fusion.

RESULTS

Genetic Analysis of S Glycoprotein Cysteine Rich Domain




Specifically for coronaviruses, the MHV S glycoprotein endodomain has been shown to

contain cysteine rich regions, which are critical for fusion of infected cells (Bos et al.,

1995; Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and Masters, 2004).

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To elucidate the role of the cytoplasmic cysteine rich domain in membrane fusion,

intracellular transport, and cell surface expression, cysteine cluster to alanine mutations

were made in the four cysteine clusters in the cytoplasmic domain of the S glycoprotein

(Fig. 3.2) (as described in materials and methods).

Effects of Mutations on S Synthesis

In order to investigate the effect of the different cluster to alanine mutations,

western immunoblot analysis was used to detect and visualize all of the mutant

glycoproteins as well as the wild type glycoprotein (Fig. 3.3). Cellular lysates prepared

from transfected cells at 48 hours post transfection were electrophoretically separated by

SDS-PAGE and the S glycoproteins were detected via chemiluminescence using a

monoclonal antibody specific for the SARS-CoV S glycoprotein. Carbohydrate addition

was shown to occur in at least four different locations of the SARS-CoV S glycoprotein

(Krokhin et al., 2003; Ying et al., 2004). Furthermore, transiently expressed S

glycoprotein in Vero E6 cells was proteolytically cleaved into S1 and S2 components

(Wu et al., 2004). The anti-S monoclonal antibody SW-111 detected a protein species in

cellular extracts from transfected cells, which migrated with an apparent molecular mass

of approximately 180 kDa, as reported previously (Song et al., 2004). All mutated S

glycoproteins produced similar S-related protein species to that of wild-type S indicating

that none of the engineered mutations adversely affected S synthesis and intracellular

processing (Fig. 3.3).

Ability of Mutant S Glycoproteins to Be Expressed on the Cell Surface

To determine if the mutant S glycoproteins were expressed on the surface of cells

efficiently, immunohistochemical analysis was used to label cell-surface expressed S

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Figure 3.2. Schematic diagram of the SARS-CoV S glycoprotein endodomain and the cysteine cluster to alanine mutations. Amino acid sequences of the carboxyl termini and the cysteine cluster to alanine mutations are shown for the wild-type as well as the mutant proteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The transmembrane portion of the endodomain is italicized and underlined. Amino acids mutated to alanines for the C1217A, C1223A, C1230A, and C1235A cluster mutations are in bold.

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Figure 3.3 Western blot analysis of the expressed mutant SARS-CoV mutant glycoproteins. Immunoblots of wild-type [So-3xF(WT)] and cysteine to alanine mutant S glycoproteins probed with monoclonal anti-SARS S antiserum. “Cells only” represents a negative control for which Vero were mock transfected and probed with the monoclonal antibody to the SARS-CoV S glycoprotein.

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under live cell conditions that restrict antibody binding to cell surfaces. In addition,

immunohistochemistry was used to detect the total amount of S expressed in cells by

fixing and permeabilizing the cells prior to reaction with the antibody (see Materials and

Methods) (Fig 3.4). An ELISA was used to quantitatively determine the relative amounts

of cell-surface and total cellular expressed S glycoprotein. A ratio between the cell-

surface localized S and total cellular S expressed was then calculated and normalized to

the corresponding ratio obtained with the wild-type S glycoprotein (see Materials and

Methods) (Fig 3.5). All cysteine cluster to alanine mutants were expressed on the cell

surface at levels comparable to that of the wild-type. Specifically, the CL2-C1217A

(91%), CL3-C1223A (87%), CL2-C1230A (101%), and CL2-C1235A (110%) were

expressed on the cellular surface by the percentages indicated when compared to the cell

surface expression of the wild-type protein.

Effect of Mutations of S-mediated Cell-to-Cell Fusion

Transiently expressed wild-type S causes extensive cell-to-cell fusion (syncytial

formation), especially in the presence of the SARS-CoV ACE2 receptor (Li et al., 2003).

To determine the ability of each cysteine cluster to alanine mutant S glycoprotein to

cause cell-to-cell fusion and the formation of syncytia, fused cells were labeled by

immunohistochemistry using the anti-FLAG antibody, and the extent of cell-to-cell

fusion caused by each mutant glycoprotein was calculated by obtaining the average size

of approximately 300 syncytia (Fig 3.6). The average syncytium size for each mutant

was then normalized to that found in wild type S transfected cells (see Materials and

Methods). The C1217A (54%), C1223A (62%), C1230A (15%), and C1235A (14%)

mutants inhibited the formation of syncytia by the percentages indicated.

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Figure 3.4 Immunohistochemical detection of cell-surface and total expression of the SARS-CoV S wild-type and mutant proteins. Vero cells were transfected with the wild-type SARS-CoV optimized S (SARS So 3xF) (E1, E2), C1217A (A1, A2), C1223A (B1,B2), C1230A (C1,C2), C1235A (D1,D2), and a wild-type SARS-CoV optimized S labeled with a 3xFLAG carboxyl tag (F1, F2), which served as a negative control. At 48 hours post-transfection, cells were immunohistochemically processed wither under live conditions to show surface expression (A1, B1, C1, D1, E1, and F1) and permeablilized conditions to show total expression (A2, B2, C2, D2, E2, and F2) with anti-FLAG antibody.

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Figure 3.5. Ratios of cell-surface to total cellular expression of mutant SARS-CoV S glycoproteins. Detection of cell surface and total glycoprotein distribution was determined by immunohistochemistry and ELISA (see materials and methods). Cell-surface expression of the S glycoprotein was measured by incubating the transfected cell monolayers with anti-FLAG antibody at room temperature before permeabilization. For total S glycoprotein detection, cells were fixed and permeabilized prior to incubation with the anti-FLAG antibody. A ratio between the surface localization and the total expression was calculated and normalized to the wild-type protein, then set to a percentage of the wild-type. The error bars represent the maximum and minimum surface to total ratios obtained from three independent experiments, and the bar height represents the average surface to total ratio as a percentage of the wild-type.

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Figure 3.6 Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments.

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DISCUSSION

Although the most important domains of the class I fusion are naturally located in

their ectodomains, it has been reported that intracytoplasmic endodomains play an

important role in intracellular transport and virus-induced cell-to-cell fusion (Bagai and

Lamb, 1996; Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Lontok, Corse, and

Machamer, 2004; Petit et al., 2005; Schwegmann-Wessels et al., 2004; Sergel and

Morrison, 1995; Tong et al., 2002; Waning et al., 2004; Yao and Compans, 1995). This

study shows that the two cysteine clusters closest to the transmembrane regions were

vital for the protein to be able to induce cell-to-cell fusion while the two cysteine clusters

closest to the carboxyl end of the protein did not drastically affect cell-to-cell fusion. Two

similar studies performed on the MHV S glycoprotein produced similar results found for

the SARS-CoV S glycoprotein.

The C1217A and C1223A are cysteine to alanine cluster mutations that target the

two cysteine rich clusters most proximal to the transmembrane region of the S

glycoprotein. Both of these proteins were expressed on cell surfaces at levels comparable

to that of the wild-type (91% and 87% of the of the wild-type protein, respectively).

However, these mutant forms had a significant impact on S glycoprotein induced cell-to-

cell fusion. Similar mutations of the cysteine clusters in the MHV S glycoprotein

produced a comparable effect on cell fusion (Chang, Sheng, and Gombold, 2000). The

C1217A cluster mutant reduced fusion activity by 55 % while the equivalent cysteine to

serine mutation in MHV reduced fusion by 56% (Chang, Sheng, and Gombold, 2000). In

addition, the C1223A cluster to alanine mutation had a substantial effect on fusion (60%

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reduction) in a similar manner to the corresponding MHV S mutant which caused a 94%

inhibition of S-mediated cell fusion. (Chang, Sheng, and Gombold, 2000).

The S2 fragment of the S glycoprotein is palmitylated on its carboxyl-terminal

region (Niemann and Klenk, 1981; Sturman, Holmes, and Behnke, 1980; van Berlo et al.,

1987). In general, cysteines proximal to the inner leaflet of the plasma membrane in viral

proteins often undergo palmitate attachment through a thioester bond (Ponimaskin and

Schmidt, 1995; Rose, Adams, and Gallione, 1984; Schlesinger, Veit, and Schmidt, 1993;

Schmidt, 1989; Sefton and Buss, 1987). Both MHV mutants corresponding to the SARS-

CoV C1217A and C1223 mutants have been shown to be acylation sites for the MHV S

glycoprotein (Bos et al., 1995). Covalent palmitic acid attachment to these cysteines may

have an impact on the conformation of the transmembrane and subsequently fusion.

Since palmytilation is able to affect an assortment of processes either mediated by or

involving fusion proteins including membrane fusion, infection of cells, and virus

assembly (Glick and Rothman, 1987; Jin et al., 1996; Melikyan et al., 2000; Naim et al.,

1992; Schroth-Diez et al., 1998; Zurcher, Luo, and Palese, 1994), it is plausible that

mutation of these cysteine clusters prevented palmytilation which in turn adversely

affected S-mediated fusion.

The C1230A and C1235A cluster mutations are the two cysteine clusters that are

closest to the carboxyl end of the S glycoprotein. Both C1230A and C1235A mutant

forms exhibited levels of S-mediated cell fusion that were similar to that of the wild-type

(85% and 88% of the wild-type protein, respectively, while they were synthesized and

expressed on cell-surfaces at levels similar to that of the wild-type S. These data suggests

that the two cysteine clusters are not vital for the proper synthesis, transport, or function

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of the S glycoprotein. Instead of participating in protein function and trafficking, these

two cysteine clusters may play a role in virion assembly or virus-protein interactions.

MATERIALS AND METHODS Cells





serum.

Plasmids

The parental plasmid used in the present study, SARS-S-Optimized, has been

previously described (Li et al., 2003). The Spike-3XFLAG-N gene construct was

generated by cloning the codon-optimized S gene, without the DNA sequence coding for

the signal peptide, into the p3XFLAG-CMV-9 plasmid vector (Sigma). PCR overlap

extension (Aiyar, Xiang, and Leis, 1996) was used to construct the cluster to alanine

mutants. To construct the truncation mutants, primers were designed that incorporated a

stop codon and a BamHI restriction site at the appropriate gene site. Restriction

endonuclease sites BamHI and Pml-I were then used to clone the gene construct into the

Spike-3XFLAG-N plasmid.

The constructed cluster mutants targeting the S cysteine rich region changed the

following sets of amino acids to alanine residues: C1217A: C(1217), C(1218); C1223A:

C(1223), C (1224), C(1226); C1230A: C(1230), C(1232); C1235A: C(1235), C(1236).

(Fig 3.2)

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Production of SARS-CoV S Monoclonal Antibodies

The monoclonal antibodies SW-111 was raised against the Spike envelope

glycoprotein of the SARS virus. A synthetic, codon-optimized gene corresponding to

SAR-CoV S glycoprotein coding sequences (Li et al., 2003) was engineered to produce

truncated, secreted proteins containing C-terminal His-tags which encode either the entire

ectodomain or just the receptor binding domain of S1. These genes were cloned into

baculoviral vectors, and the resulting virus was used to infect insect cell lines (High Five)

(Invitrogen, Carlsbad, CA). The supernatants were then tested by western blotting using

anti-6-His antibodies. Protein was partially purified from supernatants by passage

through a nickel column and then concentrated by ultrafiltration. Additional protein

derived in an analogous fashion was provided by the laboratory of Stephen Harrison.

