Genetics and Functions of Herpes Simplex Virus Type 1 (HSV

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Genetics and Functions of Herpes Simplex VirusType 1 (HSV-1) Glycoprotein K (gK) in VirusEnvelopment and Egress.Sukhanya JayachandraLouisiana State University and Agricultural & Mechanical College

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GENETICS AND FUNCTIONS OF HERPES SIMPLEX VIRUS TYPE 1 (HSV-1) GLYCOPROTEIN K (gK) IN VIRUS ENVELOPMENT AND EGRESS

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

m

The Interdepartmental Program in Veterinary Medical Sciences through the Department of Veterinary Microbiology

and Parasitology

bySukhanya Jayachandra

B-Sc., Mount Carmel Coilege-Bangalore University, 1987 M.S., Clarion University of Pennsylvania, 1990

May, 1997


UMI N um ber: 9 8 0 3 5 8 2

Copyright 1997 by Jayachandra, Sukhanya

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© Copyright 1997 Sukhanya Jayachandra

All rights reserved

ii


DEDICATION

Aiev Apicrcs'Deiv

This endeavor is dedicated to my parents

Mrs. J. Jayakumari Dr. S. R Anasuya

Mr. R Jayachandra Dr. S. V. Venkatarama Rao

Thank you for all your patience, love, sacrifice, support and for making my dreamscome true.

iii


ACKNOWLEDGMENTS

I express my heartfelt gratitude and sincere thanks to my advisor Dr. K. G.

Kousoulas for all his understanding, tutelage and continual financial support Dr.

Kousoulas's innate kindness, generosity, sound counsel, stubborn persistence and

unrelenting faith in me are responsible for the inception and successful completion of

this dissertation. The excellent state-of-the art resources his laboratory provided,

helped me attain a very diverse and in-depth knowledge of both Virology and

Biotechnology. He constantly reminded me to standup for myself and that the P in Ph.

D stood for the philosophy of perseverance, patience, persistence, perfection,

perspiration and plenty of hard work. His dedication to graduate students,

uncompromising work ethics and resourcefulness have been a model example to me.

I am greatly indebted to Dr. Johannes Storz for his historical and scientific

perspectives, guidance, expert reading and critical suggestions for improvement of this

dissertation. I am grateful to my ever patient graduate committee members, Dr. Kathy

O'Reilly, Dr. David Horohov, Dr. Ding Shih and Dr. Robert Hammer for their critical

comments and constructive criticism.

Special thanks to Dr. A. Baghian, who taught me the intricacies of cell culture

and a great deal about patience and tolerance. His tireless dedication and enthusiasm

contributed tremendously in the unraveling the "gK" story.

My sincere thanks to Laura Younger for help with all the electron microscopy

and Dr. Vladimir N.Chouljenko for invaluable help and support in various PCR and

sequencing experiments.

It is a pleasure to thank Dr. Jim Cavalcoli, Li-Ju Huang, Susan Newman who

taught me all the basic molecular biology techniques and computing and whose

cooperation, kindness, encouragement and friendship made Graduate school a

memorable and a pleasant experience.

iv


Many thanks for all the personal and professional help I have received from

Timothy P. Foster, Galena V. Rybachuk, Micah Luftig, Dr. X. Lin, Olushola Akinniyi,

Jessica Slie, Shana Dress el and Coleen Maxcy; their good humor and tolerance during

some of my stressful moments is deeply appreciated.

My gratitude and thanks to Tracy Dejean, Jodi Territo and Pat Riggleman for

taking care of my paper work and helping me negotiate the maze of University

bureaucracy; to all the VMP folks who provided access to various laboratories and

made working in this department a pleasure.

Last but not the least, special thanks and praise goes to my family, Mrs.

Padma Ramakrishna, Dr. Mahesh Jayachandra, Mrs. Menakshi Jayachandra, Naveen

Jayachandra, Magesh Mylvaghanan and to dear friends who are part of my extended

family, Dr. Lydia V. Ori and the Ori family, Ajapa Mukherjee, Ashwini Jambotkar, the

Huang family, Richard G, Dr. Shaffina and Dr. Shirish Dhume, Wanda and Melvin

Unkraut and Dr. Walter and Mrs. Seklau Wiles, all of whom who collectively kept my

body and soul together during the long and sometimes overwhelming task of being in

school.

v


TABLE OF CONTENTS

DEDICATION........................................................................................................................ iii

ACKNOWLEDGMENTS.................................................................................................iv

LIST OF TABLES ............................................................................................................vii

LIST OF FIGURES......................................................................................................... viii

ABSTRACT ...................................................................................................................... x

CHAPTER

I INTRODUCTION.......................................................................................1Statement of Problem and Hypothesis..................................................... 1Research Objectives....................................................................................1Literature Review.......................................................................................2

H HERPES SIMPLEX VIRUS TYPE-1 GLYCOPROTEIN K IS NOTESSENTIAL FOR INFECTIOUS VIRUS PRODUCTION IN ACTIVELY REPLICATING CELLS, BUT IS REQUIRED FOR EFFICIENT ENVELOPMENT AND TRANSLOCATION OF INFECTIOUS VIRIONS FROM THE CYTOPLASM TO THEEXTRACELLULAR SPACE................................................................... 45Introduction..............................................................................................45Materials and Methods........................................................................... 48Results.......................................................................................................54D iscussion................................................................................................76

m VIRAL INTERFERENCE BY GK-TRANSFORMED CELL LINES TOHSV-1 (KOS) EGRESS AND TO HSV-1 (AgK) REPLICATION...........85Introduction..............................................................................................85Materials and M ethods........................................................................... 87Results.......................................................................................................93Discussion.............................................................................................. 102

IV SITE-DIRECTED MUTAGENESIS OF THE AMINO-TERMINALOF HSV-1 GK......................................................................................... 114Introduction............................................................................................114Materials and Methods..........................................................................117Results..................................................................................................... 121Discussion.............................................................................................. 124

V CONCLUDING REMARKS.................................................................. 129S um m ary ............................................................................................... 129Future Research Challenges..................................................................131

REFERENCES................................................................................................................. 134

VITA.................................................................................................................................154

vi


LIST OF TABLES

Table 2.1 Plaquing Efficiency..................................................................................67

Table 3 .1 Plaquing Efficiency of AgK and KOS on Vero andgK-Transformed C ells............................................................................ 99


LIST OF FIGURES

Figure 1.1 HSV-1 Genome Arrangement.................................................................... 7

Figure 1.2 Schematic Representation of the HSV-1 Replicative Cycle .................. 10

Figure 1.3 Schematic Representation of HSV-1 Attachment and E ntry .................13

Figure 1.4 Diagrammatic Representation of Virion Egress......................................26

Figure 1.5 Predicted Primary Sequence of Glycoprotein K......................................39

Figure 1.6 Hydrophilic Profile, Antigenic Index and Predicted SecondaryStructure of g K ........................................................................................40

Figure 1.7 Alignment of Protein Sequences of Glycoprotein K Specified by SelectedMembers of the Subfamily Alphaherpesvirinae.......................................42

Figure 2.1 Schematic of Construction of Plasmid pSJ1724 .....................................50

Figure 2.2 Strategy for Isolation of HSV-1 AgK ..................................................... 55

Figure 2.3 PCR Detection of the AgK Genotype.......................................................56

Figure 2.4 Southern Blot Analysis of KOS and AgK Viral DN As.......................... 60

Figure 2.5 Plaque Morphology of KOS, AgK*, AgK and F-gKp Viruseson Vero Cells........................................................................................... 62

Figure 2.6 Plaque Morphology of KOS, AgK*, AgK and F-gKp Viruseson VK302 Cells........................................................................................ 64

Figure 2.7 Kinetics of Infectious Virus Production..................................................69

Figure 2.8 Electron Micrographs of Vero and HEp-2 Cells Infectedwith AgK and KOS Viruses....................................................................71

Figure 2.9 Photograph of an Autoradiogram of Immunopredpitates fromExtracts of Either KOS or AgK Virus Infected Vero C e lls .................... 75

Figure 2.10 Hydrophilic and Surface Probability Profiles of the Extra26 Amino Adds Specified by F-gKp .....................................................81

Figure 3.1 Schematic Representation of gK-Transformed Cell LinesUsed in this Study................................................................................... 89

Figure 3.2 PCR Detection of the gK Gene in Transformed Cell Lines.....................94

Figure 3.3 Northern Dot Blot Analysis of Vero and BL-1 Cellular R N A s.............95

Figure 3.4 Plaque Morphology of KOS and AgK Viruses on Vero andBL-1 cells.................................................................................................97

Figure 3.5 Kinetics of Infectious Virus Production in BL-1 and VK302 Cells — 101


Figure 3.6 Electron Micrographs of BL-1 Cells Infected withAgK and KOS Viruses...........................................................................103

Figure 3.7 Electron Micrographs of VK302 Cells Infected with AgK andKOS Viruses.......................................................................................... 105

Figure 4.1 Alignments of Protein Sequences of gK Specified by DifferentHSV-1 Syn M utants.............................................................................115

Figure 4.2 Schematic Diagram of Mutagenesis Procedure.................................... 120

Figure 4.3 Agarose Gel Electrophoresis of Viral PCR Products Restrictedwith Bbs 1............................................................................................... 122

Figure 4.4 Secondary Structure Predictions and Comparisons of KOS-Ala31and SJVal31............................................................................................ 125

ix


ABSTRACT

HSV-1 (KOS)AgK, a virus lacking the UL53 gene coding for glycoprotein K(gK)

was constructed, characterized and compared to the partially deleted gK-null virus

HSV-1 (F)-gKp. AgK and F-gK(J viruses produced microscopic plaques on Vero cells at

48 hours post infection. F-gKp plaques at 72 hours post infection contained fused cells

(syn), while AgK plaques resembled wild-type KOS plaques (syn*). AgK replicated

efficiently in exponentially dividing Vero cells whereas F-gKp yields were 10 and 1000-

fold lower than AgK yields in HEp-2 or log-phase Vero cells, respectively. Electron

micrographs of AgK-infected Vero cells revealed large numbers of capsids in the nuclei

of Vero, but not HEp-2 cells, while a small number of enveloped virions were observed

in the cytoplasm of infected Vero and HEp-2 cells that failed to translocate to the

extracellular spaces. KOS and AgK replicated inefficiently in gK-transformed cells,

with the exception of VK302 cells. Electron micrographs of the gK-transformed BL-1

cells infected with KOS revealed virions within perinuclear spaces, while there were no

virions visualized in AgK infected BL-1 cells. VK302 cells were the only gK-

transformed cells that supported the replication and cellular egress of both KOS and

AgK

To fine-map the functional domains of gK involved in membrane fusion, a long-

PCR site-directed mutagenesis system was developed that efficiently produced single

base substitutions within larger than 4 kbp DNA fragments. Utilizing this system three

different mutations were engineered and recombinant viruses containing one of the

three mutations were isolated. The mutation at position 31 resulted in a syn* virus.

This research revealed that gK is involved in nudeocapsid envelopment and

cellular egress of infectious virions. Actively replicating cells can partially compensate

for envelopment but not for the cellular egress defidendes of AgK Interference of viral

replication in gK-transformed cells suggests that the cellular gK can function in-trans to

inhibit egress by preventing certain cellular factors required for viral egress to function

x


efficiently. These results implicate that viral egress involves cellular receptors that are

modified to act as "pilots" or "sorters" for virus transport and egress in a similar

fashion to intracellular glycoprotein transport.

xi


CHAPTER I

INTRODUCTION

STATEMENT OF PROBLEM AND HYPOTHESIS

Wild-type HSV-1 infections cause limited amounts of cell fusion that are

characterized by characteristic cyto pathology of round swollen cells in natural

infections and in cell culture (Brown et al., 1973; Hoggan and Roizman, 1959; Person et

al, 1976; Spear, 1993). Mutant viruses have been occasionally isolated in natural

infections and in cell culture that cause cell fusion or syncytia characterized by

extensive fusion of cells to form multi-nucleated cells. The syncytial mutations that

cause this extensive fusion phenotype were mapped to 5 different loci in the viral

genome. Interestingly, in a majority of the independently isolated syncytial viruses, the

syncytial mutations map to glycoprotein K (gK) that is encoded by the UL53 open

reading frame. Thus, the central hypothesis for these investigations is that gK is a key

regulator of membrane fusion events during virus entry, egress, and virus-induced cell

fusion. Therefore, the absence of gK during viral infection is predicted to cause defects

in virus entry due to defects in virus to cell fusion, virion egress and virus-induced cell

fusion.

RESEARCH OBJECTIVES

The goal of this research was to establish the functional role of HSV-1

glycoprotein K (gK) in the viral replicative cycle, using novel recombination and rapid

site-directed mutagenic methods to engineer deletions and mutations into gK The

specific research aims were:

1. To determine if gK is essential for HSV-1 virus replication by genetically

constructing a gK-null virus with the entire gK gene deleted.

1


2

2. To examine the phenotypic properties of the gK-null virus, by determining the

plaque morphology, replicative kinetics and plaquing efficiency on different gK-

transformed cells.

3. To compare the genotypic and phenotypic properties of the gK-null virus with

the previously isolated gK mutant virus, F-gK0.

4. To delineate the functional domains of gK by developing a site-directed

mutagenesis system to obtain mutant viruses with specific amino add changes

in the amino-terminal portion of gK

The results from these investigations are presented as three individual chapters

following the literature review and are listed below:

1. Herpes Simplex Virus Type-1 Glycoprotein K is Not Essential for Infectious

Virus Production in Actively Replicating Cells, but is Required for Translocation

of Infectious Virions from the Cytoplasm to the Extracellular Space.

2. Viral Interference Mediated by gK-Transformed Cell Lines To HSV-1 (KOS)

Egress and to HSV-1 (AgK) Replication.

3. Long PCR Site-Directed Mutagenesis of the Amino-Terminal Of HSV-1 gK

LITERATURE REVIEW

Herpesvirus Taxonomy

Herpesviruses are widely distributed in nature infecting most animal spedes.

According to the Herpesvirus Study Group of the International Committee on the

Taxonomy of Viruses, HSV-1 also known as human herpesvirus 1 is a member of the

family Herpesviridae, subfamily Alphaherpesvirinae and the genus Simplexvirus (Roizman

et al., 1981). Animal pathogens also induded in this genus are the bovine herpesvirus

type 1 (BHV-1) and bovine herpesvirus type 2 (BHV-2). Other pathogens induded in

the subfamily Alphaherpesoirinae are varicella zoster virus (VZV) the causative agent of


3

chicken pox, pseudorabies virus (PrV) which causes Aujeszky's disease in pigs and

equine herpesvirus type 1 (EHV-1) all of which belong to the genus Varicellavirus

(Roizman and Sears, 1996).

All members of the subfamily Alphaherpesvirinae are grouped together on the

basis of certain common characteristics in that they have a broad host range, a

relatively short replicative cycle, spread rapidly in cell culture, efficiently destroy the

cells they infect and possess the capacity to establish latent infections primarily but

not exclusively in the sensory ganglia (Roizman et al., 1981; Roizman and Sears, 1996).

Historical Perspective of Herpesviral Diseases

The word "herpes" has been used as a medical term for at least 25 centimes

(Beswick, 1962). However, it is possible that in ancient times the word was used to

describe conditions as diverse as eczema and skin cancer. The word "herpes" is

derived from the Greek verb epiceiv meaning to creep, appears to have been used

initially to describe a variety of spreading cutaneous lesions. Eventually, most medical

writers seem to have used the word to describe "zoster", which are the sores that

appear around the waist in the form of a b e lt The belt-like lesions are caused by a

closely related alphaherpesvirus, varicella zoster virus (VZV). The association between

fever and mouth lesions was attributed to Herodotus (Mettler, 1947). William and

Bateman in 1814 first differentiated between labial and genital herpes infections from

herpes zoster (Mettler, 1947). Vidal in 1873 demonstrated that HSV was infectious by

human inoculation experiments (Wildy, 1973). HSV was transmitted to rabbits in 1920

and was shown to be a filterable agent the following year. Andrews and Carmicheal,

(1930) discovered that many adults had circulating neutralizing antibodies to HSV and

that the recurrent herpetic disease occurred only in such individuals. The distinction

between primary and recurrent disease was first elucidated by Burnet and Williams in

1939. In subsequent years, the clinical spectrum of HSV-induced diseases was


4

extended to indude eczema herpeticum (Seidenberg, 1941), vulvovaginitis, primary

keratoconjunctivitis (Gallardo, 1943), encephalitis and meningitis (Smith et al., 1941).

Although suggested by Lipschitz in 1921, on clinical grounds it was not until the early

1960s that the existence of two distinct antigenic types, HSV type 1 (HSV-1) and HSV

type 2 (HSV-2), was discovered (Lipschitz, 1921; Nahmais and Dowdle, 1968). A few

years later they observed that the anatomic site of isolation could be correlated

generally to antigenic type.