Standard protocols for mouse immunization were used. The animals were maintained in

the animal facility of Dana-Farber Cancer Institute, and hybridomas and monoclonal

antibodies were produced in the Dana Farber/Harvard Cancer Center Monoclonal

Antibody Core. Spleenocytes from immunogenized mice were fused with NS-1 myeloma

cells (ATCC) using standard protocols. Antibody-producing clones were identified by

Western blot analysis, using purified SARS-CoV S. The positive cells were then sub-

cloned and re-tested against the purified SARS-CoV S glycoprotein. Isotype analysis

revealed that the antibody belonged to the IgG-1 class.

Western Blot Analysis



directions. At 48 hours (h) post transfection, cells were collected by low-speed

149

centrifugation, washed with Tris-buffered saline (TBS), and lysed at on ice for 15 min in

mammalian protein extraction reagent supplemented with a cocktail of protease inhibitors

(Invitrogen/Life Technologies). Insoluble cell debris was pelleted, samples were

electrophoretically separated by SDS-PAGE, transferred to nitrocellulose membranes,

and probed with anti-SARS CoV monoclonal antibody at a 1:10 dilution. Samples were

boiled for 5 min and treated with beta mercaptoethanol. Subsequently, blots were

incubated for 1 h with a peroxidase-conjugated secondary antibody at a 1:50,000 dilution

and then visualized on X-ray film by chemiluminescence (Pierce Chemicals, Rockford,

Ill.). All antibody dilutions and buffer washes were performed in TBS supplemented with

0.135 M CaCl2 and 0.11 M MgCl2 (TBS-Ca/Mg).

Cell Surface Immunohistochemistry




fixed with iced cold methanol or left unfixed (live). Immunohistochemistry was

performed by utilizing the Vector Laboratories Vectastain Elite ABC kit (Vector

Laboratories, Burlingame, CA) essentially as described in the manufacturer’s directions.

Briefly, cells were washed with TBS-Ca/Mg and incubated in TBS supplemented with

5% normal horse serum and 5% bovine serum albumin at room temperature for 1 h. After

blocking, cells were reacted with anti-FLAG antibody (1:500) in TBS blocking buffer for

3 h, washed four times with TBS-Ca/Mg, and incubated with biotinylated horse anti-

mouse antibody. Excess antibody was removed by four washes with TBS-Ca/Mg and

subsequently incubated with Vectastain Elite ABC reagent for 30 min. Finally, cells were

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washed three times with TBS-Ca/Mg, and reactions were developed with NovaRed

substrate (Vector Laboratories) according to the manufacturer’s directions.

Determination of Cell-surface to Total Cell S Glycoprotein Expression


and processed for immunohistochemistry as described above with the exception that the

ABTS Substrate Kit , 2, 2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (Vector

Laboratories) was used instead of the NovaRed substrate. After the substrate was allowed

to develop for 30 min, 100 μl of the developed substrate was transferred, in triplicate, to a

96 well plate. The samples were then analyzed for color change at a wavelength of 405

nm. The absorbance reading from cell-surface labeling experiments obtained from live

cells were divided by the total labeled absorbance readings obtained from fixed cells

which was then normalized to the wild type protein values. The measurements were then

converted to percentages reflecting the ratio of S present on cell-surfaces versus the total

S expressed in the transfected cells.


Vero cell monolayers in six-well plates were transfected in triplicate with the

indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the







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REFERENCES

Abraham, S., Kienzle, T. E., Lapps, W., and Brian, D. A. (1990). Deduced sequence of the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site. Virology 176(1), 296-301.

Aiyar, A., Xiang, Y., and Leis, J. (1996). Site-directed mutagenesis using overlap

extension PCR. Methods Mol Biol 57, 177-91.

152






paramyxovirus-mediated membrane fusion. Mol Cell 3(3), 309-19. Binns, M. M., Boursnell, M. E., Cavanagh, D., Pappin, D. J., and Brown, T. D. (1985).

Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV. J Gen Virol 66 ( Pt 4), 719-26.



Bos, E. C., Luytjes, W., and Spaan, W. J. (1997). The function of the spike protein of

mouse hepatitis virus strain A59 can be studied on virus-like particles: cleavage is not required for infectivity. J Virol 71(12), 9427-33.



Cavanagh, D. (1995). "The coronavirus surface glycoprotein." Plenum Press, Inc., New

York, N.Y. Chang, K. W., Sheng, Y., and Gombold, J. L. (2000). Coronavirus-induced membrane


de Groot, R. J., Luytjes, W., Horzinek, M. C., van der Zeijst, B. A., Spaan, W. J., and

Lenstra, J. A. (1987). Evidence for a coiled-coil structure in the spike proteins of coronaviruses. J Mol Biol 196(4), 963-6.




Rabenau, H., Panning, M., Kolesnikova, L., Fouchier, R. A., Berger, A., Burguiere, A. M., Cinatl, J., Eickmann, M., Escriou, N., Grywna, K., Kramme, S.,

153

Manuguerra, J. C., Muller, S., Rickerts, V., Sturmer, M., Vieth, S., Klenk, H. D., Osterhaus, A. D., Schmitz, H., and Doerr, H. W. (2003). Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 348(20), 1967-76.


inhibition. Annu Rev Biochem 70, 777-810. Glick, B. S., and Rothman, J. E. (1987). Possible role for fatty acyl-coenzyme A in

intracellular protein transport. Nature 326(6110), 309-12. Gombold, J. L., Hingley, S. T., and Weiss, S. R. (1993). Fusion-defective mutants of


Holmes, K. V. (2003). SARS-associated coronavirus. N Engl J Med 348(20), 1948-51. Jin, H., Subbarao, K., Bagai, S., Leser, G. P., Murphy, B. R., and Lamb, R. A. (1996).

Palmitylation of the influenza virus hemagglutinin (H3) is not essential for virus assembly or infectivity. J Virol 70(3), 1406-14.

Krokhin, O., Li, Y., Andonov, A., Feldmann, H., Flick, R., Jones, S., Stroeher, U.,





protein of human coronavirus OC43. Virology 195(1), 195-202. Lescar, J., Roussel, A., Wien, M. W., Navaza, J., Fuller, S. D., Wengler, G., and Rey, F.




154

Lontok, E., Corse, E., and Machamer, C. E. (2004). Intracellular targeting signals contribute to localization of coronavirus spike proteins near the virus assembly site. J Virol 78(11), 5913-22.

Luytjes, W., Sturman, L. S., Bredenbeek, P. J., Charite, J., van der Zeijst, B. A.,

Horzinek, M. C., and Spaan, W. J. (1987). Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site. Virology 161(2), 479-87.





Mounir, S., and Talbot, P. J. (1993). Molecular characterization of the S protein gene of

human coronavirus OC43. J Gen Virol 74 ( Pt 9), 1981-7. Naim, H. Y., Amarneh, B., Ktistakis, N. T., and Roth, M. G. (1992). Effects of altering

palmitylation sites on biosynthesis and function of the influenza virus hemagglutinin. J Virol 66(12), 7585-8.

Niemann, H., and Klenk, H. D. (1981). Coronavirus glycoprotein E1, a new type of viral

glycoprotein. J Mol Biol 153(4), 993-1010. Parker, S. E., Gallagher, T. M., and Buchmeier, M. J. (1989). Sequence analysis reveals




155

Petit, C. M., Melancon, J. M., Chouljenko, V. N., Colgrove, R., Farzan, M., Knipe, D. M., and Kousoulas, K. G. (2005). Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion. Virology.

Ponimaskin, E., and Schmidt, M. F. (1995). Acylation of viral glycoproteins: structural

requirements for palmitoylation of transmembrane proteins. Biochem Soc Trans 23(3), 565-8.

Raabe, T., Schelle-Prinz, B., and Siddell, S. G. (1990). Nucleotide sequence of the gene

encoding the spike glycoprotein of human coronavirus HCV 229E. J Gen Virol 71 ( Pt 5), 1065-73.

Rasschaert, D., and Laude, H. (1987). The predicted primary structure of the peplomer

protein E2 of the porcine coronavirus transmissible gastroenteritis virus. J Gen Virol 68 ( Pt 7), 1883-90.

Rose, J. K., Adams, G. A., and Gallione, C. J. (1984). The presence of cysteine in the

cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for palmitate addition. Proc Natl Acad Sci U S A 81(7), 2050-4.




paramyxoviruses: capture of intermediates of fusion. Embo J 20(15), 4024-34. Schlesinger, M. J., Veit, M., and Schmidt, M. F. (1993). Palmitoylation of cellular and

viral proteins. In "Lipid Modification of Proteins" (M. J. Schlesinger, Ed.), pp. 1-19. CRC Press, Boca Raton, FL.

Schmidt, M. F. (1989). Fatty acylation of proteins. Biochim Biophys Acta 988(3), 411-26. Schroth-Diez, B., Ponimaskin, E., Reverey, H., Schmidt, M. F., and Herrmann, A.

(1998). Fusion activity of transmembrane and cytoplasmic domain chimeras of the influenza virus glycoprotein hemagglutinin. J Virol 72(1), 133-41.

Schwegmann-Wessels, C., Al-Falah, M., Escors, D., Wang, Z., Zimmer, G., Deng, H.,

Enjuanes, L., Naim, H. Y., and Herrler, G. (2004). A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but

156

absent from severe acute respiratory syndrome-associated coronavirus. J Biol Chem 279(42), 43661-6.

Sefton, B. M., and Buss, J. E. (1987). The covalent modification of eukaryotic proteins

with lipid. J Cell Biol 104(6), 1449-53. Sergel, T., and Morrison, T. G. (1995). Mutations in the cytoplasmic domain of the







Song, H. C., Seo, M. Y., Stadler, K., Yoo, B. J., Choo, Q. L., Coates, S. R., Uematsu, Y.,

Harada, T., Greer, C. E., Polo, J. M., Pileri, P., Eickmann, M., Rappuoli, R., Abrignani, S., Houghton, M., and Han, J. H. (2004). Synthesis and characterization of a native, oligomeric form of recombinant severe acute respiratory syndrome coronavirus spike glycoprotein. J Virol 78(19), 10328-35.

Stauber, R., Pfleiderera, M., and Siddell, S. (1993). Proteolytic cleavage of the murine

coronavirus surface glycoprotein is not required for fusion activity. J Gen Virol 74 ( Pt 2), 183-91.

Sturman, L. S., Holmes, K. V., and Behnke, J. (1980). Isolation of coronavirus envelope

glycoproteins and interaction with the viral nucleocapsid. J Virol 33(1), 449-62. Tong, S., Li, M., Vincent, A., Compans, R. W., Fritsch, E., Beier, R., Klenk, C., Ohuchi,




157

van Berlo, M. F., van den Brink, W. J., Horzinek, M. C., and van der Zeijst, B. A. (1987). Fatty acid acylation of viral proteins in murine hepatitis virus-infected cells. Brief report. Arch Virol 95(1-2), 123-8.

Waning, D. L., Russell, C. J., Jardetzky, T. S., and Lamb, R. A. (2004). Activation of a

paramyxovirus fusion protein is modulated by inside-out signaling from the cytoplasmic tail. Proc Natl Acad Sci U S A 101(25), 9217-22.

Wu, X. D., Shang, B., Yang, R. F., Yu, H., Ma, Z. H., Shen, X., Ji, Y. Y., Lin, Y., Wu, Y.



of human parainfluenza virus fusion proteins. J Virol 69(11), 7045-53. Ye, R., Montalto-Morrison, C., and Masters, P. S. (2004). Genetic analysis of






Zurcher, T., Luo, G., and Palese, P. (1994). Mutations at palmitylation sites of the

influenza virus hemagglutinin affect virus formation. J Virol 68(9), 5748-54.

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CHAPTER IV

GENETIC COMPARISON OF THE SARS-COV AND BCOV S GLYCOPROTEINS

INTRODUCTION

In November 2002, an outbreak of atypical pneumonia, termed severe acute

respiratory syndrome (SARS), appeared in the Guangdong Province of southern China.

The etiological agent of the disease was found to be a novel coronavirus (SARS-CoV),

which was first isolated from infected individuals by propagation of the virus on Vero E6

cells (Drosten et al., 2003; Ksiazek et al., 2003; Peiris et al., 2003). For some age groups

the mortality rates of the disease reached as high as 15% (Anand et al., 2003).

In addition to the recent impact that coronaviruses, in the form of the SARS-CoV,

have had on public health, other coronaviruses produce significant diseases and economic

impact in various animal species. These viral infections include transmissible

gastroenteritis virus (TGEV), infectious bronchitis virus (IBV) ,and murine hepatitis virus

(MHV). Of particular interest are bovine coronaviruses since their disease spectrum seem

to be similar to that of the SARS-CoV. Primarily, bovine coronaviruses were known to

be enteropathogenic viruses that caused severe diarrhea in neonatal calves. The enteric

strains of the virus were known as enteric bovine coronavirus (EBCoV). Recently,

however, respiratory bovine coronaviruses were identified as the primary cause of acute

respiratory disease “shipping fever” as well as deep lung pneumonia in two Texas-based

epizootics (Storz et al., 2000a; Storz et al., 2000b). These isolated viruses were termed

respiratory coronaviruses (RBCoV). In a similar scenario to SARS-CoV, bovine

coronavirus virions were isolated from both diarrhea fluid and intestinal samples as well

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as from nasal secretions and lung tissues of animals experiencing both respiratory and

enteric disease (Doughri et al., 1976; Mebus et al., 1973; Storz et al., 2000a; Storz et al.,

2000b). These RBCoV and EBCoV strains can be differentiated on the basis of

phenotypic variation, antigenic differences, and genetic divergence (Chouljenko et al.,

1998; Chouljenko et al., 2001; Lin et al., 2000; Storz et al., 2000a). It is not known

however, whether respiratory and enteric disease are caused by different viruses or

alternatively, whether viruses are selected from a parental strain for their ability to

replicate in specific tissues. A comparison study between respiratory and

enteropathogenic coronavirus spikes isolated from the same animal with fatal shipping

pneumonia indicated at least 7 amino acid differences including amino acid changes

within the S1 portion that may account for a potential alteration in tissue specificity

(Chouljenko et al., 1998; Gelinas et al., 2001; Hasoksuz et al., 2002; Rekik and Dea,

1994). A comparison of the RBCoV and EBCoV S glycoproteins is shown in Figure 4.1.





including the RNA replicase gene open reading frame (ORF 1ab) and the structural

proteins: nucleocapsid (N), membrane protein (M), envelope protein (E), and spike


(HE) protein is also encoded by some coronaviruses. The SARS-CoV does not contain

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Figure 4.1. Schematic diagram of the BCoV S glycoprotein denoting the differences between RBCoV and EBCoV (Chouljenko et al., 2001). A schematic diagram of the BCoV S protein is shown on top from amino acid 1 to amino acid 1363. The transmembrane region of the protein is noted with a grey box labeled “Tm”. The two heptad repeat regions are indicated with grey boxes labeled HR1 and HR2, respectively. A vertical line demarcates the location of the division between the S1 and S2 subunits of the protein with the amino acid number between S1 and S2 shown. Amino acid differences are denoted with the amino acid number shown and the corresponding residues listed below.

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the HE protein while it is found in the BCoV (Lai and Cavanagh, 1997; Rota et al.,

2003).

The SARS S glycoprotein is composed of 1255-amino-acid with an estimated

post-translational mass of approximately 180 kDa. S is a type I membrane glycoprotein

(Rota et al., 2003) that is the major protein present in the viral membrane forming the

typical spike structure found on all coronavirions. After posttranslational modifications

are completed, the SARS-CoV S glycoprotein may be cleaved into S1 and S2 subunits in

VeroE6-infected cells (Wu et al., 2004). The receptor for SARS-CoV S has been

recently identified as the angiotensin-converting enzyme 2 (ACE2) (Li et al., 2003) and

CD209L (L-SIGN) (Jeffers et al., 2004).

The bovine coronavirus spike glycoprotein is made up of 1363 amino acids with

an estimated preglycosylated mass of 151 kDa (Abraham et al., 1990; Boireau, Cruciere,

and Laporte, 1990; Jeffers et al., 2004; Parker et al., 1990; Zhang, Kousoulas, and Storz,

1991). Once fully glycosylated, the BCoV S glycoprotein has an estimated mass of 190

kDa with 19 potential sites of glycosylation (Abraham et al., 1990; Cavanagh, 1995).

After posttranslational modification, the S glycoprotein is cleaved into two subunits, the

N-terminal S1 (110 kDa) and the C-terminal S2 (100 kDa) (Cyr-Coats and Storz, 1988).

This cleavage into the two subunits occurs between amino acid 768 and 769 of the spike

glycoprotein and is thought to be mediated by cellular trypsin-like proteases (Storz, Rott,

and Kaluza, 1981).

The S glycoprotein is primarily responsible for entry of all coronaviruses into

susceptible cells through binding to specific receptors on cells and mediating subsequent

virus-cell fusion (Cavanagh, 1995). Although the exact mechanism by which the SARS-

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CoV enters the host cell has not been elucidated, it is most likely similar to other

coronaviruses. Upon receptor binding at the cell membrane, the S glycoprotein is

thought to undergo a dramatic conformational change causing exposure of a hydrophobic

fusion peptide, which is subsequently inserted into cellular membranes. This

conformational change of the S glycoprotein causes close apposition followed by fusion

of the viral and cellular membranes resulting in entry of the virion nucleocapsids into

cells (Eckert and Kim, 2001; Tsai et al., 2003; Zelus et al., 2003). This series of S-

mediated virus entry events is similar to other class I virus fusion proteins (Baker et al.,

1999; Melikyan et al., 2000; Russell, Jardetzky, and Lamb, 2001).

Although the most important domains of the class I fusion proteins are naturally

located in their ectodomains, it has been reported that their cytoplasmic endodomains

play an important role in intracellular transport and virus-induced cell fusion (Bagai and

Lamb, 1996; Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Lontok, Corse, and

Machamer, 2004; Schwegmann-Wessels et al., 2004; Sergel and Morrison, 1995; Seth,

Vincent, and Compans, 2003; Tong et al., 2002; Waning et al., 2004; Yao and Compans,

1995). Both the BCoV and SARS-CoV S glycoprotein have similar motifs in their

intracytoplasmic tails that could possibly play a role in the proper functioning of the

protein (Fig 4.2). In this study BCoV and SARS Spikes were compared with regard to

their ability to cause S-mediated cell fusion paying particular attention to comparing and

contrasting the functional domains contained within their carboxyl termini. The salient

features of this study are: 1) Transient expression of the RBCoV in Vero cells produced

significantly less cell fusion than the SARS-CoV; 2) Surprisingly, both RBCoV and

EBCoV spike proteins induced similar amounts of cell fusion; 3) The carboxyl

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Figure 4.2. Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins. The cysteine cluster and the charged rich regions of the S proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks.

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terminal domains of RBCoV S function substantially differently than their homologous

domains of the SARS-CoV.

RESULTS

Comparison of the Ectodomain of the SARS-CoV and BCoV Spike Glycoproteins

Analysis of the viral genome has demonstrated that the SARS-CoV is

phylogenetically divergent from the three known antigenic groups of coronaviruses

(Drosten et al., 2003; Ksiazek et al., 2003). Analysis of the polymerase gene alone,

however, has indicated that the SARS-CoV may be an early off-shoot from the group 2

coronaviruses (Snijder et al., 2003). Both the BCoV and the SARS-CoV S glycoprotein

contain multiple analogous motifs as well as unique motifs to their corresponding

antigenic group. Comparison of the S1 portions reveals no apparent homologies with

respect to potential domains that function in receptor binding indicating that there is

substantial divergence with respect to this function. However, a comparison between the

amino acid sequences between the SARS-CoV and BCoV S proteins reveal a variety of

regions that are conserved throughout the majority of the coronavirus families whose

exact functions have yet to be determined. One such highly conserved amino acid

sequence (KWPWYVWL) can be found proximal to the transmembrane region (Fig 4.3).

The exact function of this eight-residue sequence has yet to be elucidated, but it may play

a structural role in the stabilization of the protein in the membrane. The S2 ectodomain

portion of the coronavirus S glycoprotein was found to have a higher degree of homology

(38% homology) when compared to that of the S1 subunit (16% homology), the

transmembrane region (22% homology), or the endodomain of the S2 subunit (16%

homology). Heptad repeats (HR), a sequence motif characteristic of coiled-coils, are

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Figure 4.3. Alignment of the membrane spanning domain and endodomain of the spike glycoprotein from ten different coronaviruses (Abraham et al., 1990; Binns et al., 1985; Delmas et al., 1992; Kunkel and Herrler, 1993; Luytjes et al., 1987; Marra et al., 2003; Mounir and Talbot, 1993; Parker, Gallagher, and Buchmeier, 1989; Raabe, Schelle-Prinz, and Siddell, 1990; Rasschaert and Laude, 1987). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The carboxyl terminus (amino acids 1193 to 1255) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from nine other coronaviruses. Viruses from antigenic group I (feline infectious peritonitis virus [FIPV], transmissible gastroenteritis virus [TGEV’, human coronavirus 229E [HCoV-229E]), antigenic group II (three different mouse hepatitis virus strains [A59, JHM, and MHV2], bovine coronavirus [BCoV], and human coronavirus OC43 [HCoV-OC43]), and antigenic group III (infectious bronchitis virus [IBV]) are represented in the alignment. The membrane spanning domain and the cytoplasmic tail are denoted with arrows above the alignment. Residues conserved in at least eight of the ten coronaviruses represented are indicated by the shaded residues. Cysteines that are highly conserved throughout all of the S proteins are noted by asterisks.

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found in the S2 portion of the S molecule and are common motifs in many viral and

cellular fusion proteins (Skehel and Wiley, 1998) (Fig 4.4).These repeat regions allow the

protein to fold back upon itself. This event is critical to the function of the protein in that

it is the first step in initiating the membrane fusion event. The BCoV and SARS-CoV

HR regions are highly homologous to one another with the amino most HR region (HR1)

having 56% homology and the HR region proximal to the transmembrane sequence

(HR2) having 39% homology between the two viruses.