Herpes simplex viruses (HSV) are common human pathogens associated with a

broad spectrum of illness, ranging from asymptomatic infections to fulminant

disseminated diseases resulting in death. Two speries of Simplexviruses have been

identified, type 1 and type 2 which are very similar in biochemical composition, but

have different biological properties and can be readily distinguished from one another

by a variety of biochemical and immunological techniques. In general HSV-1 causes

orofacial infections, visceral infections in immunocomprimised hosts, and in rare cases

herpes simplex encephalitis. Based on antibody studies, it is estimated that 90% of the

population acquire HSV-1 infections before the ages 4 or 5 years, following which the

virus establishes latency for life in the peripheral ganglia of sensory and autonomic

nerves. Thereafter, recurrent self-limited attacks may occur, provoked by fever, another

viral infection, fatigue, menstruation, and other triggering factors such as UV rays and

wind. HSV-2 is more commonly associated with mucocutaneous infections of the

genital tract, and causes the majority of neonatal diseases. HSV can also infect the

cornea, causing keratitis (Whitley and Gnann, 1993).

Structure of the HSV-1 Virion

The complete virion particle is approximately 120 to 300 nm in diameter

depending on the thickness of the tegument and consists of four structural elements, 1)

a cylindrical core structure around which the viral DNA is wound in the form of a


5

torus, 2) a icosahedral capsid which is approximately 100 nm in diameter and 15 nm

thick, 3) a granular zone or tegument which surrounds the capsid, and 4) an envelope,

which is derived from the host cell as the particle buds from the nuclear membrane.

HSV is relatively sensitive to heat and must be stored at -70°C if infectivity must be

maintained for significant periods of time (Roizman and Sears, 1996).

Viral Genome: Like all other herpesviruses, the genome of HSV-1 is a single

linear double-stranded DNA molecule with a molecular mass of approximately 100 X

107 Daltons (Becker et al., 1968; Keiff et al., 1971; Plummer et al., 1969). The genome is

152 kbp with a G+C content of 68% (Becker et al., 1968; Keiff et al, 1971; McGeoch et

al, 1988) and exhibits structural properties of a class E herpes viral genome consisting

of two covalently linked components, the Long (L) and the Short (S) regions which

contain unique sequences, thus the name UL and Us- Flanking each of these unique

sequences are the inverted repeat regions (Sheldrick and Berthelot, 1975; Wadsworth et

al, 1975) (Figure 1.1). During viral replication, the UL and Us segments can invert

relative to one another resulting in four linear isomeric forms (Delius and Clements,

1976; Hayward et al., 1975). Various complementation groups were established using

conditional lethal temperature sensitive its) mutants of HSV-1, in two and three factor

crosses, enabling the functional organization of the viral genome (Schaffer et al., 1974;

Timbury and Calder, 1976). Furthermore, insertional inactivation and deletion of the

various open reading frames indicate that the HSV-1 genome encodes 84 open reading

frames (ORF), of which 60 are located in the UL region, 14 in the Usregion, 4 in each of

the repeats flanking the UL component and 1 ORF is located in the inverted repeat

bracketing the Us region (McGeoch et al., 1988; McGeoch et al, 1986; Roizman and

Sears, 1996).

Capsid: The capsid is an icosahedral protein shell approximately 15 nm in

thickness and 125 nm in diameter, consisting of 162 morphological subunits or


6

capsomeres, 150 of which are the hexons and 12 of which are composed of the pentons

(Wildy and Watson, 1962). Genetic and structural studies have demonstrated that the

assembly of the caps ids is an essential step for virion maturation. HSV-1 infected cell

extracts contain three major types of capsids designated "A" (empty), "B"

(intermediate), and "C" (full), that can be separated by velocity sedimentation through

sucrose gradients (Gibson and Roizman, 1972; Gibson and Roizman, 1974). The "A"

capsids are devoid of DNA and do not contain the internal torroid structure, which is

thought to act as the scaffholding protein for packaging of DNA. The empty "A"

capsids contains four proteins, Viral Protein 5 (VP5)/Infectious cell protein 5

aCP5)(UL19), VP19c (UL38), VP23 (UL18), and VP26 (UL35) (Cohen et al., 1980).

The major capsid protein VP5 accounts for 70% of the capsid mass and forms the

hexons and pentons, VP19c and VP23 together form the trigonal nodules called

triplexes which lie at the capsid floor and connect the capsomeres in groups of three,

and VP26 is found on the distal tips of the hexons. These capsids may be a decay

product not in the pathway of virion maturation. The "B" capsids also lacks DNA but

differ from the "A" type in that the former contains three additional proteins the

VP21/ICP35b, VP22a/ICP35e-f (UL26.5) and VP24, all of which are self-cleavage

products of the UL26 gene product (Lui and Roizman, 1991a; Lui and Roizman,

1991b). Pulse-chase experiments have shown that the "B" capsids are able to package

viral DNA to form "C" capsids, indicating that these "B" capsids may be

intermediates in virus assembly (Perdue et a l, 1976). Type "C" capsids contain the

entire viral genome and a small protein—VP22, in addition to those found in the "B"

capsids (Gibson and Roizman, 1972; Gibson and Roizman, 1974; Rixon et al., 1988).

The "C" capsids are able to mature into infectious virions.

Tegument: The tegument in HSV-1 virions is the amorphous proteinaceous

layer between the underface of the envelope and the outer surface of the capsid and is

usually represented as being variable in size and shape(Roizman and Furlong, 1974).


Reproduced

with perm

ission of the

copyright ow

ner. Further

reproduction prohibited

without

permission.

Herpes Simplex Virus Type-1 Genome

a b

TR

U l

syn 5

b a

IRL

a c

g M 8 h g B g C g K

Us c a

IRS TR

eg sJ * *-OCQHD-

syn 3 syn 1 syn 6

B -syn 2

Figure 1.1: HSV-1 Genome Arrangement.

Top line represents the prototypic arrangement of the HSV-1 genome, consisting of the unique long Ul andunique short Us regions flanked by the terminal repeats (TR) and internal repeat regions (IR).Small letters represent orientation of specific DNA elements in the repeat regions.Shown below are the relative locations of the HSV-1 specified glycoproteins. The bottom line represents the relative positions of the syncytial (sy«)loci.

-4

8

Structural proteins that are not part of the capsid or the envelope are generally

assigned to the tegument and their relative amounts are essentially constant for a

particular strain of virus. Certain tegument proteins have the ability to influence the

initiation of infection, notably the alpha-trans inducing factor (a-TTF also known as

ICP25, VP16 or Vmw65, encoded by UL48) which is required for efficient expression

of immediate-early (IE), regulatory or alpha (a) genes (Batterson and Roizman, 1983).

Other components of the tegument include the virion host shutoff protein (VHS,

encoded by UL41), VP1-2 (UL36), VP11-12 (UL46), VP13-14 (UL47) and USll

(Batterson and Roizman, 1983; Chou and Roizman, 1989; Read and Frenkel, 1983;

Roller and Roizman, 1991; Roller and Roizman, 1992; Zhang and McKnight, 1993). The

immediate-early (IE) proteins, ICPO (coded by ccO) and ICP4 (coded by a4) are

important in the transcriptional activation of viral gene expression and have been

reported to be components of the viral tegument (McLauchlan and Rixon, 1992; Yao

and Courtney, 1992). The properties of the virus tegument has been elucidated through

studies on HSV-1 light particles (L-partides) which comprise of enveloped tegument

like structures that lack nudeocapsids (McLauchlan and Rixon, 1992; Sturm et al.,

1987). The absence of capsids from L-partides suggests that the tegument proteins

have the ability to assemble into stable structures without requiring the interaction with

capsids since L-partides were produced under conditions where virion maturation did

not occur (Rixon et al., 1992). However, the processes involved in tegument assembly

and the mechanisms which direct and modulate the amount of each protein

incorporated into virion partides are unknown.

Envelope: The envelope is the outer entity of the HSV-1 virion, acquired as the

virus exits from the nudeus of infected cells and is comprised of the host cell derived

lipid bilayer and viral glycoproteins (Morgan et al., 1959).The presence of lipids was

demonstrated by analysis of virions and their sensitivity to lipid solvents and

detergents (Spear and Roizman, 1972). HSV-1 specifies 17 known or putative


9

membrane proteins, 11 of which have been proven to be glycoproteins (gB-UL27, gC-

UL44, gD-US6, gE-US8, gG-UL4, gH-UL22, gI-US7, gJ-US5, gK-UL53, gL-ULl, gM-

UL10) (Roizman and Sears, 1996; Spear, 1993). Electron micrographs of thin sections

of HSV-1 infected cells revealed an envelope with a trilaminar appearance, containing

numerous protrusions or spikes (Epstein, 1962). Evidence to date concludes that at

least three virion glycoproteins gB, gC and gD are each present in distinct and different

structures projecting from the virus envelope. There are two kinds of protrusions from

the envelope, rod-like spikes about 14 nm long and short fuzzy material (Stannard et

al., 1987). Colloidal gold coupled directly to monoclonal antibodies to gB and gD has

revealed that the rod-like spikes are composed of gB and the short fuzzy material are

composed of gD. The majority of virion glycoproteins and integral membrane proteins

that have been found in the viral envelope, play important roles in virus attachment,

penetration into cells and translocation of virions to the extracellular space (Roizman

and Sears, 1993).

The Replicative Cycle of HSV-1

The principal events required for HSV-1 infection and replication in cell culture

involves the attachment of the virus to the cell surface, penetration of the virus into the

infected cells, the movement of the capsids to the nucleus where there is a release of

viral DNA, replication of the viral DNA, viral protein synthesis, assembly of capsids,

packaging of viral DNA into the newly formed preassembled capsids and the

maturation of the virus by budding through the inner lamella of the nuclear membrane

(Figure 1.2). Most of the information about HSV viral replication and gene expression

has been obtained in cell culture systems. Characteristic cytopathic changes observed

in HSV infected cells includes ballooning of cells, chromatin condensation and

margination along the nuclear membranes, and intranuclear inclusion bodies (Morgan et

al., 1959).


10

HSV-1 Replicative Cycle

HSV-t VISION

ENTS'

iTURATION

VIRAL DNA

NUCLEAR!’MEMBRANE^

r VIRAL GLYCOPROTEINS

proteinsP proteins

MEMBRANE

Figure 1.2; Schematic Representation of the HSV-1 Replicative Cycle.

The various stages of the HSV-1 life cycle such as entry, transcription, replication, assembly, maturation and egress of HSV-1 virions are depicted.


11

Attachment In general, attachment of enveloped viruses can be defined as the

interactions between specific viral envelope proteins and receptors on the target cells.

HSV-1 is a neurotropic virus and in natural infections the virus must replicate in two

types of cells, the epithelial cells at the site of primary infection and the neural cells

where the virus establishes a latent infection; therefore the virus must have receptors on

multiple types of cells. Any constituent of the cell membrane, including carbohydrates,

lipids, and proteins, may act as viral receptors. The cell membrane components serve

normal cellular functions and are subserved by viruses for attachment and entry. The

binding of the virus to its receptor determines in part the viral tropism. Some viruses

have a narrow host range as the binding of the virus to its receptor is with high affinity

and specificity, e.g. HIV binding to the CD4 receptor (McDougal et al., 1986). Whereas,

other viruses have a broad host range and can bind to several molecules on the cell

surface. A number of cellular molecules have been identified as viral receptors, e.g., the

epidermal growth factor receptor for vaccinia virus (Eppstein et al., 1985) complement

receptor type 2 for Epstein Barr virus (Fingeroth et al., 1984), and CD4 for human

immunodeficiency virus (Dalgleish et al., 1984; Klatzmann et al., 1984; McDougal et al.,

1986).

The primary attachment of HSV-1 to cells is through viral protein interactions

with the ubiquitous heparan sulphate moieties of cell surface proteoglycans since HSV-

1 binding was greatly reduced in cells with altered or depleted cell surface heparan

sulphate proteoglycans (Gruenheid et al., 1993; Shieh et al., 1992; WuDunn and Spear,

1989). Also, heparin, a secretory product similar in structure to heparan sulphate,

inhibits the binding of virus to cells, and virions have been shown to bind to heparin-

affinity columns in physiological saline (Mettenleiter et al., 1990; WuDunn and Spear,

1989). Extensive studies have shown that viral glycoprotein, gC facilitates the initial

binding event in polarized cells (Herold et al., 1994; Herold et al., 1991; Spear et al.,

1989; WuDunn and Spear, 1989) as HSV-1 mutant viruses that lack gC are


12

significantly unpaired in their ability to bind to cells. Furthermore, HSV-1 gC exhibits

high affinity for heparin under physiological conditions (Herold et ai, 1991) and is a

major viral antigen which elicits a strong neutralizing antibody response, that can block

the binding of the virus to the cells (Fuller and Spear, 1985; Svennerholm et al.t 1991).

In nonpolarized cells, HSV-1 virus devoid of gC attach to cells, suggesting that other

components are involved. HSV-1 glycoprotein B (gB), a second high affinity heparan

sulphate glycoprotein, was found to be involved in binding to cells and may be

responsible for the gC-independent binding (Spear, 1993) (Figure 1.3).

There is growing evidence that the virus uses several receptors on the cell

surface to promote attachment and entry into cells. The basic fibroblast growth factor

receptor (FGFR) was implicated in attachment of HSV-1 in Chinese hamster ovary

(CHO) cells (Kaner, 1991). The CHO cells are normally resistant to HSV-1 infection,

however, when FGFR was expressed in CHO cells they became susceptible to

infection. Spear et al., (1991), could not confirm these results and suggested instead

that the ability of HSV to bind or to penetrate CHO cells was not influenced by the

presence or absence of FGFR. HSV-1 gD has been implicated in binding to cellular

receptor facilitating virus penetration into cells. Brunetti et a i, (1994) identified and

characterized a 275 kDa mannose-6-phosphate/ insulin-like growth factor II receptor,

using soluble forms of gD that were expressed in CHO cells.

Recently, Montgomery et al., (1996) identified a human cDNA from HeLa cells

which transfers susceptibility to HSV-1 entry to normally resistant CHO cells when

stably expressed. The HeLa cDNA codes a 228 amino add protein, with

characteristics of a Type 1 membrane glycoprotein. The protein was designated the

herpes virus entry mediator (HVEM) and based on sequence homology and cysteine

repeats in the ectodomain, this protein was included as a new member of Tumor

Necrosis Factor (TNF)/Nerve Growth Factor (NGF) receptor family. HVEM was

shown to enhance entry of a variety of HSV-1 strains in CHO cells.


13

Viral Attachment and Entry

HSV-1

HVEM

Receptor

Miootubales

Heparan Sulphate Proteoglycans

M lcrotubale Organizing Center

CYTOPLASM

NM

NUCLEUS

Figure 1.3: Schematic Representation of HSV-1 Attachm ent and Entry.

Cross-section of a eukaryotic cell depicting various components involved in virus attachement and entry.PM=plasma membrane, NM =nuclear membrane


14

HVEM was not involved in virus attachment but was shown to mediate post

attachment entry steps. Thus, heparan sulphate is the principle receptor for virus

attachment and other non-heparan sulphate receptor(s) like HVEM are involved in

post-attachment events including fusion of the virus envelope and release of the

nudeocapsids into the cytoplasm.

Virus Entry: In general, penetration of attached enveloped viruses through the

cell membrane can occur in one of two routes; 1) by direct penetration through the

plasma membrane in a pH-independent pathway or 2) through endocytosis, in a pH-

dependent pathway. Either of these pathways results in the fusion of the viral

envelope with the cellular plasma membrane or the endosomal membranes. In the pH-

independent fusion pathway, the binding of the virus to its receptor induces a

conformational change in the viral envelope glycoproteins as exemplified by HIV-1

binding to the CD4 receptor on the CD4* T-cells. The binding results in molecular

rearrangements in the HIV-1 virion glycoprotein gpl20 and in CD4 which are necessary

to establish an interaction of the fusion domain at the amino terminal of the

transmembrane glycoprotein gp41 with the target cell membrane, thus initiating fusion

(Allan, 1991). In contrast, in the pH-dependent pathway, the virus binding to its

receptor is followed by in ternalization or endocytosis of virion particles into the

endosomes, and the virion-endosomal fusion occurs within the intracellular veside. The

fusion of the viral envelope with the endosomal membrane is triggered in part by the

effect of endosomal pH changes on the conformation of the viral proteins (Hoekstra

and Kok, 1992; Sturman et a i, 1990). Many enveloped viruses (e.g. the influenza virus,

a orthomyxovirus) and a number of non-enveloped viruses (e.g. poliovirus, a

picomavirus) have been shown to be internalized via endocytosis.