Comparison of the Endodomain of the SARS-CoV and BCoV Spike Glycoproteins

A common motif that is found in the coronavirus spike protein is the presence of a

charge-rich region in the endodomain. Analysis of the endodomains indicate that both

the BCoV and SARS-CoV S glycoprotein contain a homologous charged region (Fig

4.2). The charged rich region of the SARS-CoV S protein contains six out of seven

charged residues and has been shown to be a potent endocytosis signal that may be

stabilized by the rest of the endodomain (Petit et al., 2005). The charged rich region of

the BCoV S protein contains ten out of twenty-four charged residues and is located on the

carboxyl terminus of the protein’s endodomain. Although the entire amino acid sequence

of S contains little homology between different strains of coronaviruses, all S

glycoproteins contain a cysteine-rich region in their endodomain. Contained within the

BCoV and SARS-CoV S glycoprotein endodomains are regions of eight out of eighteen

and nine out of twenty amino acids that are cysteine residues, respectively. Many of

these cysteine residues located in the S protein are conserved throughout all of the

coronavirus families (Fig 4.3).

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Figure 4.4. Alignment heptad repeat regions of the S1 subunit of the spike glycoprotein from SARS-CoV and BCoV (Abraham et al., 1990; Marra et al., 2003). A schematic diagram of the SARS-CoV S protein is shown on top from amino acid 1 to amino acid 1255. The transmembrane region of the protein is noted with a black box labeled “Tm”. The two heptad repeat regions are indicated with striped boxes and labels HR1 and HR2 respectively. A vertical line demarcates the approximate location of the division between the S1 and S2 subunits of the protein. The two regions containing the heptad repeats (amino acids 879 to 980) of the SARS-CoV S glycoprotein is shown enlarged below and is aligned with the same region of the S glycoprotein from BCoV. Residues conserved between the two viruses represented are overlayed by shaded boxes.

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Specifically for coronaviruses, the MHV S glycoprotein endodomain contains charged-

rich and cysteine-rich regions, which are critical for fusion of infected cells (Bos et al.,

1995; Chang, Sheng, and Gombold, 2000; Ye, Montalto-Morrison, and Masters, 2004).

To elucidate the role of the specific motifs found in both the BCoV and the SARS-CoV

spike proteins, a series of truncations targeting specific motifs in both proteins were made

and tested for their cell-to-cell fusion activity (Fig 4.5).

Effect of Mutations of S-mediated Cell-to-Cell Fusion

Wild-type SARS-CoV S is able to cause extensive cell-to-cell fusion (syncytial

formation) in a transient system especially when overlayed with cells expressing the

SARS-CoV ACE2 receptor (Li et al., 2003). To determine the ability of each truncation

mutant to cause cell-to-cell fusion, fused cells were first labeled by

immunohistochemistry using the anti-FLAG antibody (Fig 4.6). This

immunohistochemical analysis serves to visualize both protein expression as well as

approximate cell-cell fusion activity. The extent of cell-to-cell fusion caused by each

mutant glycoprotein was quantified by calculating the average size of approximately 300

syncytia using digital analysis software. The average syncytium size for each mutant was

then normalized to that found in wild type S transfected cells (see Materials and

Methods) (Fig 4.7). For the truncations made in the SARS-CoV S glycoprotein, a range

of different phenotypes were observed (see Chapter 2). For the T1214 (86%) and T1247

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Figure 4.5. Schematic diagram of the SARS-CoV and BCoV S glycoprotein endodomains. Amino acid sequences of the carboxyl termini are shown for the wild-type S glycoproteins as well as truncated proteins. Each SARS-CoV S truncation is coupled with its corresponding BCoV S truncation. The cysteine cluster and the charged rich regions of the S wild-type proteins are encompassed in brackets and appropriately labeled. The shaded residues represent the transmembrane portion of the endodomain. Amino acids that serve as predicted phosphorylation sites are denoted by asterisks.

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Figure 4.6. Immunohistochemical detection of total expression of the BCoV S wild-type and truncated proteins. Vero cells were transfected with T1359 (Panel A), T1355 (Panel B), T1346 (Panel C), and T1333 (Panel D), and wild-type respiratory (BCoV-Lun So 3xF) (Panel E) and enteric (BCoV-End So 3xF) (Panel F) strains of the BCoV optimized S) labeled with an amino terminus 3xFLAG tag. At 48 hours post-transfection, cells were immunohistochemically processed wither under permeabilized conditions to show total expression with anti-FLAG antibody.

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Figure 4.7. Quantitation of the extent of S-mediated cell fusion. The average size of syncytia for each mutant was determined by digitally analyzing the area of approximately 300 syncytia stained by immunohistochemistry for S glycoprotein expression using the Image Pro Plus 5.0 software package (see Materials and Methods). The fusion levels quantitated for the VERO-E6 cell line are represented by black bars while the levels for VERO cells are represented by grey bars. Error bars shown represent the standard deviation calculated through comparison of the data from each of three experiments.

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(66%) mutants, the formation of syncytia was inhibited by the percentages indicated. The

T1229 truncation had less impact on cell-to-cell fusion reducing the average size of

syncytia by 22%. In contrast, the T1238 mutant produced on the average 43% larger

syncytia than that of the wild-type SARS-CoV S.

For the BCoV Spike, transiently expression of codon optimized S is only able to

produce moderate amounts of fusion. Unlike the SARS-CoV, the receptor for BCoV has

yet to be elucidated. There is no significant difference in cell-to-cell fusion between S

proteins of the respiratory and enteric strains of the virus (Fig 4.7). Two different cell

lines, VERO and VERO-E6 were used since the BCoV receptor is not available to

augment fusion in order to gain a consensus of fusion activity. Levels of fusion were

reduced greater for VERO-E6 cells when compared to VERO cells. The T1359

truncation, which is closest to the carboxyl end of the protein and had the smallest impact

on cell-to-cell fusion, reduced the size of the syncytium by 10% and 3% for VERO-E6

and VERO cells respectively. The T1346 and T1333 truncations had similar effects on

the level fusion observed with reductions in fusion by 15% and 13% for VERO-E6 cells

and 9% and 9% for VERO cells respectively. The truncation that had the most impact on

cell-cell fusion was that T1355, which reduced syncytium formation by 17% and 9% for

VERO-E6 and VERO cells respectively.

DISCUSSION

Comparison Between the SARS-CoV and BCoV Spike Proteins

As stated above, there is significantly less homology in the S1 portion between

different strains of coronaviruses when compared to that of the S2 proteins. A possible

explanation for this lack of similarity between the two viruses in this region is that the S1

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subunit of the S protein contains a domain which is the determinant of viral tropism. The

SARS-CoV S protein that ACE2 is able to bind to a 193 amino acid fragment (amino

acids 318-510 of the SARS-CoV protein) in the S1 portion of the S protein which

indicates that this region is potentially the receptor binding domain primarily responsible

for virus binding to the target cell (Babcock et al., 2004; Chakraborti et al., 2005; Wong

et al., 2004; Xiao et al., 2003). The receptor binding domain for BCoV is currently

unknown. However for the MHV S protein, a protein highly homologous to the BCoV S,

the receptor binding domain has been localized to the amino terminal 330 amino acids of

S1 (Kubo, Yamada, and Taguchi, 1994; Suzuki and Taguchi, 1996; Taguchi et al.,

1995). It is probably because of virus receptor specificity that these regions are not very

well conserved.

The SARS-CoV S2 ectodomain was found to have a high degree of homology

(38% homologous) when compared that of the BCoV S2 ectodomain. This region is

probably more highly conserved because of the HR domains found in this portion of the

S glycoprotein. Virus infectivity depends on the folding back mechanism, which is

dependent on the two HR regions, in order to facilitate virus entry. Also, this portion of

the protein maybe limited structurally in how many amino acid changes, additions, and

deletions it can accommodate and still function efficiently to initiate the virus-to-cell

fusion event. The differences seen in the amount of fusion generated by these viruses

may be linked to structural differences found between the two proteins. Activation

leading to membrane fusion may occur more efficiently with the SARS-CoV as

compared with BCoV. Conversely, the fusion caused by the BCoV S may in fact be

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more regulated and controlled as opposed to the fusion event involving the SARS-CoV S

protein which may be somewhat deregulated.

For the endodomains of the two S proteins, there were two important domains that

were found in both proteins. These two domains were the charged rich region and the

cysteine rich domain. For the charged rich region, there was not a high degree of

homology in that same specific amino acids were present but rather the homology was

achieved through the presence of charged residues found in high concentrations in the

same areas. The charged-rich region of the murine hepatitis virus (MHV) S endodomain,

which has high sequence homology to the BCoV S glycoprotein, has been found to play a

major role in determining the ability of the protein to be assembled into virions (Ye,

Montalto-Morrison, and Masters, 2004). There are high concentrations of cysteine

residues found in both proteins’ endodomains. These cysteine residues are often

conserved throughout a majority of the coronavirus S glycoproteins. For MHV and the

SARS-CoV, studies have shown the cysteine rich domain if required for coronavirus-

induced membrane fusion (Bos et al., 1995; Chang, Sheng, and Gombold, 2000; Godeke

et al., 2000; Petit et al., 2005; Ye, Montalto-Morrison, and Masters, 2004) (Fig 4.4).

Furthermore, substituting the wild-type cytoplasmic portion of the cysteine rich region

and cytoplasmic tail with the cytoplasmic tail of the VSV-G abolished MHV S

glycoprotein mediated cell-cell fusion (Bos et al., 1995).

S-mediated Cell-to-Cell Fusion

Several studies have shown that the intracytoplasmic endodomain of the class I

fusion proteins may play an more important role in intracellular transport as well as virus-

induced cell-to-cell fusion (Bagai and Lamb, 1996; Bos et al., 1995; Chang, Sheng, and

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Gombold, 2000; Lontok, Corse, and Machamer, 2004; Petit et al., 2005; Schwegmann-

Wessels et al., 2004; Sergel and Morrison, 1995; Tong et al., 2002; Waning et al., 2004;

Yao and Compans, 1995). To compare predicted functional domains in the BCoV and

SARS-CoV S glycoproteins, serial truncations were made at sites that were predicted to

contain domains that may contribute to the function and transport of the entire protein.

The ability of the BCoV truncated proteins to produce S-mediated cell-to-cell fusion was

then quantitated and compared to its SARS-CoV S counterpart.

The T1359 truncation targeted the sequence in the BCoV S protein that had been

previously found to play a role intracellular S trafficking as well as a site that is predicted

by the NetPhos 2.0 software to be phosphorylated (Blom, Gammeltoft, and Brunak, 1999;

Lontok, Corse, and Machamer, 2004). The truncation, T1359, had a slight effect on

protein mediated fusion with a reduction from the wild-type fusion activity of 10% on

VERO-E6 cells. Another truncation introduced into the BCoV S protein was designed to

bring the charged region to a more proximal position to the carboxyl end of the protein.

This truncation, T1355, showed the biggest reduction of fusion out of all the truncation

tested for BCoV S (83% of the wild-type fusion for VERO-E6 cells).The corresponding

SARS-CoV S truncation, T1247, had a very significant impact on the proteins ability to

cause fusion. It was speculated that the T1247 truncation exposed an acidic cluster that

in turn greatly enhanced endocytosis. This enhancement of endocytosis led to a reduction

of fusion by 66%. The charged cluster in the BCoV S is less concentrated than that of the

SARS-CoV which seems to support the idea that it is exactly this concentration of

charged residues in the SARS-CoV S protein that is responsible for the substantial

aberration in fusion levels. The T1346 truncation for BCoV was designed to truncate as

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much of the charged region as possible without disturbing the cysteine rich region. The

SARS-CoV counterpart truncation targeted the charged region of SARS-CoV S

glycoprotein. The T1238 truncation of the SARS-CoV actually increase the size of

syncytia formation by 40% while the T1346 truncation made on the BCoV S protein

caused a reduction in fusion by 15% for VERO-E6 cells. This increase in fusion may be

attributed to destabilization of the endodomain by the truncation made. Stabilization of

the carboxyl terminus has been shown to decrease the fusion activity of the vesicular

stomatitis virus G glycoprotein (Waning et al., 2004), therefore, conversely, it is possible

that destabilization of the carboxyl terminus may cause an increase in fusion.

The final truncation made in the BCoV and SARS-CoV cleaved off most of the

protein’s endodomain. For the SARS-CoV, the 1214T truncation was found to not be

efficiently expressed on the surface of the cell. This inability of the protein to be

expressed on the cell surface may account for its severe reduction (15% when compared

to the wild-type) in S mediated fusion. The corresponding BCoV truncation, T1333,

reduced the size of syncytia formation by 13% in VERO-E6. This discrepancy in fusion

levels possibly may only be due to the disruption in glycoprotein transport as it is the case

with the corresponding mutations of the SARS-CoV S protein.

No differences in fusion levels were observed between the RBCoV and EBCoV S

proteins. It should be noted that between the two strains of BCoV there are seven amino

acid differences when comparing RBCoV and EBCoV (Fig. 4.1). Four of these amino

acid changes occur in the S1 portion of the protein, two of which are in the domain that

could potentially bind to the receptor on the cell surface. This change in the potential

receptor binding domain may account for the differences in virus tropism seen between

184

the two viruses (Chouljenko et al., 2001). The other three amino acid changes occur in

the S2 portion which is known to function in fusion since it contains the fusion peptide as

well as the two heptad repeat regions. These three amino acids, however, are not located

in regions known to play a role in membrane fusion. All amino acid changes in S2 occur

at locations between the two heptad repeat regions. This could potentially explain why

there are no detectable differences in level of fusion caused by the two different proteins.

A possible explanation for the differences in fusion levels may be due to the lack

of other viral proteins. In the case of SARS-CoV, the S protein seems to be the main

determinance of virus fusion activity; for the BCoV however, the hemagglutinnin-

esterase protein may play a role in virus fusion activity as well as virulence. Expressing

the S in the context of the entire virus or at least in the context of other structural proteins

may augment fusion in order to make quantification more accurate. Another potential

cause of the differences noted between the BCoV and SARS-CoV S truncations may be

due to the fundamental differences in each of the wild-type protein’s ability to cause cell-

to-cell fusion. The SARS-CoV S, when overlayed with cells expressing the ACE2

receptor, is able to induce very large syncytia formation; however, the BCoV S is not

very fusogenic. Since the BCoV receptor remains unknown, receptor mediated

enhancement of syncytia formation in is not possible. Different cell lines were used in

this study in order to establish a consensus about the levels of fusion caused by each

truncation. VERO-E6 cells were more prone to fusion than VERO cells which made

comparison of fusion levels more accurate than their VERO counterparts; therefore

VERO-E6 fusion levels were used when comparing the BCoV S and its counterpart

185

truncation. This low level of fusion activity may hamper further studies on the

functional domains of the BCoV S glycoprotein.

MATERIALS AND METHODS

Cells





serum.

Plasmids

The DNAworks program (Hoover and Lubkowski, 2002) was used to design

synthetic oligonucleotides used in a PCR-based system in order to generate a codon

optimized BCoV S glycoprotein for the respiratory strain of the virus. Since the S

proteins only differ between the respiratory and enteric strain by seven amino acids, PCR

overlap extension (Aiyar, Xiang, and Leis, 1996) was used to construct the codon

optimized enteric strain S glycoprotein. The BCoV-Lun So 3xF and BCoV-Ent So 3xF

plasmids were generated by cloning their respective codon-optimized BCoV S gene,

without the DNA sequence coding for the signal peptide, into the p3XFLAG-CMV-9

plasmid vector (Sigma). In order to construct the truncation mutants, primers were

designed that incorporated a stop codon and an Xba I restriction site at the appropriate

gene site (Fig 4.2). Restriction endonuclease sites EcoRV and XbaI were then used to

clone the gene construct into the BCoV-Lun So 3xF plasmid.

186

Total Protein Immunohistochemistry




fixed with iced cold methanol. Immunohistochemistry was performed by utilizing the

Vector Laboratories Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA)

essentially as described in the manufacturer’s directions. Briefly, cells were washed with

TBS-Ca/Mg and incubated in TBS supplemented with 5% normal horse serum and 5%

bovine serum albumin at room temperature for 1 h. After blocking, cells were reacted

with anti-FLAG antibody (1:500) in TBS blocking buffer for 3 h, washed four times with





Laboratories) according to the manufacturer’s directions.


Vero cell monolayers in six-well plates were transfected in triplicate with the

indicated plasmids utilizing the Lipofectamine 2000 reagent (Invitrogen) according to the






187





















REFERENCES Abraham, S., Kienzle, T. E., Lapps, W., and Brian, D. A. (1990). Deduced sequence of

the bovine coronavirus spike protein and identification of the internal proteolytic cleavage site. Virology 176(1), 296-301.

188

Aiyar, A., Xiang, Y., and Leis, J. (1996). Site-directed mutagenesis using overlap extension PCR. Methods Mol Biol 57, 177-91.



Babcock, G. J., Esshaki, D. J., Thomas, W. D., Jr., and Ambrosino, D. M. (2004). Amino

acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol 78(9), 4552-60.




paramyxovirus-mediated membrane fusion. Mol Cell 3(3), 309-19. Binns, M. M., Boursnell, M. E., Cavanagh, D., Pappin, D. J., and Brown, T. D. (1985).

Cloning and sequencing of the gene encoding the spike protein of the coronavirus IBV. J Gen Virol 66 ( Pt 4), 719-26.

Blom, N., Gammeltoft, S., and Brunak, S. (1999). Sequence and structure-based

prediction of eukaryotic protein phosphorylation sites. J Mol Biol 294(5), 1351-62.

Boireau, P., Cruciere, C., and Laporte, J. (1990). Nucleotide sequence of the glycoprotein

S gene of bovine enteric coronavirus and comparison with the S proteins of two mouse hepatitis virus strains. J Gen Virol 71 ( Pt 2), 487-92.



Cavanagh, D. (1995). The coronavirus surface glycoprotein. In "The Coronaviridae" (S.

G. Siddell, Ed.), pp. 73-113. Plenum, New York. Chakraborti, S., Prabakaran, P., Xiao, X., and Dimitrov, D. S. (2005). The SARS

Coronavirus S Glycoprotein Receptor Binding Domain: Fine Mapping and Functional Characterization. Virol J 2(1), 73.

Chang, K. W., Sheng, Y., and Gombold, J. L. (2000). Coronavirus-induced membrane


189

Chouljenko, V. N., Kousoulas, K. G., Lin, X., and Storz, J. (1998). Nucleotide and predicted amino acid sequences of all genes encoded by the 3' genomic portion (9.5 kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses. Virus Genes 17(1), 33-42.

Chouljenko, V. N., Lin, X. Q., Storz, J., Kousoulas, K. G., and Gorbalenya, A. E. (2001).

Comparison of genomic and predicted amino acid sequences of respiratory and enteric bovine coronaviruses isolated from the same animal with fatal shipping pneumonia. J Gen Virol 82(Pt 12), 2927-33.

Cyr-Coats, K. S., and Storz, J. (1988). Bovine coronavirus-induced cytopathic expression

and plaque formation: host cell and virus strain determine trypsin dependence. Zentralbl Veterinarmed B 35(1), 48-56.



Doughri, A. M., Storz, J., Hajer, I., and Fernando, H. S. (1976). Morphology and

morphogenesis of a coronavirus infecting intestinal epithelial cells of newborn calves. Exp Mol Pathol 25(3), 355-70.




inhibition. Annu Rev Biochem 70, 777-810. Gelinas, A. M., Boutin, M., Sasseville, A. M., and Dea, S. (2001). Bovine coronaviruses


Godeke, G. J., de Haan, C. A., Rossen, J. W., Vennema, H., and Rottier, P. J. (2000).

Assembly of spikes into coronavirus particles is mediated by the carboxy-terminal domain of the spike protein. J Virol 74(3), 1566-71.



190

Holmes, K. V. (2003). SARS-associated coronavirus. N Engl J Med 348(20), 1948-51. Hoover, D. M., and Lubkowski, J. (2002). DNAWorks: an automated method for

designing oligonucleotides for PCR-based gene synthesis. Nucleic Acids Res 30(10), e43.

Jeffers, S. A., Tusell, S. M., Gillim-Ross, L., Hemmila, E. M., Achenbach, J. E.,

Babcock, G. J., Thomas, W. D., Jr., Thackray, L. B., Young, M. D., Mason, R. J., Ambrosino, D. M., Wentworth, D. E., Demartini, J. C., and Holmes, K. V. (2004). CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 101(44), 15748-53.



Kubo, H., Yamada, Y. K., and Taguchi, F. (1994). Localization of neutralizing epitopes

and the receptor-binding site within the amino-terminal 330 amino acids of the murine coronavirus spike protein. J Virol 68(9), 5403-10.


protein of human coronavirus OC43. Virology 195(1), 195-202. Lai, M. M., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Adv Virus

Res 48, 1-100. Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran,


Lin, X. Q., Chouljenko, V. N., Kousoulas, K. G., and Storz, J. (2000). Temperature-

sensitive acetylesterase activity of haemagglutinin-esterase specified by respiratory bovine coronaviruses. J Med Microbiol 49(12), 1119-27.



Luytjes, W., Sturman, L. S., Bredenbeek, P. J., Charite, J., van der Zeijst, B. A.,

Horzinek, M. C., and Spaan, W. J. (1987). Primary structure of the glycoprotein E2 of coronavirus MHV-A59 and identification of the trypsin cleavage site. Virology 161(2), 479-87.

191



Mebus, C. A., Stair, E. L., Rhodes, M. B., and Twiehaus, M. J. (1973). Neonatal calf




Mounir, S., and Talbot, P. J. (1993). Molecular characterization of the S protein gene of

human coronavirus OC43. J Gen Virol 74 ( Pt 9), 1981-7. Parker, M. D., Yoo, D., Cox, G. J., and Babiuk, L. A. (1990). Primary structure of the S

peplomer gene of bovine coronavirus and surface expression in insect cells. J Gen Virol 71 ( Pt 2), 263-70.

Parker, S. E., Gallagher, T. M., and Buchmeier, M. J. (1989). Sequence analysis reveals




Petit, C. M., Melancon, J. M., Chouljenko, V. N., Colgrove, R., Farzan, M., Knipe, D.

M., and Kousoulas, K. G. (2005). Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion. Virology.

Raabe, T., Schelle-Prinz, B., and Siddell, S. G. (1990). Nucleotide sequence of the gene

encoding the spike glycoprotein of human coronavirus HCV 229E. J Gen Virol 71 ( Pt 5), 1065-73.