HSV-1, like other herpesviruses, enter cells by fusion of the viral envelope with

the plasma membrane in a pH-independent pathway since low pH and inhibitors that

block endocytosis do not inhibit entry of the virus (Wittels and Spear, 1991).


15

Furthermore, fusion of the virus to the plasma membrane can be blocked by

neutralizing antibodies and enhanced by the chemical fusogen polyethylene glycol

which can overcome various kinds of block to infectious HSV penetration (Fuller et al.,

1989; Fuller and Spear, 1985; Fuller and Spear, 1987).

The fusion activity of enveloped viruses to cellular membranes in most of the

different viruses studied to date has a common mechanism, in that a single gene

product confers fusion activity. This mode of mechanism is well-characterized for

influenza virus where the surface protein responsible is the hemagglutinin (HA).

Exposing HA-containing membranes to low pH conditions triggers rapid fusion and in

natural infections this occurs in the endosomes (Stegmann et al., 1989; White, 1992). In

contrast, the mechanism of fusion of the herpesvirus viral envelope with the plasma

membrane is not mediated by any one glycoprotein and no fusion protein has been

identified. The envelope of HSV-1 virion contains at least 11 different glycoproteins,

several of which project as distinct spikes from the membrane surface. Therefore, it

seems possible that binding and the subsequent virus-cell fusion may require a cascade

of multiple HSV-1 specified envelope glycoproteins interacting with several different

sites or receptors on the cell surface. At least four viral glycoproteins, gB, gD, gH and

gL have been shown to be essential for virus entry into cells at a step subsequent to

viral attachment and have all been implicated in the pH-independent fusion of the

viral envelope with plasma membrane (Cai et al., 1988a; Cai et al., 1988b; Ligas and

Johnson, 1988; Roop et al., 1993; Sarmiento et al., 1979; Sarmiento and Spear, 1979). gL

is required for the proper expression and incorporation of gH into virions, but whether

it plays a more specific role in entry has not been determined (Hutchinson et al.,

1992a). The roles of these glycoproteins and the specific events in penetration and

entry is yet to be fully understood. However, deletion mutants of gB, gD, gH and gL

attach to host cells but do not enter (Cai et al., 1988a; Cai et al., 1988b; Desai et al.,

1988; Forrester et al., 1992; Ligas and Johnson, 1988). Also ts mutants with ts lesions in


16

the genes that encode gB and gD attach to but fail to penetrate cells (Desai et al., 1988;

Manservigi et al., 1977; Sarmiento et al., 1979; Sarmiento and Spear, 1979).

Furthermore, gD present on the plasma membrane of cells that constitutively express

this glycoprotein interferes with the penetration of superinfecting HSV (Campadelli-

Fiume et al., 1990), suggesting a role for gD in penetration of cells. By direct analysis of

these HSV-1 mutant viruses deleted in the individual glycoproteins and from the entry

kinetic studies, a possible scenario for virus-cell fusion is emerging where the individual

glycoproteins are involved at separate and different points in entry in a sequential

fashion. According to this model, the initial attachment of HSV-1 to the cell surface

heparan sulphate is mediated by gC and or/gB followed by interaction of gD with

cellular receptors in a stable attachment step. The stable interaction is resistant to

heparin washes and is proposed to be a prerequisite for virus fusion with the plasma

membrane (McClain and Fuller, 1994). These interactions triggers fusion between viral

envelope and the plasma membrane and gH is involved in initiation of this fusion

bridge but not in the entry of the nudeocapsid into the cytoplasm (Fuller and Lee,

1992) (Figure 1. 3). Handler et al., (1996) used chemical cross-linking to demonstrate

that the five envelope glycoproteins, gB, gC, gD, gH, gL were dose enough to each other

on the virion envelope where they could be cross-linked into homodimeric and

heterodimeric forms and these together could partidpate in the formation of a fusion

machine as proposed by White, 1992 (Figure 1.3).

Intracellular Transport of Nudeocapsids to the Nudeus: Replication of the

viral DNA requires the transport of the nudeocapsid to the nudear pore, where the

viral genome is released from the capsid into the nudeoplasm. The cytosolic transport

of capsids has been reported to be mediated by cellular cytoskeletal microtubules

(Dales and Chardonnet, 1973; Kristensson et al., 1986). Recently, Sodeik et al., (1996)

used microtubule depolymerizing agents in conjunction with immunofluroscence and

immunoelectron microscopy and proposed that the nudeocapsids use dynenin, a


17

minus-end directed microtubule dependent motor, to move along the microtubules

towards the centriole from where they are transported to the nudeopore (Figure 1.3).

Cascade Synthesis of Viral Proteins and Their Coordinated Regulation: The

HSV-1 lytic replication cycle has been described as a coordinated process that involves

the sequential transcription and temporal regulation of at least three viral gene classes:

aplha (a) or immediate early (IE), beta ((1) or early, gamma (y) or late. Early studies

defined the time of appearance and thus the kinetic classification of infected cell

proteins (ICP) on the basis of requirements for either de novo proteins or DNA

synthesis (Honess and Roizman, 1974; Honess and Roizman, 1975). Five a genes,

ICPO, ICP4, ICP22, ICP27 and ICP47, have been identified that are expressed in cells

soon after infection. The transcription of these a genes does not require prior viral

protein synthesis occurring before viral DNA replication. However, the transcription of

these a genes are stimulated by a protein component of the virus particle, ICP25 (also

called a gene frans-indudng factor, a-TIF, Vmw65 or VP16). During productive

infection, the a-TIF forms a complex with the cellular transcription factor Oct-1 and at

least one additional cellular protein resulting in interaction with the octamer binding

sites present in the promoters of the a genes. Two of the five a genes, ICP4 and ICP27

are essential for viral replication in cell culture (Dixon et al., 1983; Sacks et al., 1985).

The accumulation of functional a gene products activates the P genes, many of which

are enzymes involved in nucleotide metabolism and DNA replication, resulting viral

DNA synthesis begins shortly after their expression. The process leads in turn to the

activation of the y genes, which can be further designated into the y 1 and y 2 kinetic

class of genes. The expression of y 1 genes is enhanced by viral DNA, but significant y

1 gene expression can occur prior to, or in the absence of viral DNA replication.

Finally the y 2 genes are transcribed, whose expression is stringently dependent on

onset of virus DNA replication. Many of the HSV y gene products are structural

components of the mature virus particles and are transcribed at early as well as at late


18

times after infection. The sequential and regulated pattern of HSV protein synthesis is

influenced at both the transcriptional and post-transcriptional levels (Roizman and

Sears, 1996).

Viral DNA Replication: Upon entry into the nucleus the linear double stranded

DNA genome circularizes and only a small portion of the viral DNA that enters the cell

undergoes replication. Comparison of the terminal structures of mature DNA and the L

and S junction sequences suggests that the circularization of the double stranded linear

DNA molecule takes place by direct ligation of the ends which was observed by

analyzing replication intermediates and from electron microscopic examination of viral

DNA isolated from infected cells (Davison and Wilkie, 1981; Mocarski and Roizman,

1982). Subsequent replication of the circularized DNA yields large head-to tail

concatemers consisting of tandem repeats of the viral genome. Available data suggest

that HSV-1 DNA replicates through rolling circle mechanisms (Becker et al., 1978; Jacob

et al., 1979). However, the simple rolling circle mechanism does not account for

intermediate branched structures or the rapid amplification of viral DNA seen in the

initial hours after infection (Hammerschmidt and Sugden, 1990). Therefore there are

several additional models proposed for the generation of concatermers. Martinez et al.,

(1996) eluded to a DNA replication model, where the initial rounds of replication

occurs by theta (6) replication that is initiated at one or more of the origins of

replication and at late times of infection the rolling circle mode of replication takes

over, generating the head-to-tail concatermers. Another interpretation of this model is

that the initial theta-type replication could be followed by a recombination event where

an inversion occurs between the replicated copy of an inverted repeat unit and an

unreplicated copy, resulting in two replicating forks being developed that generates the

concatemers. A third model proposed for replication is similar to that seen in T4

phages where strand invasion provides the 3' hydroxyl primers for initiation of DNA

replication which can be converted to the replication forks necessary for the generation


19

of the concatemers which the mature into unit-length molecules by site-specific cleavage

(Martinez et al., 1996).

The HSV-1 genome contains three cis acting origins (oil) of DNA replication and

genome replication may originate in either direction from any of three origins of

replication (Friedman and Becker, 1977; Hirsch et al., 1977; Stow, 1982; Stow and

McMongale, 1983; Weller et al., 1985). One of the origins, orit maps to the middle of

the L component, between the promoters of the f) genes specifying the major DNA

binding protein ICP8 and the DNA polymerase (Weller et al., 1985). The ot̂ consists

of an 144 bp, A+T rich, palindromic region that is unstable when cloned into E.coli. The

other two origins, orisl and oris2 that map to th e 'd sequences flanking the S

component of the genome, between the promoter elements of the a genes, ICP4 and

ICP22 (orisl) (Locker et al., 1982; Weller et al., 1985) or between the a genes ICP4 and

ICP47 (oris2) (Barnett et al., 1983; Deb and Doelberg, 1988; Mocarski and Roizman,

1982; Stow, 1982; Stow and McMongale, 1983). Cell culture studies have

demonstrated that the oriL and at least one of the oris can be deleted (Longnecker and

Roizman, 1986; Polvino-Bodnar et al., 1987) or both oris sequences can be deleted

without affecting viral replication (Igarashi et al., 1993). The strategic location of the

three origins of DNA replication between the transcription initiation sites in the HSV

genome may be regulated by local changes that occur due to the initiation of

transcription in this region (Hubenthal-Voss et al., 1987).

Also aiding in the replication process are seven virus specified proteins that are

essential and sufficient for HSV-1 origin-dependent plasmid amplification in a

transient transfection assay (Wu et a i, 1988). The seven essential viral DNA

replication genes are the UL5, UL8, UL9, UL29, UL30, UL42 and UL52. Briefly, the

UL9 gene product binds specific sequences within the origins of replication of HSV-1

(Elias and Lehman, 1988; Elias et al., 1986) and also possesses helicase activity

(Bruckner et al., 1991; Fiererand Challberg, 1992). UL29 encodes ICP8, a single


20

stranded DNA binding protein (Quinlan and Knipe, 1983). The UL30 and UL42 gene

products compose a heterodimeric DNA polymerase in which UL30 codes the

catalytic subunit and UL42 encodes the subunit that acts to increase the processivity

of the enzyme (Chartrand et ai, 1980; Coen et al., 1984; Hay and Subak-Sharpe, 1976;

Honess et d ., 1984; Keir, 1968; Keir and Gold, 1963; Powell and Purifoy, 1977). The

HSV-1 DNA polymerase is unusual in that it is sensitive to compounds such as

phosphonoacetate, phosphonoformate and nucleoside analogs like acydoguanosine.

Finally, the UL5, UL8 and UL52 gene products form a heterotrimeric complex in which

each protein is present in equimolar ratios and function with both 5' and 3' helicase

and primase activities (Crute and Lehman, 1991; Crute et d ., 1989). The seven

replication proteins localize to gobular nuclear structures termed the replication

compartments which are found adjacent to NDlOs, nuclear domains present in all cell

types whose function is unknown (Maul et al., 1996). These central nudear structures

have been defined by the presence of the single-stranded DNA binding protein, ICP8

(de Bruyn Kops and Knipe, 1994).

In addition, the HSV-1 genome codes for a number of proteins involved in

nucleic add metabolism that play important roles in processing, deavage, and

packaging of the genomic viral DNA. Most of these proteins are not essential for viral

replication in cell culture. Some of these virally encoded nudeic add metabolism

proteins are the alkaline nuclease, thymidine kinase, ribonudeotide reductase, uracil

DNA glycosylase and the dUTPase reductase (Roizman and Sears, 1996).

The alkaline nudease is a phosphoprotein which has DNAse activity and is

encoded by the UL12 ORF (Keir and Gold, 1963; McGeoch et d., 1988). Through the

study of ts mutants, the DNase activity of this enzyme was demonstrated to be

essential for viral growth (Francke et d ., 1978). Shao et d ., 1993 reported that HSV-1

mutant viruses that lack the alkaline nudease gene may be defective in genome

maturation and for the production of competent "C" capsids and thus the capsids


21

accumulate in the nucleus. Available data indicate that nonlinear branched viral DNA

replication structures arise in cells infected with either wild-type virus or with the

alkaline nuclease-null mutant viruses and that the alkaline nuclease enzyme was

required for the efficient processing of complex nonlinear viral DNA replication

intermediates that arise during replication in these viruses (Martinez et al., 1996).

The viral thymidine kinase (TK) encoded by the UL23 ORF was found to be

essential for viral replication in natural infections, but is dispensable in cell culture. The

TK has been designated as a deoxypyrimidine kinase, though it functions to

phosphoryiate purine pentosides and a wide variety of nucleoside analogs. The unique

property of the viral TK along with its broad substrate range is the basis of antiviral

treatments such as acyclovir and gangicydovir (Roizman and Sears, 1996).

HSV-1 ribonudeotide reductase is a complex enzyme made up of a large and

small subunit encoded by the UL39-ICP6 and UL40 ORFs, respectively (Bacchetti et

a l, 1986; Frame et al., 1986; Huang et al., 1988). The enzyme generates a pool of

substrates for DNA synthesis by reducing ribonudeotides to deoxyribonudeotides

(Roizman and Sears, 1996). The activity of this enzyme is dispensable for viral

replication in actively dividing cells maintained at 37°C but is essential in non-dividing

or stationary phase cells maintained at 39.5°C, indicating to a cellular homolog that

can complement the viral function in actively dividing cells (Goldstein and Weller,

1988).

The viral uracil DNA glycosylase encoded by the UL2 ORF was described by

Caradonna and Cheng (1981), and it has since been shown to be highly conserved in all

other herpesviruses (Cardonna et al., 1987; McGeoch et al., 1988; Wohlrab and Francke,

1980; Worrad and Cardonna, 1988). TheDNA repair and proof reading enzyme

excises uracil residues in DNA that result from the misincorporation of dUTP or

spontaneous deamination of cystosine. The UL50 ORF encodes the HSV-1

deoxyuridine triphosphate nucleotidohydrolase (dUTPase), an enzyme catalyzing the


22

hydrolysis of deoxyuridine triphosphate (dUTP) to deoxyuridine monophosphate

(dUMP) which is eventually converted to deoxythymidine monophosphate (dTMP) via

methylation of dUMP by thymidylate synthetase, thus providing a pool of nucleotide

substrates that are incorporated into the newly synthesized viral DNA (Caradonna

and Cheng, 1981). Also dUTPase maintains the low intracellular concentrations of

dUTP so that uracil cannot be incorporated into the newly synthesized DNA.

Assembly of Capsids: Assembly of HSV-1 capsids involves capsid formation,

DNA encapsidation and packaging. Capsid assembly occurs exclusively in the nuclei

of infected cells (Darlington and Moss, 1968; Schwartz and Roizman, 1969). Genetic

and biochemical studies suggest that the HSV-1 capsids are made up of seven

polypeptides, which are encoded from six viral genes. VP5, VP19c, VP22a, VP23,

VP24, and VP26 are the products of the UL19, UL38, UL26.5, UL18, UL26, and

UL35, respectively. Cell and virus-free assembly assays have demonstrated that only

six of the capsid proteins, namely, VP5, VP19c, VP21, VP22a, VP23, and VP24, are

required for proper icosahedral capsid assembly, suggesting that no additional viral

genes are required (Newcomb et al., 1994; Thomsen et al., 1995; Thomsen et al., 1994).

Recently, capsid assembly in the nucleus was reported to occur in distinct "dense

nuclear structures" designated as "assemblons" which are distinct from the viral DNA

replication centers. Immuno-confocal microscopy revealed that assemblons localized to

the periphery of the nucleus and contained the capsid proteins and the newly

discovered capsid assembly accessory protein, the UL43.5 gene product (Ward et al.,

1996). The steps which precede capsid assembly are not fully understood, however,

chemical-cross linking studies reveal that protein-protein interactions play a critical

role in this assembly (Hong et al., 1996; Thomsen et al., 1995). Capsid assembly is

considered to begin when the capsid proteins are transported to the nucleus. The

efficient localization of VP5 to the nucleus depends on its interaction with VP22a, the

scaffolding protein specified by the UL26.5 (Matusick-Kumar et al., 1994; Nicholson et


23

al., 1994). After transport to the nucleus, the VP5/VP22a complexes come together by

virtue of the ability of VP22a to interact with itself, forming a structure to which the

other capsid proteins attach to form a large cored "B" capsid (Thomsen et al., 1995).

Using three-dimensional cryoelectron microscopic reconstruction, Newcomb et al.,

1996, showed that capsid assembly begins with a partial capsid and proceeds through

a closed, spherical, procapsid intermediate to the closed, stable icosahedral form seen

in the mature "C" capsid. Three types of capsids "A", "B", and "C" capsids are

produced in HSV-1 infected cells (described in virion structure), which differ from each

other in their capsid protein composition and their ability to package DNA Studies

with ts mutants have identified eight HSV-1 genes UL6, UL15, UL25, UL28, UL32,

UL33, UL36 and UL37 whose products are the non-capsid, accessory proteins

required for the assembly of the DNA containing "C" capsids (Roizman and Sears,

1996). These gene products have been implicated in the various processes involved in

the cleavage and packaging of viral DNA.

Concurrent or following viral DNA replication large DNA concatemers are

synthesized and cut into unit length virion DNA at specific recognition sequences, and

packaged into the preassembled "B" capsids, with the concomitant loss of the

scaffolding protein, VP22a. Cleavage and packaging are tightly linked processes

(Ladin et al., 1980; Sherman et al., 1992). The inverted repeats, ab and a'b' present at

the terminals of the UL and the inverted repeats a'c7 and ca of the Us segments of the

viral genome have a variable number of "a" sequences repeats. These "a" sequences

contain two highly conserved sequence elements termed pacl and pac 2 that are the

signals that direct site-specific cleavage for encapsidation of the progeny viral

genomes. Furthermore, the "a" sequences are flanked by direct repeats of a twenty

nucleotide sequence termed direct repeat 1 (DR1) and is composed of two unique

sequences the Uband Uc that are separated by internal repeated arrays that vary in

copy number and sequence between the HSV-1 isolates (Davison and Wilkie, 1981;


24

Mocarski and Roizman, 1981; Varmuza and Smiley, 1985). The "a" sequences serves

as the recognition sequence for the machinery that processes the concatemers to unit

length DNA. The cleavage-packaging reaction involves two site-specific breaks that are

made on either side of the "a" sequences, at defined distances from the signals located

in the Ub and Ueregions (Varmuza and Smiley, 1985). Two models for viral

encapsidation were put forward by (Deiss et al., 1986), from the analysis of amplicons.

In the first model, the cleavage-packaging proteins attaches to the Ue sequences on the

concatemeric DNA, initiates the first break and scans the viral genome across the UL

towards the Us component until it reaches the Ub sequence and the second cleavage

occurs. Alternatively, the cleavage-packaging complex attaches to the Ue regions of the

"a" sequences scans the genome till it encounters the next Ue-Ub- At this point there is

a duplication of the "a" sequences by a process of gene conversion, following which the

final cleavage occurs in the DR1 of two adjacent "a" sequences (Roizman and Sears,

1996).

Maturation of Virion Particles: The final step in the replicative cycle of HSV-1

is the process of virus maturation, or budding, or egress whereby the newly assembled

capsids acquire the tegument, the bilayer lipid envelope and are transported to the

extracellular spaces. The production of infectious particles is critically dependent on

two steps: first, capsid proteins must selectively encapsidate the viral genome from a

pool of other viral and cellular nucleic adds, and second the capsid must be enveloped

by a membrane containing the viral envelope proteins. Defects in certain tegument

proteins like the UL48 gene product, the a-TIF and the UL11 gene product (encodes a

myristilated tegument protein located on the underside of the viral envelope) also

inhibit viral egress (Ace et al., 1988; Baines and Roizman, 1992; Weinheimer et ai,

1992). Another, tegument protein, ICP34.5, has also been implicated in viral egress, as

null mutants lacking this protein are retained in the perinudear nudear space of

infected cells. In addition, the ICP34.5-null mutants in strain HSV-1 (17) replicate as


25

well as the parental wild-type HSV-1 (17) virus in actively dividing cells, whereas in

stationary phase cells the mutant has a defect in viral maturation (Brown et al., 1994).

Deletion mutants in other tegument proteins such as, the products of the UL41, UL47,

US9 genes do not severely affect viral maturation (Fenwick and Everett, 1990; Post

and Roizman, 1981; Zhang et a i, 1991).

The envelopment of HSV occurs as the nudeocapsid transits through the

nudeus acquiring the envelope from the inner nudear membrane (Nii et al., 1968;

Roizman and Furlong, 1974). The exact mechanism by which the assembled capsid

triggers the acquisition of the tegument and envelope is unknown; however, budding

has been observed at specialized patches at the inner lamella of the nudear membrane

(Roizman and Furlong, 1974). The path the virions follow thereof, as they traverse the

cytoplasm to the extracellular space is poorly understood. Based on the immuno-

electron microscopic examination of static thin sections of virus infected cells,

immunofluorescence, subcellular fractionation and by studying the genetic and

biochemical aspects of viral glycoprotein transport, two models of viral egress have

been proposed (Figure 1.4).

The first and more widely accepted model (Figure 1.4, model B) suggests that

virions use the cellular secretory pathway being transported within vacuoles or

vesides, derived from the outer lamella of the nudear membrane or the endoplasmic

reticulum (ER), which then fuse sequentially with the Golgi dstemae or with Golgi

derived membranes carrying Golgi enzymes, eventually reaching the plasma membrane

where they are released. The model comes from observations of infectious, enveloped,

extracellular HSV-1 virions with glycoproteins on their surface which have complex N-

or Olinked oligosaccharides indicative of Golgi processing. However, the glycoproteins

found on the surface of the nudear membranes are of the immature forms.


26

Virion Egress

VESICLES ©CYTOPLASM

%*9®?®® . ®®®|capsids

PM

NUCLEUS

Figure 1.4: Diagram matic Representation of V irion Egress.

Cross-section of a eukaryotic cell depicting the two models (A and B) of virion maturation and egress. Events are explained in the text. NM=nuclear membrane, PM=plasma membrane, ER=endoplasmic reticulum.


27

Little is known about whether these mature glycoproteins are added at the time

of initial envelopment and undergo subsequent Golgi level processing during virus

egress or how cytoplasmic enveloped virus transits through the plasma membrane.

Evidence to support the model comes from studies where monensin, an ionophore, that

hinders full processing of glycans, reduces the egress of HSV-1 (Johnson and Spear,

1982). Also, ammonium chloride inhibits the accumulation of mature glycoproteins

although, the synthesis of precursor glycoproteins were unaffected (Kousoulas et al.,

1982). Furthermore, high yields of infectious virions accumulate in the cytoplasm of cell

lines defective in the processing of high mannose to mature glycoproteins (Banfield and

Tufaro, 1990; Campadelli-Fiume et al., 1982). The study of various mutant HSV-1

viruses with mutations or deletions in many viral specified glycoproteins and

membrane proteins revealed 1) Temperature-sensitive(fs) gH mutants accumulate in the

cytoplasm of infected cells, however, virions that do get released to the extracellular

space, lack gH and are thus non-infectious (Desai et al., 1988). 2) Viruses with mutant

gD successfully infect cells that express wild-type gD , however, there was an

accumulation of unenveloped capsids in the cytoplasm, suggesting that in these cells

the virion envelope fused with the cytoplasmic membranes (Campadelli-Fiume et al,

1991). 3) Mutant viruses with deletions in the membrane protein specified by the UL20

ORF accumulate in the perinuclear spaces of infected cells, such as Vero and HEp-2

cells, however, in human 143 TK- cells this defect was compensated (Baines et al.,

1991). Interestingly, Vero and HEp-2 cells upon infection with wild-type virus results

in the fragmentation and dispersal of the Golgi apparatus, without any detrimental

effect on the production of infectious viral particles (Campadelli et al., 1993). Thus, the

UL20 protein was shown to be essential for viral egress in these cells (Avitabile et al.,

1994). 4) HSV-1 mutants lacking glycoproteins E (gE), or glycoprotein I (gl), form small

plaques in human fibroblasts and these glycoproteins in wild-type infections have been


28

implicated to play a pivotal role in the basolateral cell-to-cell spread of the virus

(Balan et al., 1994; Dingwell et al., 1994).

The second model (Figure 1.4, model A) originally proposed by Stackpole,

(1969), suggested a pathway where the virus gets enveloped at the inner nuclear

lamella, traverses the perinuclear space and the envelope fuses with the outer nuclear

lamella, releasing the nudeocapsid into the cytoplasm (Figure 1.4). The process is

followed by a second re-envelopment at the endoplasmic reticulum (ER) and/or the

Golgi and the final release into the extracellular space by the fusion of enveloped virion

carrying vesides with the plasma membrane. The major lines of evidence to support

this model show: 1) The examination of confocal and electron micrographs of axonal

transport of virions in the neurons revealed the presence of nudeocapsids in the

cytoplasm (Penfold et al., 1994). The authors implicated that the glycoproteins and

unenveloped nudeocapsids are transported through separate pathways, the

glycoproteins using the neuritic transport vesides while the nudeocapsids utilized the

axonal anterograde micro tubular transport, respectively (Penfold et al., 1994). 2)

Studies with recombinant gH viruses with specific endoplasmic reticulum retention

signals (KKXX) in gH, localized gH and gL to the inner nudear membranes of infected

cells, while the transport of all other glycoproteins were not affected. Also, the

infectivity of the recombinant virus was reduced by 100-fold when compared to the

wild-type virus, however, the number of virion partides present in the extracellular

fluids were similar to that of wild-type (Browne et al., 1996). The consequence of

retaining gH in the ER showed that the progeny virus acquired their envelopes at

another subcellular compartment (Le. at the Golgi or a post-Golgi compartment),

because the secreted virions did not contain detectable levels of gH and were non-

infectious. By targeting gH to the Golgi compartments, the virion infectivity was

restored suggesting that the excreted virions incorporated gH (Browne et al., 1996). 3)

Previous reports of maturation and egress of virus in other alphaherpesviruses


29

systems, like pseudorabies virus (PrV) and varicella zoster virus (VZV), indicate that

these viruses acquire their final envelope from the frans-Golgi network and their

envelope glycoproteins are transported to the fnzns-Golgj network independently of the

nudeocapsids (Zhu et al., 1995).

Regardless of which pathway the nudeocapsids take as they traverse the

cytoplasm, it is certain that intracellular virion particle transport is regulated by a

number of different proteins and other factors of cellular and viral origins.

HSV-1 Specified Glycoproteins and Membrane Proteins

The HSV-1 genome specifies at least 17 putative membrane proteins that are

either integral membrane proteins or proteins associated with cellular membranes.

These are the ULl-gL, ULlO-gM, UL11, UL20, UL22-gH, UL24, UL27-gB, UL34,

UL43, UL44-gC, UL53-gK, US4-gG, US5-gJ, US6-gD, US7-gI, US8-gE (Spear, 1993;

Roizman and Sears, 1996). Presently, eleven of these proteins have been identified as

glycoproteins which are encoded by ORFs in the UL (ULl-gL, ULlO-gM, UL22-gH,

UL27-gB, UL44-gC and UL53-gK) and Us (US4-gG, US5-gJ, US6-gD, US7-gI, US8-gE)

segments of the viral genome (Figure 1.1). Initially, the major glycoproteins were

designated according to their mobility on SDS-polyacrylamide gels (PAGE) as VP8,

VP7, VP8.5, VP18-19 (Heine et al., 1974; Spear and Roizman, 1972) however, they

were renamed as gB, gC, gD and gE based on their antigenic differences from studies

with polyvalent sera (Baucke and Spear, 1979; Spear, 1976). Most of the glycoproteins

belong to either the ^ or the y kinetic class of proteins and are regulated as such. The

glycoproteins, gB, gC, gD, gE, gG, gH, gL gj, and gK, share some common characteristics

in that they all have 1) a deavable signal sequence at the amino-terminal of the protein;

2) possess membrane spanning hydrophobic domains near their carboxy-termini; and

3) they all contain sites for N-linked glycosylation.


30

Some of these glycoproteins, gC, gE, gG, gl, gj and gM are nonessential and are

dispensable for virus infection and replication in most cells in culture as mutant viruses

lacking these glycoproteins showed no discernible phenotypic abnormalities when

compared to the wild-type strains (Balan et al., 1994). gC encoded by UL44, though

not essential for virus production in cell culture (Ryechan et al., 1979), mediates the

initial attachment of HSV-1 virions to the cells by binding to the heparan sulphate

moieties on the cell surface (Fries et al., 1986; Herold et al., 1991; Langeland and

Moore, 1990). gC serves multiple accessory functions including its ability to function as

a receptor for the complement component, C3b (Friedman et al., 1984). gC is also a

major viral antigen which elicits a strong humoral and cellular immune response and

thus protects HSV-1 infected cells from lysis by antibody plus complement; however,

the most important protection is against lysis mediated by complement alone (Fries et

ai, 1986; Harris et al., 1990). Glycoproteins gE and gl, encoded by the US8 and 1)57,

respectively, are involved in the basolateral spread through the junctions of cultured

polarized cells and the complex of gE and gl acts as a receptor for immunoglobulin G

(IgG) (Centifanto-Fitzgerald et al., 1982; Dingwell et al., 1994). The function of gG

specified by the US4 ORF is ill-defined. gG is found in the virion envelope and mutants

lacking this glycoprotein have reduced virulence in experimental animals, but the

mechanism involved is unclear (Balan et al., 1994; Longnecker et al., 1987; Longnecker

and Roizman, 1987; Weber et al., 1987). The US5 ORF was first identified by McGeoch

et al, (1986) who noted the predicted translation product contained a potential N-

terminal signal, a transmembrane domain and a single N-glycosylation site. The gene

product was designated as gj; however, no function has yet been assigned to this

putative protein (Roizman and Sears, 1996). The product of the UL10 ORF is gM, a

glycoprotein present in the virions and found associated with the plasma membranes

of infected cells. The protein has the characteristics of a glycoprotein containing the

required N-glycosylation sites and the transmembrane regions; however, the protein


31

lacks a deavable signal sequence (Baines and Roizman, 1993). The dispensable viral

structural components, gC, gE, gl, gG, gj and gM play important roles in vivo in

infection of diverse cell types, movement of virus through tissue, or protection from

host immune responses (Balan et al.f 1994; Roizman and Sears, 1996).

gB, gD, gH, gK and gL are glycoproteins essential for virion infectivity. gB, gD

and gH-gL are required for viral attachment and entry (Cai et al., 1988a; Cai et al.,

1988b; Campadelli-Fiume et al., 1990; Forrester et al, 1992; Hutchinson and Johnson,

1995; Ligas and Johnson, 1988; Roizman and Sears, 1996; Roop et al., 1993; Spear,

1993). The UL27 ORF codes for gB which is one of the most abundantly expressed

glycoprotein component of the virion envelope. The fully glycosylated form of gB is a

homodimer and is one of the most highly conserved glycoproteins among the different

members of the herpesvirus family (Pereira, 1994). Analysis of ts mutants, antibody

resistant mutants and gB null mutants demonstrated that gB is essential for virus

penetration (Cai et al., 1987; Cai et al., 1988b; DeLuca et al., 1982; Highlander et al.,

1989; Little et al., 1981; Sarmiento and Spear, 1979) and certain mutations in gB

compromise the intercellular spread of the virus. Furthermore, point mutations in the

carboxy terminal of gB cause extensive syncytia in cell culture (Bzik et al., 1984a; Bzik

et al., 1984b; Cai et al., 1988b). gD encoded by US6 ORF is involved with interaction

with cellular receptors and is essential for viral entry (Ligas and Johnson, 1988), and

has been implicated in cell fusion (Spear, 1993), superinfection restriction (Johnson and

Spear, 1989), and neuroinvasiveness (Izumi and Stevens, 1990). Purified gD elicits a

neutralizing antibody response and a protective immune response to lethal challenge

with virus. This property of gD has lead to development of human subunit vaccines

(Langenberg et al., 1995). The UL44 ORF encodes the glycoprotein H (gH) that can be

detected in HSV-1 infected cells and in purified virions (Buckmaster et al., 1984) and

like gB is ubiquitous among herpesviruses (McGeoch et al., 1986). gH is essential for

viral penetration into cells at a step subsequent to viral attachment (Fuller et al., 1989).


32

Mutant viruses devoid of gH are noninfectious, and cells pre-adsorbed with gH-null

virions are resistant to superinfection with wild-type virus, indicating that gH is

required for membrane fusion but not for receptor binding (Forrester et d ., 1992).