192

Rasschaert, D., and Laude, H. (1987). The predicted primary structure of the peplomer

protein E2 of the porcine coronavirus transmissible gastroenteritis virus. J Gen Virol 68 ( Pt 7), 1883-90.






paramyxoviruses: capture of intermediates of fusion. Embo J 20(15), 4024-34. Schwegmann-Wessels, C., Al-Falah, M., Escors, D., Wang, Z., Zimmer, G., Deng, H.,

Enjuanes, L., Naim, H. Y., and Herrler, G. (2004). A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus. J Biol Chem 279(42), 43661-6.

Sergel, T., and Morrison, T. G. (1995). Mutations in the cytoplasmic domain of the







Storz, J., Lin, X., Purdy, C. W., Chouljenko, V. N., Kousoulas, K. G., Enright, F. M.,

Gilmore, W. C., Briggs, R. E., and Loan, R. W. (2000a). Coronavirus and

193

Pasteurella infections in bovine shipping fever pneumonia and Evans' criteria for causation. J Clin Microbiol 38(9), 3291-8.



Storz, J., Rott, R., and Kaluza, G. (1981). Enhancement of plaque formation and cell

fusion of an enteropathogenic coronavirus by trypsin treatment. Infect Immun 31(3), 1214-22.

Suzuki, H., and Taguchi, F. (1996). Analysis of the receptor-binding site of murine

coronavirus spike protein. J Virol 70(4), 2632-6. Taguchi, F., Kubo, H., Takahashi, H., and Suzuki, H. (1995). Localization of

neurovirulence determinant for rats on the S1 subunit of murine coronavirus JHMV. Virology 208(1), 67-74.







Wong, S. K., Li, W., Moore, M. J., Choe, H., and Farzan, M. (2004). A 193-amino acid

fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279(5), 3197-201.

Wu, X. D., Shang, B., Yang, R. F., Yu, H., Ma, Z. H., Shen, X., Ji, Y. Y., Lin, Y., Wu, Y.


Xiao, X., Chakraborti, S., Dimitrov, A. S., Gramatikoff, K., and Dimitrov, D. S. (2003).

The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun 312(4), 1159-64.

194

Yao, Q., and Compans, R. W. (1995). Differences in the role of the cytoplasmic domain of human parainfluenza virus fusion proteins. J Virol 69(11), 7045-53.

Ye, R., Montalto-Morrison, C., and Masters, P. S. (2004). Genetic analysis of




Zhang, X. M., Kousoulas, K. G., and Storz, J. (1991). Comparison of the nucleotide and

deduced amino acid sequences of the S genes specified by virulent and avirulent strains of bovine coronaviruses. Virology 183(1), 397-404.

195

CHAPTER V

CONCLUDING REMARKS

SUMMARY

The SARS-CoV is a recently discovered coronavirus that is able to produce very

high mortality rates in subsets of the population (Anand et al., 2003). A key target for

analysis of how the SARS-CoV was able to achieve such a high mortality rate is the viral

spike (S) glycoprotein. Unfortunately, the ability to transiently express the S protein had

been hindered by the proteins massive size of 1255 amino acids (Marra et al., 2003)

along with the predicted high degree of secondary structure of the RNA encoding the

protein.

Recently, however, the protein has been codon optimized in order to allow the

expression of the S protein using a transient system (Li et al., 2003). This process of

codon optimization, in which not only are all codons replaced with the specific codon

most commonly used to encode the amino acid but the secondary structure of the RNA is

significantly reduced, has allowed the efficient in vitro expression of the protein. The

work included in this dissertation has focused on the genetic alteration and manipulation

of the optimized SARS-CoV S protein in order to elucidate the role of several putative

functional and conserved domains in SARS-CoV S mediated cell-cell fusion as well as

intracellular transport. In addition, we generated an optimized clone of the bovine

coronavirus (BCoV) S glycoprotein in order to compare similar mutations made in both

proteins.

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These investigations have shown that there are several regions that are

indispensable for the proper functioning and transport of this protein. The amino

terminal heptad repeat as well as regions adjacent to the heptad repeat are important

structural domains for the correct functioning of the protein in S-mediated cell-cell

fusion. In order to investigate these regions, mutations designed to collapse the alpha

helical structure of these domains were introduced into the protein. These mutations

produced a severe impact on S-mediated cell-cell fusion but allowed for the appropriate

synthesis and transport found in the wild-type protein.

The role of the SARS-CoV endodomain was investigated by the construction of a

series of carboxyl terminal truncations as well as cluster to alanine mutations targeting

specific conserved motifs in the endodomain of the protein. The endodomain of the

protein is able to affect the function of the proteins commonly thought to be associated

with the ectodomain (Bagai and Lamb, 1996; Bos et al., 1995; Chang, Sheng, and

Gombold, 2000; Lontok, Corse, and Machamer, 2004; Schwegmann-Wessels et al., 2004;

Seth, Vincent, and Compans, 2003; Tong et al., 2002; Waning et al., 2004; Yao and

Compans, 1995). Endo-domain truncations of the SARS-CoV S protein specifically

targeted the acidic cluster and the cysteine rich clusters found within the SARS-CoV S

endodomain. The SARS-CoV S protein is divergent from other coronavirus S proteins in

that the acidic cluster is a separate domain from the cysteine rich domain as opposed to

the overlapping of these domains as seen in other coronaviruses such as mouse hepatitis

virus (MHV). Cluster to alanine mutations were introduced targeting the acidic cluster,

the cysteine rich domains, and a predicted phosphorylation site in the endodomain.

Mutations affecting regions of the endodomain led to a wide array of phenotypes. These

197

phenotypes include a decrease in S-mediated cell-cell fusion, an increase in S-mediated

cell-cell fusion, altered endocytosis patterns, altered protein recycling, and altered

intracellular protein transport. This wide variety of results demonstrate how incredibly

important the endodomain of type I membrane fusion proteins are in the regulation of

protein function and transport.

The bovine coronavirus (BCoV) is able to produce a disease state similar to that

of the SARS-CoV in cattle (Storz et al., 2000a; Storz et al., 2000b). Given this

resemblance to SARS, a comparison study between the two S glycoproteins was

performed to better understand any homologies shared between the two proteins. This

was accomplished by constructing a panel of BCoV S truncations and comparing their

fusion activity to the activity of a panel of SARS-CoV S truncations. The absence of

correlation in impact of protein function between the truncations may point to domains

that are unique to the SARS-CoV S. However, there were correlations between some

SARS-CoV S truncations with similar truncations of the MHV S glycoprotein (Chang,

Sheng, and Gombold, 2000). Further studies of the BCoV S glycoprotein are needed in

order to make any supported claims about the functional domains of the BCoV S

endodomain.

In conclusion, this dissertation work has capitalized on the recent development of

the codon optimization of the SARS-CoV and BCoV S glycoproteins in order to

introduce targeted mutations and truncations to both of these proteins. Currently, the

work produced herein has contributed to the understanding of potent motifs in the S

endodomain in their functioning in S-mediated cell-cell fusion as well as intracellular

transport.

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CURRENT AND FUTURE RESEARCH CHALLENGES

The high mortality rate for patients infected with the SARS-CoV and the probable

link between this high mortality with the S glycoprotein make the study of the protein

tremendously important to the field of viral pathogenesis. The SARS-CoV is the most

recently identified human coronavirus whose possible impact on worldwide public health

has led to an intense research effort in the field. The elucidation of functional domains

found in the protein may ultimately lead to new drug development as well as new

potential vaccine development for the disease.

These investigations clearly show that the mutations introduced into the predicted

functional domains of the protein had a significant impact on protein transport and

function. One aspect of particular interest would be these mutants impact on viral

assembly. Virus like particles have been produced for other coronaviruses using transient

systems (Corse and Machamer, 2003; de Haan et al., 1998; Huang et al., 2004; Vennema

et al., 1996). If a virion like particle assembly system could be established, each of the

mutants and truncations could be assayed for their ability to be incorporated into the

virion particles using immunoelectron microscopy. Through the elucidation of the

SARS-CoV packaging signal, which is already known for several coronaviruses

(Cologna and Hogue, 2000; Escors et al., 2003; Fosmire, Hwang, and Makino, 1992;

Izeta et al., 1999; Woo et al., 1997), a potential virus packaging system could also be

established. Once incorporated into the virus like particles, the mutants and truncations

could also be assayed for their ability of mediating virus entry by using a reporter dye

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enclosed by the particle or potentially by packaging a reporter gene within the virus like

particle. Another use of the particle packaging system could be in vaccine development

and production.

The construction of an infectious cDNA clone (Almazan et al., 2000; Gonzalez et

al., 2002; Yount, Curtis, and Baric, 2000; Yount et al., 2003) may provide a tool to

observe the constructed mutants and truncations constructed in the research presented in

this dissertation ability to function in the context of the entire virus. Specific

measurements concerning viral entry kinetics, viral replication, virus pathology, and viral

egress could be ascertained for the entire panel of mutants constructed. However it

should be noted that the incorporation of mutations in the cDNA clone could potentially

be problematic because of the enormous size of the infectious clone. A potential solution

for this problem would be to create an infectious cDNA clone without the S glycoprotein

where the S glycoprotein could be provided in trans. This would allow the S mutants to

be made on a separate more manageable plasmid. This S-null infectious clone would

also be substantially more safe and easier to handle than a fully infectious clone. An

alternate possibility would be to construct an infectious clone using a bacterial artificial

chromosome (BAC) system in which mutations could be introduced into the BAC using a

Sac B/Rec A mutagenesis system . The introduction of the mutations in the context of

the virus could potentially lead to very significant finding which would significantly

progress the area of coronavirus research.

REFERENCES Almazan, F., Gonzalez, J. M., Penzes, Z., Izeta, A., Calvo, E., Plana-Duran, J., and

Enjuanes, L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 97(10), 5516-21.

200

Anand, K., Ziebuhr, J., Wadhwani, P., Mesters, J. R., and Hilgenfeld, R. (2003). Coronavirus main proteinase (3CLpro) structure: basis for design of anti-SARS drugs. Science 300(5626), 1763-7.





Chang, K. W., Sheng, Y., and Gombold, J. L. (2000). Coronavirus-induced membrane


Cologna, R., and Hogue, B. G. (2000). Identification of a bovine coronavirus packaging

signal. J Virol 74(1), 580-3. Corse, E., and Machamer, C. E. (2003). The cytoplasmic tails of infectious bronchitis

virus E and M proteins mediate their interaction. Virology 312(1), 25-34. de Haan, C. A., Kuo, L., Masters, P. S., Vennema, H., and Rottier, P. J. (1998).


Escors, D., Izeta, A., Capiscol, C., and Enjuanes, L. (2003). Transmissible gastroenteritis

coronavirus packaging signal is located at the 5' end of the virus genome. J Virol 77(14), 7890-902.

Fosmire, J. A., Hwang, K., and Makino, S. (1992). Identification and characterization of

a coronavirus packaging signal. J Virol 66(6), 3522-30. Gonzalez, J. M., Penzes, Z., Almazan, F., Calvo, E., and Enjuanes, L. (2002).

Stabilization of a full-length infectious cDNA clone of transmissible gastroenteritis coronavirus by insertion of an intron. J Virol 76(9), 4655-61.



Izeta, A., Smerdou, C., Alonso, S., Penzes, Z., Mendez, A., Plana-Duran, J., and

Enjuanes, L. (1999). Replication and packaging of transmissible gastroenteritis coronavirus-derived synthetic minigenomes. J Virol 73(2), 1535-45.

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Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran, M., Sullivan, J. L., Luzuriaga, K., Greenough, T. C., Choe, H., and Farzan, M. (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426(6965), 450-4.





Schwegmann-Wessels, C., Al-Falah, M., Escors, D., Wang, Z., Zimmer, G., Deng, H.,

Enjuanes, L., Naim, H. Y., and Herrler, G. (2004). A novel sorting signal for intracellular localization is present in the S protein of a porcine coronavirus but absent from severe acute respiratory syndrome-associated coronavirus. J Biol Chem 279(42), 43661-6.



Storz, J., Lin, X., Purdy, C. W., Chouljenko, V. N., Kousoulas, K. G., Enright, F. M.,

Gilmore, W. C., Briggs, R. E., and Loan, R. W. (2000a). Coronavirus and Pasteurella infections in bovine shipping fever pneumonia and Evans' criteria for causation. J Clin Microbiol 38(9), 3291-8.





202

Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten, D. J., and Rottier, P. J. (1996). Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15(8), 2020-8.



Woo, K., Joo, M., Narayanan, K., Kim, K. H., and Makino, S. (1997). Murine

coronavirus packaging signal confers packaging to nonviral RNA. J Virol 71(1), 824-7.


of human parainfluenza virus fusion proteins. J Virol 69(11), 7045-53. Yount, B., Curtis, K. M., and Baric, R. S. (2000). Strategy for systematic assembly of

large RNA and DNA genomes: transmissible gastroenteritis virus model. J Virol 74(22), 10600-11.

Yount, B., Curtis, K. M., Fritz, E. A., Hensley, L. E., Jahrling, P. B., Prentice, E.,

Denison, M. R., Geisbert, T. W., and Baric, R. S. (2003). Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 100(22), 12995-3000.

203

APPENDIX A

ADDITIONAL WORK

VIRUS LIKE PARTICLE AND PACKAGING SYSTEM

The etiological agent for the recently recognized severe acute respiratory

syndrome (SARS) was discovered to be the SARS coronavirus (SARS-CoV) (Drosten et

al., 2003; Ksiazek et al., 2003). An enveloped positive stranded virus, this coronavirus

displays a host range divergent from other members of the coronavirus family. This

divergence from typical coronavirus may play a role in its dramatically increased

pathogenicity. Most coronaviruses can replicate in either respiratory and/or

gastrointestinal tract (Holmes, 2001; Li et al., 2003), however the SARS-CoV has

evolved the ability to replicate in both places during the viral infection.

Another coronavirus of particular interest is the bovine coronavirus (BCoV).

BCoV has a significant impact on the cattle industry resulting in serious economic losses.

Infection by the virus cause epidemics of acute diarrhea in calves and adult cattle (Mebus

et al., 1973; Saif et al., 1988; Taniguti et al., 1986; Tsunemitsu et al., 1991; Weisberg,

1975) which results in reduced milk production in dairy cows and/or death (Saif et al.,

1988; Takahashi, Inaba, and Sato, 1980; Taniguti et al., 1986).

The typical coronavirus encodes for four structural proteins. The protein

responsible for binding and attachment is the spike (S) glycoprotein. It is the largest

encoded structural protein found in the coronavirus genome. It functions as a typical type

I membrane glycoprotein. The most abundant protein contained in the virion envelope is

the matrix (M) glycoprotein. It is a triple spanning membrane protein that has a long

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carboxyl terminal cytoplasmic domain inside the virion envelope and a short amino

terminal domain located outside of the virion (Holmes, 2001; Locker et al., 1992;

Narayanan et al., 2000). The other protein that comprises the virion membrane is the

envelope (E) protein. This protein is expressed at significantly lower levels than the that

of the M protein (Godet et al., 1992; Liu and Inglis, 1991; Siddell, 1995b; Yu et al.,

1994). The final structural protein found in the typical coronavirus is the nucleocapsid

(N) protein. The N protein is an internal phosphoprotein that interacts with the genomic

RNA to form the virion core. BCoV, as with the SARS-CoV, encoded for these four

structural proteins that are typically found in coronaviruses. Unlike the SARS-CoV

however, the BCoV also has a hemmagglutinin-esterase protein encoded into its genome

that is only found in a certain subset of coronaviruses.

It has been shown that the M and E proteins are instrumental in virion assembly.

In fact, studies have revealed that expression of the M and E proteins are enough to

produce noninfectious membrane bound particles of similar size and shape to the normal

wild-type virion (Corse and Machamer, 2003; de Haan et al., 1998; Huang et al., 2004;

Vennema et al., 1996). Expressing S, along with M and E, produced VLPs that were not

only similar in size and shape but were also infectious. This indicates that the S protein is

not required for virion assembly but is required for infectivity. Similar roles of structural

proteins have also been elucidated for other coronaviruses including the infectious

bronchitis virus (IBV) (Corse and Machamer, 2000). For the SARS-CoV, the expression

of M and E does not lead to particle formation; however the expression of M and N is

able to produce VLPs (Huang et al., 2004).

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In this study, attempts were made to create virus like particles (VLPs) for BCoV

and to repeat the study in which VLPs for the SARS-CoV were synthesized. These

particles could then be used to create potential vaccines for the viruses that the structural

proteins were derived from. The definitive goal of this series of experiments was to

produce BCoV or SARS-CoV particles in which a foreign gene could be packaged.

Particularly for BCoV, particles created in this packaging system could be loaded with a

gene that encodes for a cytolytic protein or a protein that is able to elicit a targeted

immune response. Since BCoV has a predilection for rectal cells, a BCoV virus like

particle packaged with a gene encoding a cytolytic protein could be used as treatment

against rectal (colon) cancer.

For the BCoV, the M, E and S proteins were cloned into an expression vector

under the cytomegalovirus promoter. Once cloned into the vector, the genes were then

transfected into cells in order to check for the appropriate protein synthesis. The M and E

proteins were able to be efficiently expressed; however, the S glycoprotein was not able

to be expressed by transient expression. Several different methods were attempted in

order to correct the problem of the S protein not able to be expressed. Among the

methods employed were cloning the S protein into T7 vaccinia driven expression system,

cloning the vector into a variety of different vectors under different promoters, and

separately cloning the S1 and S2 subunits of the S protein into two different vectors and

transfecting the two subunits into the same cell. None of these methods were able to

produce transiently expressed S protein without killing the cells. It is probable that the S

protein was so large that the RNA encoding the protein produced from the transfected

plasmid was unable to get out of the nucleus. Since coronaviruses are purely

206

cytoplasmic, no mechanism evolved that would allow such a large RNA to be transported

out of the nucleus. Another problem is that the RNA encoding S protein has an

extremely high degree of 2° structure which would further inhibit the RNA from exiting

the nucleus.

Recently, the SARS-CoV S protein was able to be transiently expressed through

the technique of codon optimization (ACE2). Codon optimization takes advantage of the

degenerate code used to encode amino acids. Certain codons are more prevalent for

encoding the same amino acid than other degenerate codons. Codon optimization is the

process of reconstructing the nucleotide sequence in such a way that all of the codons

used in the optimized gene are the most prevalent used codons. The optimization

process, usually done with the assistance of computing software, can also produce a

sequence that not only uses all of the optimal codons but abolishes most of the 2°

structure associated with the RNA. In order to be able to express the BCoV S protein, a

condon optimized version of the gene was synthesized which allowed transient

expression of the protein.

Once all of the proteins were able to be efficiently expressed, different

combinations of the structural proteins were transfected into a variety of cell lines. The

essential combinations transfected for the SARS-CoV were M and N and M, N, and S,

while for BCoV the most important combinations were M and E and M, E, and S. Once

the plasmids were cotransfected, the cells were allowed to incubate for 48 hours before

processing them for visualization using electron microscopy. VLPs were not able to be

visualized using this method.

207

A possible reason that no VLPs were able to be visualized could be that the only

codon optimized proteins used in the experiment was the S glycoprotein. In the previous

study in which SARS-CoV proteins cotransfected were able to produce VLPS, all of the

structural proteins used were codon optimized. This codon optimization would lead to

much higher expression of protein from all of the structural proteins which would also

lead to higher levels of VLP production. Our combination of plasmids may not have

been able to produce enough levels of protein in order to be able to visualize VLP

formation. Codon optimization of the BCoV M and E and the SARS-CoV N and M may

be able to increase expression levels so that enough protein is produced to be able to form

coronavirus like particles. Another possible explanation could be the difficulty in

visualizing the particles themselves. Coronavirus particles are approximately the same

size and shape as a wide variety of cellular structures. This in combination with low

protein expression or inefficient transfection levels may make it very difficult to visualize

coronavirus like particles using electron microscopy. Different transfection reagents in

combination with the use of cells more susceptible to transfection may be able to alleviate

this particular problem.

BOVINE CORONAVIRUS INFECTIOUS CLONE

The development of full-length cDNA clones has remarkably advanced the ability

to perform molecular genetic analysis on the structure and function of RNA viruses. This

cDNA clone, once transfected into a permissive cell line, becomes the source of

infectious RNA transcripts (Ahlquist et al., 1984; Boyer and Haenni, 1994). These

infectious clones have been constructed for several viruses including caliciviruses,

arteriviruses, flaviviruses, picronavirures, and alphaviruses whose positive sensed RNA

208

genomes that range in size from approximately 7-15 kb in length (Agapov et al., 1998;

Davis et al., 1989; Racaniello and Baltimore, 1981; Rice et al., 1989; Rice et al., 1987;

Sosnovtsev and Green, 1995; Sumiyoshi, Hoke, and Trent, 1992; van Dinten et al., 1997).

These clones have made new methods and approaches in studying RNA viruses available

that have not been previously available before.

The family Coronaviridae has the largest RNA viral genomes found in nature

(Lai and Cavanagh, 1997; Siddell, 1995a). It is this enormous length of the coronavirus

genome and stabilization issues with plasmids encoding coronavirus replicase sequences

have made the construction of a full-length cDNA clone very cumbersome (Ahlquist et

al., 1984; Eleouet et al., 1995; Enjuanes and Van der Zaijst, 1995; Lai and Cavanagh,

1997; Siddell, 1995a). These problems were overcome when a full-length cDNA clone

of the transmissible gastroenteritis virus (TGEV) was constructed and stably cloned into a

bacterial artificial chromosome (BAC) vector (Almazan et al., 2000).

In order to be able to take advantage of molecular genetic analysis, a full-length

cDNA clone of the bovine coronavirus (BCoV) was attempted to be constructed using

recombinant technology. Specifically, a cDNA clone could have a dramatic impact on

studies involving the comparison of two strains of BCoV that were found to have

different tropisms. This comparison study was between a respiratory strain of BCoV and

an enteric strain of BCoV that were isolated from the same animal with fatal shipping

pneumonia. It has been suggested that the difference in viral tropism is due to slight

variation in the S1 subunit of the spike (S) glycoprotein (Chouljenko et al., 1998; Gelinas

et al., 2001; Hasoksuz et al., 2002; Rekik and Dea, 1994). There are only 4 amino acids

in the S1 subunit of the S protein that are different between the respiratory and enteric

209

strains of BCoV. It is known that the main determinance of tissue tropism is attributed to

the S1 portion of the S protein. It is possible that a single amino acid change in the S1

subunit may shift the BCoV tropism from the respiratory tract to the enteric tract. Once

constructed, mutagenesis of the infectious clone could isolate with amino acid(s) that

plays a role in this difference in tropism.