Monoclonal antibodies to gH neutralize virus infectivity and inhibit cell fusion by

syncytial viruses (Buckmaster et d ., 1984; Gompels and Minson, 1986). Furthermore,

modifications in the cytoplasmic tail of gH abolishes fusion caused by syncytial strains

(Wilson et al., 1994). Also, gH has been implicated in viral egress as certain ts mutants

with mutations in gH accumulate in the cytoplasm of infected cells, however, virions

that are excreted to the extracellular space are devoid of gH and are thus non

infectious (Desai et al., 1988). Transient expression of gH in COS cells reveal the

intracellular retention, and the presence of precursor carbohydrates, suggesting that gH

may interact with another protein during maturation (Roberts et d ., 1991). A new

glycoprotein L (gL) specified by UL1 has been characterized using antipeptide sera

(Hutchinson et d ., 1992a). gL when expressed, behaves similar to gH, in that the

protein is not properly processed and expressed in the absence of other HSV-1

specified proteins. However, co-expression of both gL and gH enabled both proteins to

be processed and transported to the cell surface, indicating direct interaction of these

proteins in a gH-gL complex that is present in the virion envelope and the plasma

membrane of infected cells, and enables the virus in entry (Hutchinson et al., 1992a).

(Glycoprotein K encoded by the UL53 ORF will be reviewed later). Most of the

glycoproteins of HSV-1 play important roles in pathogenicity, mediating both entry of

the virion into host cells, via attachment and penetration and cell-to cell spread of

virus.

Based on sequence data, the other predicted intrinsic membrane proteins

specified by the HSV-1 genome are gene products of the UL20, UL24, UL34, UL43

and UL45 ORFs (McGeoch et al., 1988; Spear, 1993). The UL20 encodes a membrane

protein, associated with purified virions, the nuclear membranes and with the


33

cytoplasm of infected cells (Baines et al., 1991; Ward et al., 1994). The UL20 gene

product plays a vital role in translocation of the enveloped virions from the perinuclear

space to the cytoplasm in Vero and HEp-2 cells. However, deletion of this gene from

HSV-1 causes the mutant virus to cause cell fusion in certain other cell lines such as

143TK cells (Baines et al., 1991). Little is known about the function of the UL24 and

UL43 gene products except that they are dispensable for viral replication in some cells

in culture (MacLean et al., 1991). Mutations and deletions in UL24 give rise to the

syncytial phenotype, in which massive cell fusion was observed (Jacobson et al., 1989).

The UL34 gene product is a membrane phosphoprotein essential for viral replication in

cell culture as numerous attempts to delete it have been unsuccessful and is

phosphorylated by the HSV-1 protein kinase specified by the US3 ORF (Purves et al.,

1992; Roizman and Sears, 1996). The UL43 ORF gene product has not yet been

defined, but immunofluorescence studies have associated this protein with the Golgi

(Roizman and Sears, 1996). Interestingly, the UL43.5 ORF gene product which is

antisense to UL43 has been defined as a 38 kDa y protein that colocalizes with capsid

proteins in the nucleus of infected cells and functions as an accessory protein in capsid

assembly (Ward et ai, 1996). Visalli and Brandt (1993), characterized the UL45 gene

product as a membrane protein that functions to facilitate cell to cell fusion in HSV

infected cells as frameshift mutations in this gene result in a loss of immunodetectable

UL45 and the suppression of cell fusion caused by the syn3 mutation in gB (Haanes et

al., 1994).

Glycoprotein Trafficking

Previously mentioned studies of HSV envelopment and egress need to be

considered in the context of the intracellular macromolecular transport mechanisms.

HSV-1 like other animal viruses utilizes the host cell glycosylation machinery to

synthesize and process oligosaccharides attached to viral glycoproteins. The first steps


34

in the eukaryotic secretory pathway is the targeting and translocation of nascent

protein precursors across the ER membrane and to the Golgi complex where post-

translational modifications occur. The processed glycoproteins are stored in

specialized secretory vesicles which then fuse with the cell surface during exocytosis

(Palade, 1975). The glycoproteins are synthesized as non-glycosylated precursors on

the rough ER (RER) and their N-terminal signal sequences target them to the lumen of

the RER, where oligosaccharide chains are added to the precursor proteins by

oligosaccharide transferases. The oligosaccharides are added en bloc from dolichol lipid

precursors in the lumen of the ER. There are two kinds of glycosylation in eukaryotic

cells, the N-linked glycosylation, where the sugars are linked via a N-glycosidic bond to

the Asn residue in the sequence Asn-X-Ser/Thr where X is any amino acid other than

Pro. The second kind of glycosylation occurs via a O-glycosidic bond to the hydroxyl

groups present in Ser and Thr residues (Hubbard and Ivatt, 1981; Rothman, 1994). N-

linked glycosylation is one of the most common post-translational modifications of

viral glycoproteins. The best studied model to date is the G protein of vesicular

stomatitis virus (VSV) (Rothman, 1994). Several approaches have been used to define

the functional roles of N-linked carbohydrate addition to glycoproteins. These

approaches include the use of agents that interfere with glycosylation, elimination of

each N-linked glycosylation site by site-directed mutagenesis, and the use of glycan-

processing deficient cell lines (Rothman, 1994). The oligosaccharides on the

glycoproteins play a significant role in initiation and maintenance of folding into

biologically active conformations, protecting polypeptides from proteolytic attack and

influencing the antigenicity and immunogenidty of the glycoproteins. Although the

functional contribution of the carbohydrates varies with the glycoprotein in question, it

has been generally accepted that glycosylation is necessary to aid proteins in attaining

and maintaining correct tertiary structures and in undergoing intracellular transport

(Kiely et al., 1976). The glycoprotein transport continues in electron dense vesicles


35

budding from the RER which rapidly fuse with the ds-Golgi dstemae via a vesicle

targeting mechanism (Rothman, 1994). This vesicle shuttle allows transport between a

pair of membrane-bound compartments. One member of the pair, the donor

compartment, produces the transport vesicle and the other, the acceptor compartment,

receives the vesicle with the immature glycoprotein. The Golgi complexes are the sites

for sequential removal and addition of oligosaccharide residues that results in a final

high mannose glycoprotein (Hubbard and Ivatt, 1981; Spear, 1976). Transport of these

vesicles continues through the Golgi dstemae and eventually, the vesides bud from the

frans-Golgi network, reach the plasma membrane and fuse with i t Brefeldin A, a fungal

metabolite that causes the retrograde movement of transport vesides from the Golgi to

the ER resulting in the dissolution of the Golgi stacks, also inhibits HSV-1 egress from

the cells (Cheung et al., 1991). Also, HSV-1 virions accumulate in gro29 cells, a cell-line

defective in Golgi processing and protein transport (Banfield and Tufaro, 1990; Tufaro

et al., 1987). Accumulation of virions in the cytoplasm was also observed in HSV-1

infected cells treated with monesin, an ionophore, that interferes with the processing

and transport of N-linked and O-linked HSV-1 glycoproteins (Johnson and Spear,

1982). Furthermore, wild-type HSV-1 infection of certain cell lines, such as Vero and

HEp-2, results in fragmentation and dispersal of the Golgi in the cytoplasm.

(Campadelli et al., 1993). Immunofluorescence studies against specific Golgi enzyme

markers revealed that these cytopathological changes occur late in HSV-1 infection and

may be caused by the overflow of viral glycoproteins as they traffic through the Golgi

complex (Campadelli et al., 1993). The UL20 gene product was required for egress of

infectious virions from Golgi fragmented cells (Avitabile et a i, 1994). Recently, genetic

approaches were used to eluddate the mechanism by which viral glycoproteins and

virions are trafficked through the celL Recombinant HSV-1 viruses with ER targeting

signals for gH were constructed, and it was shown that gH along with gL were not

processed and virions that were released from recombinant virus infected cells were


36

devoid of gH and were noninfections (Browne et al., 1996). HSV-1 exploits the host cell

secretory machinery, however, it is unclear at the present time whether the mechanisms

for transport of viral proteins is s im i l a r to that utilized for the transport of infectious

virions.

Virus - Induced Cell Fusion

Two important aspects of HSV-1 infection of cells in culture are viral entry and

viral spread. Both these processes involve distinct fusion events between the virus and

cellular membranes that occur dining viral entry and cell to cell fusion during viral

spread. A combination of virus-encoded membrane proteins and host cellular factors

contribute to both kinds of fusion events. Viral entry was reviewed in viral replicative

cycle. This section will focus on virus mediated cell to cell fusion which occurs dining

the transfer of virus from an infected to an uninfected neighboring cell as evidenced by

the formation of plaques in the presence of neutralizing antibody, hi cell culture, wild-

type HSV-1 infections cause limited amounts of fusion, but, mutant viruses have been

isolated both in cell culture and in natural infections that cause extensive cell fusion

resulting in giant, multinucleated cells or syncytia (Brown et al., 1973; Hoggan and

Roizman, 1959; Person et a i, 1976; Spear, 1993). The mutations causing the syncytial

phenotype (syn) have been mapped to at least 5 distinct loci of the HSV-1 genome

(Figure 1.1). Using intertypic HSV-1 x HSV-2 mutants, Ryechan et al., (1979) were the

first to identify two major syn lod, the syn 1 and syn 3 loci, which were later shown to

be located in the genes coding for gK and gB, respectively (Bond and Person, 1984;

Bzik et al., 1984b; Debroy et al., 1985; Hutchinson et al., 1992b; Manservigi et al., 1977;

Pogue-Geile et al, 1984; Ryechan et al., 1979). The syn 2 domain was originally

mapped to the UL1 ORF which encodes gL (Little et ai, 1981), however, the mutation

has since been remapped to the gK gene (Roop et al., 1993). The syn 4 locus was

mapped to the UL20 ORF a gene that encodes a membrane protein, involved in virus


37

egress in some cell types (Baines et al., 1991). Cells infected with mutant UL20-null

viruses form syncytia suggesting that this protein modulates fusion activity in infected

cells (Baines et al., 1991). The syn 5 locus affects the UL24 ORF, but its gene product

has not been characterized (Jacobson et al., 1989; Tognon et al., 1991). Finally, the

terminal repeats flanking the UL regions of the viral genome contains the syn 6 locus,

nestled between the genes encoding the ICPO and y 34.5 genes (Romanelli et al., 1991)

The best characterized syn loci are the syn 3 and syn 1 loci present in gB and

gK respectively. The syn 3 mutation affects the carboxy-terminal of gB and is the result

of an Arg to His change at amino ad d 875 in the (published) sequence of HSV-1 (KOS)

(Bzik et a l, 1984b; DeLuca et al., 1982; Kousoulas et al., 1982). In addition, certain

other point mutations between the amino add residues of 816 and 817 also cause the

syncytial phenotype (Bzik et al., 1984b). Studies using mutagenesis and truncations of

gB suggest that the carboxy-terminal 28 amino adds of gB are not essential for the

virus, although truncations cause extensive cell fusion (Baghian et al., 1993). To date

the majority of the syncytial mutants that have been characterized and mapped have

mutations in the synl locus present in the UL53 ORF encoding gK (Pogue-Geile et al.,

1984). These non-lethal amino add changes in gK are concentrated in two domains,

one at the amino-terminal and the other at the carboxy-terminal of the protein (Figure

4.1) (Dolter et al., 1994). In addition to the above mentioned proteins, the possibility

that other viral membrane proteins are indirectly involved in the fusion process emerges

from the observations that monoclonal antibody to at least three glycoproteins, gD

(Fuller and Spear, 1985), gH (Gompels and Minson, 1986) and gE, were found to block

fusion of the plasma membranes of infected cells. Furthermore, gE, gl, and gM and the

UL45 ORF, though dispensable for viral entry, are required for effident syncytium

formation by certain syncytial strains that contain the syn3 locus (Davis-Poynter et al.,

1994; Haanes et al., 1994; Visalli and Brandt, 1991). Interestingly, many gC negative

mutants are syncytial and conversely, many syncytial mutants have secondary


38

mutations which inactivate gC (Bond and Person, 1984; Heine et al., 1974; Zezulak and

Spear, 1984) suggesting that gC modulates cell fusion, the exact mechanism of which is

unknown at the present time. Thus, Roizman, (1968) put forward a hypothesis that

postulated the existence of a complex of proteins involved in cell fusion, and

mutations or deletions of components of this complex could destabilize the complex

resulting in extensive cell fusion.

HSV-1 Glycoprotein K (gK)

This section details a review of the experimental work to date on glycoprotein K.

gK is the product of the UL53 ORF (Figure 1.5), the ninth HSV-1 glycoprotein to be

described (Hutchinson et al, 1992b). The UL53 ORF nucleotide sequence was first

described by Debroy et al. (1985) as the gene that encodes a very hydrophobic protein

involved in virus induced cell fusion (Figure 1.6). Analysis of the UL53 coding

sequences reveals a G+C content of 61% compared to 67% for the total genome (Keiff

et a l, 1971). In vitro transcription and translation studies revealed that the 338 amino

add protein encoded by UL53 has characteristics of an integral membrane

glycoprotein, (as depicted in Figure 1. 5) in that it has 1) signal sequence at the N-

terminus of the protein that is deaved between the Gly and Ala residues at amino

adds 30 and 31; 2) three potential N-linked glycosylation sites present in the amino

one-third of the protein, and 3) four hydrophobic domains, possibly functioning as

transmembrane regions to anchor the protein (Ramaswamy and Holland, 1992). In vivo

and in vitro studies of gK report that the processed UL53 gene product has a molecular

weight ranging 36 kDa to 40 kDa. There are conflicting reports regarding the kinetic

class (immediate-early, early, or late) to which gK belongs. Debroy et al., (1985)

reported UL53 mRNA was not detected in infected cells in the presence of

phosphonoacetic add (PAA), a viral DNA synthesis inhibitor, indicating the UL53

gene product to be a y protein stringently dependent upon viral DNA synthesis.


Reproduced

with perm

ission of the

copyright ow

ner. Further


without

permission.

SYN MUTATION-j SIGNAL SEQUENCE |

40* * * * *M L A V R S L Q H L S T V V L I T A Y G L V L V W Y T V F G A S P L H R C I Y A

N-CLYCOSYLATION N-GLYCOSYLATION* I * I * * *V R P T G T N N D T A L V W M K M N Q T L L F L G A P T H P P N G G W R N H A H

* * * * *I C Y A N L I A G R V V P F Q V P P D A T N R R I M N V H E A V N C L E T L W Y

T R V R L V V V G W F L Y L A F V A L H Q R R C M F G V V S P A H K M V A P A T HYDROPHOBIC DOMAIN 1

* * * * * Y L L N Y A G R I V S S V F L Q Y P Y T K I T R L L C E L S V Q R Q N L V Q L F

E T D P V T F L Y H R P A I G V I V G C E L I V R F V A V G L I V G T A F I S RHYDROPHOBIC DOMAIN 2

* * * * *G A C A I T Y P L F L T I T T W C F V S T I G L T E L Y C I L R R G P A P K N A

HYDROPHOBIC DOMAIN 3 .* * * * *

D K A A A P G R S K G L S G V C G R C C S I I L S G I A M R L C Y I A V V A G VHYDROPHOBIC DOMAIN 4

* * *V L V A L H Y E Q E I Q R R L F D V— ' 338Figure 1. 5: Predicted Primary Sequence of Glycoprotein K.

The protein sequence is shown with its signal peptide, the syn mutation at amino acid 40,the two N-linked glycosylation sites and the hydrophobic domains are underlined.

sO

40

■s.x

Hydrophiiidty Window Size Scale - Kyte-Doolittle

2 .00

^ CF Helix •5 CF Sheet = CF Turns ~ RG Helix' ” RG Sheet »- RG Turns •S CfRg Hlx' 0 CfRg Sht « CfRg T m vt

Figure L 6: Hydrophilic Profile, Antigenic Index and Predicted Secondary Structure of Glycoprotein K.

Analysis was performed using the Protein Toolbox application of the MacVector software (IBI-Kodak). CF = Chou-Fasman secondary structure prediction;RG = Robson-Gamier secondary structure prediction.


97

41

However, Hutchinson et al., (1992b) reported that gK was not sensitive to PAA

treatment and was regulated most likely as an early (a or {Dprotein. gK being a

hydrophobic protein has been difficult to detect in cell culture and in vitro translated

gK aggregates on heating (Ramaswamy and Holland, 1992). Immunopredpitations and

immunofluorescence studies with antibodies generated to synthetic peptides derived

from the antigenic portions of the primary gK sequence, detected low levels of gK in

infected cells. Furthermore, immunofluorescence microscopy of HSV-1 infected cells

and gK expressed from a recombinant adenovirus vector, localized gK to the

perinuclear, nuclear membranes and ER, but gK could not be detected in the plasma

membrane of infected cells. Also, gK was not detected in purified virion particles.

Interestingly, gK expressed in the baculovirus system had an apparent molecular

weight similar to that expressed in infected cells and was detected both in the nuclear

and plasma membranes of infected insect cells (Ghiasi et al., 1994). gK seems to be a

multi-functional protein involved in virus-induced cell fusion as a number of missense

syn mutations that cause the syncytial phenotype map to gK (Bond and Person, 1984;

Bond et al., 1982; Debroy et al., 1985; Dolter et al., 1994; Little and Schaffer, 1981;

Pogue-Geile and Spear, 1987; Read et al., 1980). Moreover, gK may be functioning in

viral entry as indirect evidence shows that gC negative viruses with the synl mutation

in gK are resistant to the inhibitory effects of heparin, infect gD expressing cell lines,

and thus they overcome gD-mediated interference (Pertel and Spear, 1996). These gD

expressing cell lines are normally resistant to wild-type HSV-1 infections and the block

appears to be at the level of penetration as the virus binds normally to these cells

(Warner et al., 1996). gK has also been implicated in intracerebral pathogenicity QQ of

HSV-1 as pathogenic strains of HSV-1 have mutations that map to the UL53 ORF

(Moyal et al., 1992). In an effort to determine if gK elicits neutralizing antibody and

cell-mediated immune responses like some of the other HSV-1 specified glycoproteins,


42

ALIGNMENT OF GLYCOPROTEIN K SPECFIED BY SELECTED ALPHAHERPESVIRUSES

HSV-1(KOS) HSV-1 (mP) HSV-2(HG52) BHV-1EHV-1(Ab4p)PRVVZVC o n s e n s u s

..MLaVRsLQ

..MLaVRsLQ

..MLaVRsLQ

.MLLGgRtv.

.MLLGgRta.

.MLLGgRpL. mqaLGIkte. -M-LGVR-LQ

HLStvVLITA HLStvVLITA HLttvIFITA nLaaLaLlTA yLSvLgLITA HLlvLgvmgA HFiiMcLlsg HLS—LVLITA

YgLvLVWYTvYgLvLVWYTvYgLvLaWYibhlaLalWaplYaaFtlWYTlYagLgaYYathavFtlWYTaY-L-LVWYT-

FgAsPLHrci FgAsPLHrci FgAsPLHrci aapLrerc.. taqLhnpcvy varLPhpwy rvkFehecvy F-ALPLH--

50yAVRpTGTNN yAVRpTGTNN yAVRpaGaqN .AlavraTga atVsi.dskd aAlplgedaa attvi....N -AVR-TGTNN

HSV-1(KOS) HSV-1(mP) HSV-2(HG52) BHV-1EHV-1 (Ab4p)PRVVZVC o n s e n s u s

51dtALVWmkmN dtALVWmkmN dtALVWmliN ngsLrWelrd giAakWevYN ggApdWeaFN ggpvVWgsYN — ALVW— YN

QTLlFlGaPTQTLlFlGaPTQTL1F1GPPTpgaVYvwggasTiVYayPenaXalYvaPnensLIYv.tfvQTL-Y-GPPT

hPPN. .gGwr hPPN..gGwr aPPg. .gawtn.... natlgakrFsdGISt.... dalSnhstFldGIS —PPNF— G-S

nHahlCyANLnHahlCyANLpHarVsdANiaadapCRhavgfdyVCReNwP ........gydysCReNL -H CRANL

100IAGRWP Fqv IAGRWPFqv IeGRaVsLpa VqhippgLld Vneskldvlk

lsGdtmvkta I AGRWP--

HSV-1(KOS) HSV-1(mP) HSV-2(HG52) BHV-1EHV-1(Ab4p)PRVVZVC o n s e n s u s

101PPDAtnrRIm PPDAMnrRIm hPaAMsrRVra gdEALHgRVR nmkeLHdkVR . ..ALrdRaR istpLHdkIR PPDALH—R-R

nVHeAvNClenVHeAvNClenVHeAbNCleaVagARdCrAiVvgtRNCrAvVyarRdCrAiVlgtRNChA-VH-ARNC-A

tLWYtrvRLVtLWYtrvRLVaLWdtQMRLVYLWsaQaRsaYLWsvQLqMIYLWdvhFRLaYFWCvQLkMIYLWY-QLRLV

WgWFLYLAFWgWFLYLAFWgWFLYLAFllAWLLYvAFtgAWLiYiAFaVAWLLYaAFffAWFvYgmYWAWFLYLAF

150VaLhQrRcMF VaLhQrRcMF VaLhQrRcMF VyLRQeRRMF lcLRQeRRLL VyaRQeRRMF lqFRriRRMF V-LRQ—RRMF

Figure 1.7: contd.

Figure 1. 7: Alignment of Protein Sequences of Glycoprotein K Specified By Selected Members of the Subfamily Alphaherpesvirinae.

Sequences were aligned using the University of Wisconsin Genetic Computer Group software package. Dots indicate gaps, upper case letters represent identical amino adds to the consensus sequence and lower case letters represents mismatch amino acids. HSV-1 = Herpes simplex virus type 1, HSV-2= Herpes simplex virus type 2, BHV-1= Bovine herpes virus type 1, EHV-1 = Equine herpesvirus type 1,PRV= Pseudorabies virus, VZV= Varicella zoster virus. Strain names are represented in parenthesis.


43


151GVvsPAHkMVGVvsPAHkMVGVvsPAHsMVGIcRndaDFlGpfRnqnEFlGpfRdpaEFlGpfRsscELIGV-RPAHE-V

aPATYlLNYAaPATYlLNYAaPATYlLNYAsPggYtLNYAsPtgYtFNYAtPekYtLNYA3Pt3YsLNYv-PATY-LNYA

GRIVSSVFLQGRIVSSVFLQGRIVSSbFLQaaalaaVvghtytlattvLkasVlaatvigtRVISnlLLgGRIVSSVFLQ

YPYTKitRLL YPYTKitRLL YPYTKitRLL gPYTKLARLM thYTKFAlLL C3YTKFAWyM YPYTKLARLL YPYTK—ARLL

200CELSVqRQnLCELSVqRQnLCELSVqRQtLCELSaRRrALCEaSlRRvALaELatRRaALCDvSmRRdgMCELSVRRQAL


201vQLFETDPVTvQLFETDPVTvQLFETDPVTavnFrlDPlgsrtFkrDPIgsrdLreDPITskvFnaDPIs-QLFETDP-T

FLYHRPAIGVFLYHRPAIGVFLYHRPAIGVcaWrRPglllFLCehsAalaLahrhPtliaFLYmhkgVtlFLYHRPAIGV

iVGcELivRF iVGcELMLRF iVGcELLLRF pllaEgLaRL UGlEvgthF UllELgLRL lmllEviahi —VG—ELLLRF

VAvGLIVGTAVAvGLIVGTAVA1GLIVGTAgARiaaassvVARlLWGTvgARmalftTlssgciVllTlVARGLIVGTA

250fISRGaCAIT fISRGaCAIT HSRGaCAIT glthaPCAaa tlvhtPCsqi gVtRaPCAlv gVaytPCAll -ISRGPCAIT


251YPLFLTITTWYPLFLTITTWhPLFLTITTWYPLYLklwaWYPiYLklasWFPLYaralvWYPtYirllaWYPLYLTITTW

cFVSTIgLTEcFVSTIgLTEcFVSilaLTEvhValfaglEgFVvaVtivEiFVlaVgalEwVcTlaivE-FVSTI-LTE

LycILRRgPA LycILRRgPA LyfILRRgsA LvslLyRkPA ivaliyekPp LlaatlphiA LisyvRpkPt L— ILRR-PA

PKNADkAAAP PKNADkAAAP PKNAEpAApr rrrggsAvAg . .ktgssAnP ..rvsgAtAt kdNhlnhint PKNAD—AAAP

300GR SKG1GR SKG1GR SKGwdggdgGesGI ptp..athGV parsdGgraaG ...... GIGR GSKGI

HSV-1 (KOS) HSV-1(mP) HSV-2(HG52) 3HV-1EHV-1(Ab4p)PRVVZVC o n s e n s u s

301SGVCGRCCSiSGVCGRCCSiSGVCGRCCSirkVCvnCCSTkGlCtsCCSTlGVCGaCCSTrGICttCCaTSGVCGRCCST

ILSGIAmrLcILSGIAVrLcILSGIAVrLclLaGllVKalVLanlcgKLvVLaGIfaKalVMSGlAIKcf-LSGIAVKL—

YIAWAGWLYIAWAGWLYIAWAGWLYlAalvGgViYlllViGaVsYlcllvGgVLYIvIfAiaVvYIAWAGWL

VaLHYEQEIQ VaLHYEQEIQ VaLrYEQEIQ aLLHYEhnlr ILLHYEQrIQ lFLHYEr..h IFMHYEQrVQ V-LHYEQEIQ

350rRLFdv.... rRLFdv.... rRLFdlm. . . lRLLGaqt.. igLLGeSfsSitiFG....vsLFGeSenS qkh -RLFG-S— S --

Figure 1.7


44

gK was expressed in a baculovirus system, and the recombinant viruses were injected

into mice. gK-vacdnated mice were partially protected against lethal HSV-1 challenge,

despite the lack of a significant neutralizing antibody response. The mice vaccinated

with gK and occularly challenged with a stromal-disease-produdng strain of HSV-1

developed significantly more ocular disease than did identically challenged mock-

vaccinated mice (Ghiasi, 1996)

Initial attempts to delete the gK gene were unsuccessful indicating gK was

essential for viral replication (McLean et al., 1990). Recently a partially deleted HSV-1

mutant, F-gK0 was generated in which the ICP6::lacZ cassette was inserted into the

Hpa I site 336 nucleotides from the start of the UL53 ORF, thus allowing for the

expression of the amino-terminal one-third of the gK polypeptide (Hutchinson and

Johnson, 1995). F-gK{5 failed to replicate in Vero cells. The authors, on the basis of

electron microscopic studies, concluded that F-gKf) virions became enveloped by

budding through the inner lamellae of the nuclear membrane, but failed to translocate

from the perinuclear space resulting in the appearance of high numbers of unenveloped

capsids in the cytoplasm of cells. The hypothesis was proposed that in the absence of

gK viral envelopes fused in an unregulated manner with cytoplasmic membranes

resulting in the accumulation of unenveloped virions in the cytoplasm of Vero cells.

Significant homologs of gK have been identified in other herpesviruses including

herpes simplex virus -2 82.8%, (DebRoy, 1990), varicella zoster virus (Davison and

Scott, 1986), equine herpes virus-1 (Zhao et al., 1992), Marek's disease virus, 49.7%

(Ren et al., 1994) infectious laryngo tracheitis virus, 64.8%, (Johnson et al., 1995) and

bovine herpesvirus -1,46%, (Khadr et al., 1996) (Figure 1.7).


CHAPTER n

HERPES SIMPLEX VIRUS TYPE-1 GLYCOPROTEIN K IS NOT ESSENTIAL FOR INFECTIOUS VIRUS PRODUCTION IN ACTIVELY REPLICATING CELLS, BUT IS REQUIRED FOR TRANSLOCATION OF INFECTIOUS VIRIONS FROM THE

CYTOPLASM TO THE EXTRACELLULAR SPACE

INTRODUCTION

Herpes simplex virus entry, nucleocapsid envelopment, egress, and transfer of

infectious virions from infected to uninfected cells involve fusion of cellular and viral

membranes that are thought to be regulated by membrane-associated virus-specified

proteins and glycoproteins (Roizman and Sears, 1996; Spear, 1993). Syncytial

mutations (syn) can arise in at least 4 different regions of the viral genome (Figure 1.1).

Syncytial mutations in UL53(gK) gene are more frequently isolated than syncytial

mutations in any other genes (Bond and Person, 1984; Bond et al., 1982; Debroy et al.,

1985; Dolter et al., 1994; Little and Schaffer, 1981; Pogue-Geile and Spear, 1987; Read

et al., 1980). Studies with anti-gK serum raised against peptides that represented

immunogenic portions of gK detected the glycoprotein in the nuclear and perinuclear

space, but not in the plasma membrane of infected cells. Furthermore, based on studies

with these anti-peptide sera, it is believed that gK is not part of the viral envelope

(Hutchinson et al., 1992b; Hutchinson and Johnson, 1995). Studies with virus mutants

have demonstrated that gD and the gH-gL complex are also required for virus entry

and cell fusion (Cai et al., 1988a; Cai et al., 1988b; Davis-Poynter et al., 1994; Forrester

et al., 1992; Ligas and Johnson, 1988). Moreover, evidence exists that gE, gL gM and the

UL45 protein are required for virus-induced cell fusion, because syncytial mutants in

gB do not cause fusion in the absence of these proteins (Balan et al., 1994; Davis-

Poynter et al., 1994; Haanes et al., 1994). However, others have reported that mutants

that lack gE (Neidhardt et al., 1987) or gl (Dingwell et al., 1994) retain the syncytial

phenotype, implying that the syncytial mutations were not located within the gB gene.

45


46

Finally, cell factors have been shown to influence fusion of infected cells (Bzik et al.,

1984a; Serafini-Cessi et al., 1983; Shieh and Spear, 1994).

HSV-1 Nudeocapsid Envelopment and Virion Egress

Herpesvirus capsids acquire their envelope by budding through the inner lamellae

of the nuclear membrane (Compton and Courtney, 1984; Morgan et al., 1959; Schwartz

and Roizman, 1969). The release of infectious virions from infected cells requires the

transport of infectious virions from the perinuclear to the extracellular space. The

translocation most likely occurs within transport vesicles derived from the outer

lamellae of the nuclear membrane and the contiguous ER (Campadelli-Fiume et al.,

1991; Compton and Courtney, 1984; Darlington and Moss, 1968; Morgan et al., 1959;

Roizman and Sears, 1996; Schwartz and Roizman, 1969). An alternative model has

also been proposed that involves de-envelopment at the outer nuclear membrane

followed by envelopment at the RER (Nii, 1992; Nii et al., 1968; Roizman and Sears,

1996; Stackpole, 1969; Whealy et al., 1991). Regardless of which model of transport is

correct, it is certain that intracellular virion particle transport is regulated by a number

of different proteins and other factors of cellular and viral origins. Specifically, in cells

defective in the processing of high-mannose precursor glycoproteins to mature

glycoproteins, or when Golgi-dependent functions are inhibited by the ionophore

monensin, infectious virions accumulate in the cytoplasm but are not transported into

the extracellular space (Banfield and Tufaro, 1990; Campadelli-Fiume et al., 1990;

Johnson and Spear, 1982) indicating that Golgi-dependent glycoprotein maturation is

vital for virion egress. gH must play an important role in egress of infectious virions,

since ts mutant viruses with mutated gH are retained within cells, while non-infectious

virions that lack gH are released by infected cells (Desai et al., 1988). Furthermore,

targeting gH to the RER with a specific retention motif KKXX results in virions released

by infected cells that are not infectious, while infectious virions that contain gH are


47

retained in the cytoplasm (Browne et al., 1996). HSV-1 virions de-envelope in gD

expressing cells, indicating that gD can interfere with intracellular transport

(Campadelli-Fiume et al., 1991). The UL20 protein seems to be involved in a post

envelopment virion transport step, since UL20-null mutant viruses envelope in the

inner lamellae of the nuclear membranes, but are retained within the perinuclear space

of Vero cells. However, certain other cells are able to complement the UL20-null defect

and allow UL2Q-null virions to overcome this early block in the translocation pathway

indicating that cellular factors are involved (Avitabile et al., 1995; Baines et al., 1991;

McLean et al., 1990; Ward et al., 1994). Finally, gE and gl seem to play an essential role

in the basolateral spread of viruses in polarized cells (Dingwell et al., 1994; Roizman

and Sears, 1993).

To characterize the role of gK in virus replication and membrane fusion, a HSV-1

mutant virus AgK that lacks the entire UL53(gK) gene was constructed and

characterized. AgK virus is genotypically different from the previously reported F-gKp

mutant virus that was generated by insertion of an ICP6::lacZ cassette into the Hpa I

site 336 nucleotides from the start of the UL53 ORF, thus allowing expression of the

amino terminal one-third of the gK polypeptide (Hutchinson and Johnson, 1995). F-

gKP failed to replicate in Vero cells, and on the basis of electron microscopic studies, it

was concluded that F-gKp virions were able to envelope by budding through the inner

lamellae of the nuclear membrane, but failed to efficiently translocate from the

perinuclear space resulting in the appearance of high numbers of unenveloped capsids

in the cytoplasm of cells. By contrast to FgK-p, AgK virus replicated in sub-confluent

Vero and HEp-2 cells, and predominantly enveloped capsids were observed in the

cytoplasm of infected cells. In stationary Vero cells, AgK accumulated capsids in the

nucleus, and there was a concomitant decrease in the average number of enveloped

capsids in the cytoplasm. In addition, FgK-P produced partially syncytial plaques,

while AgK did not cause virus-induced cell fusion. These results indicate that gK is


48

required for efficient virus envelopment and translocation of infectious virions to

extracellular spaces, and it further suggests that the observed phenotypic properties of

FgK-0 may be due to expression of the amino-terminal portion of gK.