A full-length infectious clone was constructed for both the respiratory and enteric

strains of BCoV. This infectious clone was then transfected into a variety of cells

including HRT-18G, VERO, BHK, and COS-7. These cells were tested for their ability

to produce infectious virus after being transfected with the cDNA clone using

immunohistochemistry, indirect immunofluorensence, and observing the cells for the

cytopathic effect of the virus. Once transfected, several rounds of passaging the potential

virus were made in order to increase virus production to detectable levels. Also in order

to prevent false positive results, two silent mutations were introduced into the viral

genomes so that they could be distinguished from the wild-type virus. All attempts to

transfect the cDNA clone and produce infectious virus turned up negative by

immunohistochemistry, indirect immunofluorensence, and signs of cytopathic effect

created by the virus. One method employed to try and augment the efficiency of virus

production was the use of a T7 in vitro transcription system. First, several sequences in

the cDNA clone were mutated because they were know to interfere with T7 transcription.

The mutations placed in these areas were silent mutations so that the same amino acids

would be used in translation of the cDNA clone. Once the infectious clone was free of

possible interfering sequences, the T7 system was used to transcribe that cDNA into

RNA that could then be directly transfected into the cell lines. This was thought to make

210

virus production more efficient since the RNA has already been transcribed. All attempts

at isolating infectious virus were unsuccessful.

One potential problem in producing infectious virus from both the cDNA and

RNA transfection system could have been the cell lines used. The most commonly used

cell line for BCoV propagation is HRT-18G (Storz et al., 1996). This cell line, while

being the best at virus production has very poor transfection efficiency. HRT-18G cells

are difficult to transfect under optimal conditions much less when dealing with such a

large construct. The enormous size of the genetic material in conjunction with the given

inability of the cells to be efficiently transfected, make infectious virus recovery virtually

impossible. Other cells lines that are able to be efficiently transfected are not very

hospitable for virus production. Another problem may be the lack of actual transcription

or translation of the genetic construct. Such an enormous RNA would have a very high

degree of 2° structure which may prevent translation or transcription. Also, the cDNA

copy of the virus is unlikely to be efficiently transcribed or translated. During viral

replication, viral proteins may play a role in enhancing transcription and/or translation.

Without the aid of these proteins, expression of the viral genome may not be efficient

enough to produce an amount of virus that could be isolated.

REFERENCES

Agapov, E. V., Frolov, I., Lindenbach, B. D., Pragai, B. M., Schlesinger, S., and Rice, C. M. (1998). Noncytopathic Sindbis virus RNA vectors for heterologous gene expression. Proc Natl Acad Sci U S A 95(22), 12989-94.

Ahlquist, P., Frech, R., Janda, M., and Loesch-Fries, L. S. (1984). Multicomponent RNA

Plant Virus Infection Derived from Cloned Viral cDNA. PNAS 81, 7066-7070. Almazan, F., Gonzalez, J. M., Penzes, Z., Izeta, A., Calvo, E., Plana-Duran, J., and

Enjuanes, L. (2000). Engineering the largest RNA virus genome as an infectious bacterial artificial chromosome. Proc Natl Acad Sci U S A 97(10), 5516-21.

211

Boyer, J. C., and Haenni, A. L. (1994). Infectious transcripts and cDNA clones of RNA

viruses. Virology 198(2), 415-26. Chouljenko, V. N., Kousoulas, K. G., Lin, X., and Storz, J. (1998). Nucleotide and

predicted amino acid sequences of all genes encoded by the 3' genomic portion (9.5 kb) of respiratory bovine coronaviruses and comparisons among respiratory and enteric coronaviruses. Virus Genes 17(1), 33-42.

Corse, E., and Machamer, C. E. (2000). Infectious bronchitis virus E protein is targeted to

the Golgi complex and directs release of virus-like particles. J Virol 74(9), 4319-26.

Corse, E., and Machamer, C. E. (2003). The cytoplasmic tails of infectious bronchitis

virus E and M proteins mediate their interaction. Virology 312(1), 25-34. Davis, N. L., Willis, L. V., Smith, J. F., and Johnston, R. E. (1989). In vitro synthesis of

infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant. Virology 171(1), 189-204.

de Haan, C. A., Kuo, L., Masters, P. S., Vennema, H., and Rottier, P. J. (1998).




Eleouet, J. F., Rasschaert, D., Lambert, P., Levy, L., Vende, P., and Laude, H. (1995).

Complete sequence (20 kilobases) of the polyprotein-encoding gene 1 of transmissible gastroenteritis virus. Virology 206(2), 817-22.

Enjuanes, L., and Van der Zaijst, B. A. M. (1995). Molecular basis of transmissible

gastroenteritis coronavirus (TGEV) epidemiology. In "The Coronaviridae" (S. G. Siddell, Ed.), pp. 337-376. Plenum Press, New York, NY.

Gelinas, A. M., Boutin, M., Sasseville, A. M., and Dea, S. (2001). Bovine coronaviruses


212

Godet, M., L'Haridon, R., Vautherot, J. F., and Laude, H. (1992). TGEV corona virus ORF4 encodes a membrane protein that is incorporated into virions. Virology 188(2), 666-75.



Holmes, K. V. (2001). Coronaviruses. 4th ed. In "Fields virology" (D. M. Knipe, P. M.

Howley, D. E. Griffin, M. A. Martin, R. A. Lamb, B. Roizman, and S. E. Straus, Eds.), pp. 1187–1203. Lippincott Williams & Wilkins, Philadelphia, PA.





Lai, M. M., and Cavanagh, D. (1997). The molecular biology of coronaviruses. Adv Virus

Res 48, 1-100. Li, W., Moore, M. J., Vasilieva, N., Sui, J., Wong, S. K., Berne, M. A., Somasundaran,


Liu, D. X., and Inglis, S. C. (1991). Association of the infectious bronchitis virus 3c

protein with the virion envelope. Virology 185(2), 911-7. Locker, J. K., Rose, J. K., Horzinek, M. C., and Rottier, P. J. (1992). Membrane assembly

of the triple-spanning coronavirus M protein. Individual transmembrane domains show preferred orientation. J Biol Chem 267(30), 21911-8.

Mebus, C. A., Stair, E. L., Rhodes, M. B., and Twiehaus, M. J. (1973). Neonatal calf


Narayanan, K., Maeda, A., Maeda, J., and Makino, S. (2000). Characterization of the

coronavirus M protein and nucleocapsid interaction in infected cells. J Virol 74(17), 8127-34.

213

Racaniello, V. R., and Baltimore, D. (1981). Cloned poliovirus complementary DNA is infectious in mammalian cells. Science 214(4523), 916-9.



Rice, C. M., Grakoui, A., Galler, R., and Chambers, T. J. (1989). Transcription of

infectious yellow fever RNA from full-length cDNA templates produced by in vitro ligation. New Biol 1(3), 285-96.

Rice, C. M., Levis, R., Strauss, J. H., and Huang, H. V. (1987). Production of infectious

RNA transcripts from Sindbis virus cDNA clones: mapping of lethal mutations, rescue of a temperature-sensitive marker, and in vitro mutagenesis to generate defined mutants. J Virol 61(12), 3809-19.

Saif, L. J., Redman, D. R., Brock, K. V., Kohler, E. M., and Heckert, R. A. (1988).

Winter dysentery in adult dairy cattle: detection of coronavirus in the faeces. Vet Rec 123(11), 300-1.

Siddell, S. G. (1995a). "The coronaviridae." (S. G. Siddell, Ed.) Plenum Press, New

York, NY. Siddell, S. G. (1995b). The small-membrane protein. In "The Coronaviridae" (S. G.

Siddell, Ed.), pp. 181-189. Plenum Press, New York, N.Y. Sosnovtsev, S., and Green, K. Y. (1995). RNA transcripts derived from a cloned full-

length copy of the feline calicivirus genome do not require VpG for infectivity. Virology 210(2), 383-90.

Storz, J., Stine, L., Liem, A., and Anderson, G. A. (1996). Coronavirus isolation from

nasal swap samples in cattle with signs of respiratory tract disease after shipping. J Am Vet Med Assoc 208(9), 1452-5.

Sumiyoshi, H., Hoke, C. H., and Trent, D. W. (1992). Infectious Japanese encephalitis

virus RNA can be synthesized from in vitro-ligated cDNA templates. J Virol 66(9), 5425-31.

Takahashi, E., Inaba, Y., and Sato, K. (1980). Epizootic diarrhea of adult cattle associated

with a coronavirus-like agent. Vet Microbiol 5, 151-154. Taniguti, S., Iwamoto, H., Fukuura, H., Ito, H., Gekai, N., and Nagato, Y. (1986).

Recurrence of bovine coronavirus infection in cows. J Jpn Vet Med Assoc 39, 298-302.

214

Tsunemitsu, H., Yonemichi, H., Hirai, T., Kudo, T., Onoe, S., Mori, K., and Shimizu, M. (1991). Isolation of bovine coronavirus from feces and nasal swabs of calves with diarrhea. J Vet Med Sci 53(3), 433-7.

van Dinten, L. C., den Boon, J. A., Wassenaar, A. L., Spaan, W. J., and Snijder, E. J.

(1997). An infectious arterivirus cDNA clone: identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci U S A 94(3), 991-6.

Vennema, H., Godeke, G. J., Rossen, J. W., Voorhout, W. F., Horzinek, M. C., Opstelten,

D. J., and Rottier, P. J. (1996). Nucleocapsid-independent assembly of coronavirus-like particles by co-expression of viral envelope protein genes. Embo J 15(8), 2020-8.

Weisberg, L. A. (1975). The syndrome of increased intracranial pressure without

localizing signs: a reappraisal. Neurology 25(1), 85-8. Yu, X., Bi, W., Weiss, S. R., and Leibowitz, J. L. (1994). Mouse hepatitis virus gene 5b

protein is a new virion envelope protein. Virology 202(2), 1018-23.

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APPENDIX B

PERMISSION TO INCLUDE PUBLISHED WORK

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VITA

Chad Michael Petit was born on March 26, 1978, to Kevin and Gwyneth Petit in

Metairie, Louisiana. Growing up, Chad had interests in art and music. He fulfilled these

interests by learning how to play the guitar, trumpet, and baritone and also by being in the

gifted and talented art program in high school. In 1996, Chad graduated 11th in his class

of 300 from Hahnville High School. Chad was then awarded a chancellor’s aid

scholarship to attend Louisiana State University and pursued a major in biochemistry.

While attending LSU, Chad served as the treasurer for the Student Affiliation of the

American Cancer Society, a member of the student election board, and was a member of

the Louisiana State University Tiger Marching Band. Upon graduation, Chad was

awarded three Bachelor of Science degrees in the fields of biochemistry, chemistry, and

microbiology by earning 190 total credit hours. Always having an interest in viruses,

Chad joined the laboratory of Dr. K. G. Kousoulas to pursue a doctoral degree in the area

of research of coronaviruses.

Documents

Genetics and functions of the SARS coronavirus spike protein