MATERIALS AND METHODS

Cells and Viruses

African green monkey kidney (Vero), human epidermoid larynx carcinoma (HEp-

2) and rabbit skin cells were obtained from ATCC (Rockville, MD). Cells were

propagated and maintained in Dulbecco's Modified Eagles Media (DMEM; Sigma

Chemical Co., S t Louis, MO) supplemented with 10% heat inactivated fetal bovine

serum (FBS). V27 cells carry a stably integrated copy of the HSV-1 (KOS) ICP27 gene

and was kindly provided by Dr. D. M. Knipe, Harvard Medical School. These cells

were propagated in DMEM supplemented with 10% FBS (Rice and Knipe, 1990). The

VK302 cell line, a gK transformed cell line, was obtained from Dr. D. C Johnson,

Oregon Health Sciences University, and were maintained in DMEM lacking histidine

(GIBCO Laboratories, Grand Island, NY) and supplemented with 03 mM histidinol

(Sigma Chemical Co. St. Louis, MO). The cells were passaged once in DMEM plus 10%

FBS without selection prior to infection with virus (Hutchinson and Johnson, 1995).

HSV-1 (KOS), the parental wild-type strain used in this study was originally obtained

from Dr. P. A Schaffer (Dana-Faber Cancer Institute, Boston, MA). Viruses HSV-

l(KOS) d27-l has a 1.6 kb BamH I-Stu I deletion of the ICP27 gene was kindly

provided by Dr. D. M Knipe, Harvard Medical School and was propagated in V27

cells (Rice and Knipe, 1990). The gK mutant, F-gKp was a kind gift by Dr. D. C.

Johnson, Oregon Health Sciences University, and was propagated in VK302 cells

(Hutchinson and Johnson, 1995) (Figure 2.2).


49

Reagents

Restriction enzymes, DNA modification enzymes and the Spe I* linker were

obtained from New England BioLabs (Beverly, MA). Deoxynudeotide triphosphates,

sequencing buffers and T7 DNA polymerase were from United States Biochemical

Corp. (Cleveland, OH) and were used in the dideoxy-chain termination method

according to manufacturer's instructions. Monoclonal antibodies mAb HS-B1 against

gB, mAb HS-Cl against gC, and mouse monospecific polyclonal antisera against gD

were produced in our laboratory by following standard procedures (Baghian et al.,

1993; Baghian et al., 1990). mAb 52-S against gH was obtained from ATCC (Rockville,

MD). 35S-methionine (13.62 mCi/mL) was from ICN Radiochemicals (Irvine, CA), and

35S-dATP sequencing grade(12.5 m Q/m L) was from DuPont/NEN (Wilmington,

DE).

Plasmids

Plasmid pSG28 contains the EcoR IE-K fragments of HSV-1 (KOS) inserted into

pBR325 was a gift from Dr. R. M. Sandri-Goldin (University of California, Irvine, CA)

and was described previously (Goldin et al., 1981). The 3.2 kbp EcoR I-BamH I

fragment, which contains the carboxy-terminal two-thirds of the UL52 ORF and the

complete UL53 ORF, was removed from pSG28 and was inserted into the multiple

cloning site (MCS) of plasmid pUCl9 to generate plasmid pSJ1710 (Figure 2.1).

Plasmid pSJ1711 was derived from pSJ1710 and it contains an Spe I* linker

(CTAGACTAGTCTAG, that carries stop codons in all three reading frames and the

recognition sequence for restriction enzyme Spe D, at the unique Bbs I site (within the

UL53 ORF at nucleotide 75 from the start codon). A plasmid that contains the HSV-1

(KOS) BamH I-B fragment in pUC19 was kindly provided by Bill Goins (University of

Pittsburgh).


Reproduced

with perm

ission of the

copyright ow

ner. Further


without

permission.

o.i

KOS.......t«i*

BamHI

Construction of Plasmid pSJ1724u . u

0.2“t— 0.1 0.4 —t—

O.S 0.6 0.7_____

0.8■I..J

0.9 1.0 —1

Ec'oRI

UL52 UL53-gK t r / / / / y y / ^ / / / / / / /

UL54-ICP27

Kpnl Bbsl HpalTBamHI

pSJ1711

pSJ1723

UL52

Eco^l"

Ecofol

(~3.2 kbp) UL53-gK

BamHI

" v j j j f r r r ^

Kpnl BUsl HpiHpalI UL54-ICP27 (-5.6 kbp)

Spe I * pSJ1722 ,BamHI ~ 1

KpnlTX/ZZ/ZZZ/ZZiZZZAf

Spi I* Hpal BamHI

Pst I

Pst I

pSJ1724EcoR I

UL52 UL53-gK 1068 bp deletion

UL54-ICP27

Pst I

Figure 2.1: Schematic of Construction of Plasmid pSJ1724.

The relevant DNA fragments that were used to construct plasmids pSJ1711, pSJ1722, pSJ1723 and pSJ1724 are shown, the cloning scheme is described in the text.

o

51

Plasmid p6J1722 was derived from the BamH I-B by excising the BamH I-Pst I

fragment that contains the HSV-1 (KOS) UL54 ORF (coding for ICP27) and cloning it

into the MCS of pUC19. Plasmid pSJ1723 was derived by removing the BamH I-Hind

m fragment from pSJ1722 and inserting into BamH I-Hind HI site of pSJ1711, such as to

reassemble the UL52, UL53 and UL54 ORF in sequence. Finally/ the pSJ1724 was

derived by eliminating a 1 kbp Spe I-BamH I fragment from pSJ1723 that deleted the

entire UL53 ORF but kept the UL52 and UL54 ORFs intact (Figure 2. 2).

Construction of the gK-Null Virus (AgK)

Rabbit skin cells were transfected with plasmid pSJ1724 using the calcium chloride

precipitation method (Chen and Okayama, 1987) and 18 hours post transfection cells

were infected with d27-l (ICP27 null) virus, and the cells were lysed, and virus stocks

were made 24 hours post infection (h.p.i.). Viral recombinants were propagated on

Vero cells. Twenty five plaques were picked and plaque purified five times on Vero

cells.

Rescue of the gK-Null Virus Revertant Virus (AgK*)

Primers PI and P4 (Figure 2.3) were used to generate the 3.9 kbp PCR fragment

using wild-type KOS viral DNA as the template. Vero cells were transfected with the

wild-type KOS fragment using the calcium chloride precipitation method (Chen and

Okayama, 1987) and 24 hours post transfection, cells were infected with AgK virus.

Cell cultures were incubated at 37°C for an additional 48 hours at which point virus

stocks were prepared. Recombinant virus stocks were plated on subconfluent Vero cell

monolayers at different dilutions and allowed to replicate for 48 hours. Large wild-

type plaques were picked and plaque purified three times.


52

Viral DNA

Viral DNA was prepared in Vero cells as described previously (Kousoulas et al,

1984). Briefly, Vero cells were infected with plaque purified virus at an multiplicity of

infection (m.o.L) of 5. At 3 days post infection cells were lysed with 1% Nonidet * P40

(NP40) (Particle Data Laboratories Ltd. Elmhurst, IL.), 0.5% deoxycholic add (DOC)

(Sigma Chemical Co. St. Louis, MO) in 10 mM Tris, 1 mM ethylenediamine tetra acetic

add (EDTA) (both chemicals from Sigma Chemical Co. S t Louis, MO). Lysates were

treated with RNase (lOmg/mL) for 10 min. at 37°C, followed by addition of 1%

sodium dodecyl sulfate (SDS) (Sigma Chemical Co. St. Louis, MO) and proteinase K

(lOmg/mL) at 55°C, overnight. DNA was purified through two phenol ether

extractions and predpitated with 3M sodium acetate and ice cold ethanol To get

ultra-pure viral DNA, further purification through two Nal gradients was performed

(Kousoulas et al, 1984).

PCR Detection of gK-Null Virus

PCR was performed using the XL PCR kit supplied from Perkin Elmer as

described (Chouljenko et al., 1996). Viral DNA was prepared as described earlier.

Approximately 100 ng of viral DNA was used in a total reaction volume of 100 |i l

PCR primers were PI (5' -GGC AAC GAC GAC GGG GAC TGG T-3'), P2 (5'-TGG

GTC CTC CTA CAG CTA), P3 (5' -GGC ACA GAC AAG GAC CAA TCA GAC

ACC A-3'), P4 (5’ -TCT CTC CGA CCC CGA CAC CAA-3') (Figure 2. 4). All the

primers were synthesized in our laboratory with a Perkin Elmer/Applied Biosystems

DNA Synthesizer, Model 394, using the phosphoramidite chemistry. The XL-PCR

reaction conditions were as follows: 94°C for 5 sec, 68° C for 8 min. for 30 cydes.

Southern Blot Analysis of Viral DNA

DNA from either KOS or AgK-infected Vero cells were extraded as described

previously. The purified DNA was digested with restriction endonuclease BamH I


53

(New England Biolabs, Beverly, MA), electrophoresed on a 0.8% agarose gel,

transferred by Southern blot method (Southern, 1975), to positively charged nylon

membranes and hybridized with a biotin labeled probe. The probe was derived from a

3.2 kbp PCR fragment which contains portions of the UL52 ORF, the complete UL53

ORF and a portion of the UL54 ORF. The PCR fragment was purified and labeled with

biotin-dUTP using the NEBlot phototope kit (New England Biolabs, Beverly, MA.)

which incorporates biotin into the probe through a random primer reaction.

Prehybridization, hybridization and detection of the biotinylated probe was achieved

using the Southern-Light^^ chemiluminescent detection system according to

manufactures instructions (Tropix, Inc. Bedford, MA).

Visualization of Intracellular Virus Particles in Vero and HEp-2 cells Using Electron Microscopy

Vero and HEp-2 cells were infected with AgK or wild-type KOS virus at an

m.o.i. of 10 and were incubated at 37°C for 48 hours. The infected cells were washed

three times with IX phosphate buffered saline (PBS) and fixed with 2% glutaraldehyde

in 0.1 M sodium cacodylate (CAC) buffer, pH 7.4 for 1 hour at room temperature.

Cells were then washed three times with 0.1 M CAC buffer supplemented with 5%

sucrose, pH 7.4 at room temperature and fixed with a 1% osmium tetroxide, 1%

potassium ferrocyanide solution for 1 and 1 /2 hours. Cells were further washed three

times at room temperature with 5% sucrose in 0.1 M CAC buffer, stained with 2.5%

uranyl acetate in distilled water and refrigerated overnight Finally cells were washed

in distilled water and dehydrated using gradient steps of ethanol, then infiltered with

epon-araldite resin and polymerized for 24 hours at 60°C Sample blocks were

sectioned, mounted on grids and at least five sections and 10 different infected cells

per section were examined using a Philips 410 electron microscope for the presence of

viral particles (Cavalcoli et al., 1993).


54

Kinetics of Infectious Virus Production

Sub-confluent Vero and HEp-2 cells were infected with either AgK, F-gKf), KOS, or

AgK* viruses at an m. o. L of 10. After a 60 min. adsorption at 4° C, unadsorbed

viruses were removed and cultures were washed with media. Fresh pre-warmed

medium was added to the cells, and cultures were incubated at 37°C for the duration

of the study. At selected times, the supernatants and the cell pellets were harvested

and collected in separate tubes. The samples were frozen and thawed three times,

sonicated on ice by pulsing 8 times with a microtip probe attached to a Branson

sonifier, cell disruptor 200 (Dansbury, CD and end-point plaque assays were carried

out on Vero cells.

Radiolabeling and Immunopredpitations

Conditions for radiolabeling and immunopredpitation were as described

previously (Baghian et al., 1992). Briefly, subconfluent Vero cells were infected at an m.

o. i. of 5 and labeled with 50 jxCi/mL 35s-methionine/cysteine mixture (ICN

Radiochemicals, Irvine, CA), from 6 to 24 h.p.L in methionine/cysteine-free media.

Cellular extracts were prepared and antibodies against gB, gC, gD and gH were used in

immunopredpitations following standard procedures (Baghian et al., 1993; Pereira et

al., 1980). Immunopreripitates were electrophoresed inSDS-PAGE gels as described

previously (Laemmli, 1970; Pereira et al., 1980).

RESULTS

Construction of the gK-null virus HSV-1 (KOS) AgK

HSV-1 (KOS)d27-l, a host-range mutant virus that carries a deletion in the ICP27

gene rendering the virus unable to replicate in Vero cells, while infectious virions can be

produced by complementation in the V27 cell line that is permanently transformed

with the ICP27 gene (Rice and Knipe, 1990) (Figure 2.2).


55

TR03 0.703 0.60.1 03 Hi

UL53-gK UL54-ICP27

BamHI EcoRI Kpnl Bbsl Hpal BamHI

UL52 1058 aa

UL54-ICP27 512 aaR)Cell lines

gD promoter UL53-eK VK302 r - •

UL54-ICP27 V-27 —mmmmmmm

VirusesUL52 UL53-gK UL54-ICP27

“ 5V-1 i ----- ---(KOS)

UL52d27-l -------1-------(ICP27 null virus)

Plasmid

pSJ1724 —UL52 /

F Z / 77Z\ /

V / \

/ \/ \

UL53-gK UL54-ICP27ZZZZZZ2—C v

deletion \\ /

Y

/ \x uL ^-gr. UL54-ICP27/ \

EZ1UL53-gK

1068 bp deletion

Figure 2.2: Strategy for Isolation of HSV-1 AgK.

A) The top line represents the prototypic arrangement of the HSV-1 genome with unique long (UL) and unique short (US) regions flanked by the terminal repeats (TR) and internal repeats (IR) regions. Shown below is the region of HSV-1 (KOS) genome (between map units of 0.7 and 0.8) containing the UL52,UL53 and UL54 open reading reading frames with relevant restriction endonuclease sites and the transcripts from the UL52, UL53 and UL54 ORFs.B) Relevant genomic regions of the cell lines VK302 and V-27, viruses HSV-1 (KOS), HSV-1 (d27-l) and the plasmid pSJ1724 employed in this study. Hypothetical recombination between homologous regions of d27-l and plasmid pSJ1724 that will result in AgK virus is depicted by dashed lines.


56

Figure 2.3: PCR Detection of the AgK Genotype.

A) Region of KOS, d27-l (ICP27 null) and the AgK (gK null) viral genomes containing the UL52, UL53 and UL54 open reading frames with relevant restriction endonuclease sites and the relative positions of the PCR primers. Open parentheses represent the portions of genes that were deleted.B) List of the predicted PCR products from amplification with each of the indicated PCR primer pairs. C) Agarose gel electrophoresis of ds DNA PCR products. Lane 1: lambda phage DNA digested with HindUI (marker). Lanes 2,3 and 4: Viral DNA of KOS, AgK-1 and AgK-2 viruses amplified with PCR primer pair PI and P2, respectively. Lanes 5,6 and 7: Viral DNA of KOS, AgK-1 and AgK-2 viruses amplified with PCR primer pair P3 and P4, respectively. Lane 8: Molecular mass marker (1 kb ladder).


57

PCR DETECTION OF gK NULL VIRUS, AgK

UL52 PL PL UL53-gK UL54-ICP27

A) |---1 ' I' - - | 'BamHI EcoRI v w RVi«r u _r BamHKpn I Bbsl Hpal BamHI P3 P4

d27-1 UL52 UL53-gK UL54-ICP27I C P 2 7 n u l l I - w /S * * * ** * * * MVbp

UL52 UL53-gK . UL54-ICP27AgK 1 —T * 1068 Bp H — -------gKnull deletion

B)Prim er

P airKOS AgK d27-l

P1/P4 3936 bp 2868 bp 2171 bp

P2/P3 1604 bp 535 bp noamplification

C) 1 2 3 4 5 6 7 8

3-9 kl 2-8 kb

1-6kb

535b—


58

Plasmid pSJ1724 was constructed to have a deletion of the entire portion of gK that

specified the mature gK Progeny recombinant viruses were expected to be generated as

a result of homologous recombination between the d27-l viral genome and the

transfected plasmid pSJ1724 in which the deletion in the ICP27 gene of the d27-l

genome was rescued by the intact ICP27 gene on the plasmid pSJ1724. ICP27 rescue

could only occur if the gK deletion was also transferred into the recombinant viral

genomes, since there were no homologous sequences between the gK and ICP27 genes

that could be used to independently rescue the ICP27 deletion (Figure 2.2).

Recombinant virus stocks plated on exponentially growing Vero cells, produced

two types of plaques on Vero cells; a wild-type size plaque composed of more than

100 infected cells and a smaller size plaque where 6*15 cells were recruited to form the

plaque. Putative recombinant viruses that formed large and small plaques on Vero cells

were isolated, plaque purified three times and the viral genomes from both kinds of

plaques were tested by diagnostic PCR.

Different sets of PCR primers were designed to detect either the gK-null or the

ICP27-null genotype. Primers PI and P2 were located within the UL52 coding

sequence, whereas primers P3 and P4 were located within the UL54-ICP27 coding

sequence (Figure 2. 3A). Primer P3 was located within the portion of UL54-ICP27 that

was deleted, while primer P4 was located outside the deleted region (Figure 2 .3A).

The sizes of the expected PCR products from KOS, AgK or d27-l viral DNAs are

shown in Figure 2 .3B. PCR testing of the viral DNA samples obtained from

representative large plaques produced on Vero cells showed the presence of two viral

genotypes, the gK-null (AgK) and ICP27-null (d27-l) indicating that these plaques were

produced by Vero cells by mixed infections with these two viruses. Primer pairs P1/P4

and P2/P3 amplified DNA fragments of 3936 bps and 1604 bps, respectively, when

wild-type KOS was used as the control template DNA (Figure 2 .3C, lane 2 and lane

5). Viral DNA extracted from plaque-purified viruses obtained from the small plaques


59

were subjected to diagnostic PCR. Primer pairs P1/P4 and P2/P3 amplified DNA

fragments of 2868 bp (Figure 2 .3C, lane 3,4) and 535 bp (Figure 2. 3C, lane 6,7),

respectively/ from the recombinant viral stocks indicating that these viral genomes

contained the expected deletion of the gK gene, while there were no contaminating

ICP27-null (d27-l) genotypes detected (Figure 2 .3 0 . Six such viral isolates obtained

from well-isolated and plaque-purified viral stocks were proven to contain exclusively

gK-null viruses. These virus isolates were designated AgK-1 through 6.

The genotype of representative gK-null viruses, AgK-1 and AgK-2, was confirmed

by Southern blotting (Figure 2.4). Viral DNA from KOS and AgK were digested with

the BamH I restriction endonuclease, separated by agarose gel electrophoresis and

transferred to a nylon membrane. The membrane was probed with a 3.2 kbp

biotinylated DNA fragment that contains the 3' third of UL52 ORF, the entire UL53

ORF and a portion of UL54 ORF (Figure 2.4B). The probe hybridized to two wild-

type KOS DNA fragments, the BamH I-L fragment (53 kbp) which contains the UL52

and UL53 ORFs and the BamH I-B fragment (10.1 kbp) which contains the UL54 ORF

(Figure 2 .5a lane 5). As expected, the labeled probe hybridized to a single DNA

fragment of AgK of approximately 14.4 kbp. The DNA fragment represented BamH I-L

+ BamH I-B (5.3 kbp + 10.1 kbp = 15.4 kbp) - deletion in the gK gene (1.0 kbp) = 14.4

kbp. DNA sequencing revealed that the BamH I site joining the BamH I-L and -B DNA

fragments was lost in the engineered gK deletion resulting in the fusion of the BamH I-L

and-B DNA fragments (data not shown).

Rescue of the AgK Virus by DNA Fragments Spanning the gK Gene

To ascertain that the phenotype of the deletion mutant was due solely to the

deletion of the gK gene, marker-rescue experiments were performed with DNA

fragments that spanned the entire gK gene. Primers P2 and P3 were used to generate a

1. 6 kbp PCR fragment using wild-type KOS viral DNA as the template.


60

A)

B)

14.4K b-----

10-1 kb

5-3kb

UL52 UL53-gK UL54-ICP27

rBamHI KpnI Bbsl Hpal BamHI

B iotinylated probe(3.2 kb)

Figure 2.4: Southern Blot Analysis of KOS and AgK Viral DNAs.

A) Lanes 1 and 5 are plasmids pBam HI-B and pRB126 digested with BamHI. Digestion of these plasmids with BamHI results in the BamHI-B (10.2 kbp) and BamHI-L (5.3 kbp) fragments respectively. Lanes 2,3 and 4 represent KOS, AgK-1 and AgK-2 viral DNAs digested with restriction endonuclease, BamHI. Lane 6, lambda phageDNA digested with restriction enzyme, HindHI.B) Location of the 3.2 kbp biotinylated probe relative to the viral genome.


61

The PCR amplified DNA fragment was transfected into Vero cells and 24 hours post

transfection, cells were infected with AgK virus. Approximately, 4% of the plaques

appeared larger than the typical small plaques produced by the AgK virus. A typical

virus plaque produced by the AgK revertant virus, designated AgK* is shown in Figure

2.5B. Virus stocks were prepared from 4 different large plaques, and plaque-purified

three times. The DNA from these plaques was extracted and subjected to diagnostic

PCR All 4 viral DNAs were positive for the presence of the entire gK gene, and

indistinguishable from the wild-type KOS genotype. Furthermore, single step

replication kinetics of the AgK* virus was similar to that of the wild-type KOS.

Plaque Morphology of AgK and F-gKp Mutant Viruses on Different Cell Lines

The AgK virus was plated on exponentially growing Vero and HEp-2 cells (60-

70% confluent monolayers) and the resultant viral plaques were examined

microscopically three days post infection. AgK formed smaller plaques on HEp-2 cells

and Vero cells than the wild-type KOS virus (Figure 2 .5A and 2 .5C). The number of

infected cells that were found in these viral plaques ranged between 10-20, and the

plaques became first visible at 48 h.p.i., while KOS viral plaques were first visible at 24

h.p.L The average plaque size of AgK became larger with time and contained on the

average 40-50 cells at 72 h.p.L

Similar experiments were performed using confluent monolayers of Vero and HEp-

2 cells (cells seeded at 80-90% confluency). AgK produced small plaques on both cell

lines which involved 3-5 cells at 48 h. p. L F-gK(3 produced similar size plaques on

Vero cells, in agreement with a previous report (Hutchinson and Johnson, 1995). At

late times post infection, side-by-side comparison of AgK and F-gKp plaques on Iog-

phase Vero cells revealed that F-gKp plaques contained some fused cells, while AgK

plaques did no t The phenotypic difference was even more pronounced at high m.o.L

with F-gKP producing small syncytia, while AgK did not cause any cell fusion.


62

Figure 2. 5: contd.

Figure 2. 5: Plaque Morphology of KOS, AgK% AgK and F-gKP Viruses on Vero Cells.

Panels A and B represent KOS and AgK* virus and panels C and D represent AgK and F-gK(3 virus infected Vero cells, respectively. Cells were infected at an m. o. i. of 0.2 PFU/cell and photographed through a phase contrast microscope at 72 h.p.i.


63

Figure 2.5


64

Figure 2. 6: contd.

Figure 2. 6: Plaque Morphology of KOS, AgK*, AgK, and F-gK(3 Viruses on VK302 Cells.

Panels A and B represent KOS and AgK* virus and panels C and D represent AgK and F-gKp virus infected VK302 cells, respectively. Cells were infected at an m. o. i. of 0. 2 PFU/cell and photographed through a phase contrast microscope at 72 h.p.i.


65

Figure 2. 6


66

The KOS and revertant AgK" plaques formed on VK302 cells were similar to those

obtained on Vero cells (Figure 2 .5A and 2 .5C). AgK produced larger, non-syncytial

plaques on VK302 (150 to 200 cells/plaque) when compared to Vero cells, however

these viral plaques were smaller than KOS or AgK" suggesting that AgK was not fully

complemented by VK302 cells (Figure 2 .6C). F-gKp produced partially syncytial, large

plaques on VK302 (Figure 2 .6D) as reported previously (Hutchinson and Johnson,

1995).

Propagation and Plaquing Efficiency of AgK and F-gKp on Different Cell Lines

A series of experiments were performed to compare the replication properties of

AgK and F-gKp mutant viruses in different cell lines. An equal number of infectious

virions obtained by titration on permissive cells, actively dividing Vero and VK302

cells for AgK and F-gKp, respectively, were used to infect both Vero and VK302 cells.

Virus stocks were collected and titered on log-phase Vero and VK302 cells. The results

of these experiments are shown in Table 2.1 A. AgK titers were approximately 100-fold

(2 logs) lower than KOS titers in actively replicating Vero cells, and 1,000,000-fold (6

logs) lower than KOS titers in stationary phase Vero cells (Table 2.1 A). Virus titers

obtained by propagation of AgK and F-gKp on Vero cells and subsequently titered on

VK302 cells were 100-fold(2 logs) and 10,000 (4 logs)-fold lower than KOS titers,

respectively (Table 2. IB). Furthermore, the average plaque size of AgK was larger (150

to 200 cells/plaque) than the average plaque of AgK produced on Vero cells (Figure 2.

5 and Figure 2. 6). F-gKp, and AgK viruses propagated on VK302 cells and titered on

VK302 cells produced approximately equal to KOS virus titers, indicating that VK302

cells complemented both gK-null viruses efficiently (Table 2. IB). The number of

infectious F-gKP virions produced by propagation of the virus on VK302 cells was

approximately 4-logs lower on Vero than on VK3Q2 cells, while AgK virus stocks

prepared on VK302 cells had similar titers on Vero and VK302 cells (Table 2. IB).


67

Table 2.1: Plaquing Efficiency.

2. laVirus* Cells

GrownOn

CellsTitered

On

Genotype Phenotype TiterStationaryPhaseCelis(PFU/mL)

TiterLog

PhaseCelis(PFU/mL)

KOS Vero Vero gK' gK' syn" 5.5 x 10y 1.8 x lO*1*VK302 gK* gK" syn* 7.7 x 10’ 8.0 x 10’

AgK Vero Vero g ^ gfCsyn" 3.2 x 103 2.9x10*VK302 g ^ gK" syn* 7.7 x 107 7.1 x 107

FgK-P Vero Vero g ^ gK- syn 3 x 102 1.8 x 10*VK302 gK- gfCsyn 6.5 x 10s 4.5 x 107

2.1bVirus* Cells

GrownOn

CellsTitered

On

Genotype Phenotype Titer(PFU/mL)

Titer Relative to

KOS on VK302 Cells

KOS VK302 Vero g ^ gK" syn" 6 x 10iU 11VK302 gK' gK" syn" 5.1 x 10’ 1

AgK VK302 Vero g ^ gK- syn" 1.3 x 10’ 0.25VK302 g ^ gfC syn" 6x 10’ 1.17

FgK-P VK302 Vero gK- gfCsyn 4.5 x 10s .00008VK302 g ^ gBCsyn 7 x 10’ 1.37

a Virus stocks from Vero cells infected with either KOS or AgK viruses, and VK302 cells infected with F-gKp virus were made and their plaquing efficiencies were obtained on the indicated cells. PFU titers represent an average of three independent experiments with individual titers varying less than 2-fold. Stationary phase cells were approximately 2.5 x 106 cells per 9.62 cm , and log-phase cells were 8 x 10s cells per 9.62 cm2 well, respectively at the time of exposure to viruses.


68

Kinetics of AgK and F-gKfi Infectious Virus Production

The kinetics of AgK infectious virus production was examined by measuring the

number of PFU at different times after infection. Equal numbers of subconfluent Vero

and HEp-2 cells were infected with mutant AgK and the wild-type KOS virus

respectively at an m.o.i. of 10. At various times post virus adsorption the cells were

harvested, viral stocks were prepared from cellular extracts and from the supernatant

fluids of infected cell cultures. The total number of plaque forming virions were

determined for each sample by titration on log phase Vero cells (Figure 2.7A). The

infectious virus production kinetics of AgK on Vero cells was markedly different from

the wild-type KOS virus, and the final number of infectious virions was approximately

two logs lower than that of KOS (Figure 2 .7A). Approximately 1000 infectious AgK

virions were found in the supernatant fluids at late times after infection, while the

number of infectious KOS virions released from infected Vero and HEp-2 cell

monolayers increased with time after infection reaching a maximum number that was

approximately 500,000-fold (5 logs) higher than the number of extracellular, infectious

AgK virions (Figure 2. 7A and 2 .7B).

A side-by-side comparison of the growth kinetics of AgK and F-gK0 virus was

performed (Figure 2 .7 0 . An equal number of infectious virions were used to infect

subconfluent Vero cell monolayers and the amount of virus produced at different times

was assessed by titrations on their respective permissive cell lines. AgK produced 2-

logs higher number of infectious virions than F-gK0. Infectious AgK and F-gKfl

accumulated exclusively within infected cells, while only a small fraction of the total

number of infectious virions was found in the extracellular fluids (Figure 2 7 0 .

Electron Microscopy

Conventional fixation-embedding electron microscopic analysis was undertaken to

identify the intracellular localization of AgK virus in Vero and HEp-2 cells.


permission

of the copyright

owner.

Further reproduction

prohibited w

ithout perm

ission.

1 1 -

9 -

8 -

4 -

2-

I I I T " T ~1— 1 I I I— I—0 4 8 12 16 20 24 28 32 36 40 44 480

12

11

10

- 9

- 8

7

6

5

4

3

2

1

0

HOURS POST INFECTION HOURS POST INFECTION HOURS POST INFECTION

Figure 2.7: Kinetics of Infectious Virus Production.

Subconfluent Vero and HEp-2 cells containing approximately 8 x 10 5 cells per 9. 62 cm2 at the time of infection(panels A and B) were infected at an m. o. i. of 10 with either KOS or AgK viruses. Subconfluent Vero cells (panel C) were infected at an m. o. i. of 10 with either AgK or F-gKp viruses. At various times after infection, virus stocks were prepared from the supernates and cell extracts and were titered on Vero cells. Symbols: □ , extracellular KOS virus; O , cell associated KOS virus; A, extracellular AgK virus; O , cell associated AgK virus; extracellular F-gKp virus; H , cell associated F-gKp virus.

70

Intracellular virus was examined only in cells with morphologically intact nuclear

membranes to avoid studying late secondary cytoddal effects. The nuclei of all cells

were viewed at both 24 and 72 h.pi. appeared normal, exhibiting margination of the

chromatin along the nuclear membrane indicating that there was no destruction of

nuclear structures. In AgK-infected Vero cells there was an accumulation of naked (A

capsids), partially filled (B capsids) and completely DNA-filled capsids (C capsids)

capsids in the nucleus appearing in paracrystalline arrays proximal to the nuclear

membrane (Figure 2.8). Cells infected with the AgK virus revealed dilated perinuclear

vesicles, mitochondria and endoplasmic reticulum within the cytoplasm and very little

cytoplasmic virus was evident at 24 h. p. L However, at 72 h.p.L a small number of

completely enveloped capsids were visualized in the cytoplasm of infected cells (Figure

2.8B) while there were no virions observed in intercellular spaces. In contrast,

enveloped virus particles were detected in the cytoplasm and in tercellu lar spaces of

KOS infected Vero cells (Figure 2 .8C and 2 .12D). Electron micrographs of AgK-

infected HEp-2 cells failed to reveal an unusually high number of capsids in the nuclei

of infected cells (Figure 2 .8E and 2 .8F), while enveloped virions were visualized in the

cytoplasm in clusters enclosed within vesicles (Figure 2 .8B). Intracellular spaces did

not contain any virion particles.

Characterization of AgK-Specified Glycoprotein Synthesis and Transport to the Cell Surface

Subconfluent Vero cell cultures were infected with AgK virus at an m.o.L of 5 and

labeled with a ^methionine/cysteine mixture from 6-24 h.p.L Subsequently, infected

cells were collected and cell extracts were prepared. Specific antibodies against

glycoproteins gB, gC, gD, and gH were used in immunopredpitation experiments to

detect their presence in AgK-infected cell extracts. Immunopredpitates were analyzed

by polyacrylamide gel electrophoresis and autoradiographed.


71

Figure 2. 8: Electron Micrographs of Vero and HEp-2 Cells Infected with AgK and KOS Viruses.

Subconfluent Vero cells were infected with AgK virus (panel A and B), KOS virus (panel C and D) and HEp-2 cells were infected with AgK virus (panel E and F). All cells were infected at an m. o. i. of 10 PFU/cel1, incubated at 37°C for 72 hours and prepared for electron microscopy. The arrows in panel A point to arrays of viral capsids in the nucleus. Arrow in panel B point to a enveloped AgK virus in the cytoplasm. Arrows in panel C mark the extracellular KOS virus. Arrows in panel D point to KOS virions in the perinuclear space, cytoplasm and extracellular space. In panel F the arrows mark AgK viruses found in the cytoplasm and unenveloped capsids in cytoplasmic vesicles, n (nucleus), c (cytoplasm), e (extracellular space), scale bar = 0.5 pm.


72

Figure 2. 8


73


74

Figure 2. 8


75

1 2 3 4 5 6 7 8 910

94kO—

67 KD—

43k0—

30kD -----

20kD—

Figure 2. 9: Photograph of an Autoradiogram of Immunoprecipitates from Extracts of Either KOS or AgK Virus Infected Vero Cells.

Lane 1: protein marker, Lanes 2 ,3 ,4 and 5 are KOS infected cellular extracts immunoprecipitated with anti -gB, -gC, -gD and anti-gH antibodies, respectively. Lanes 6,7,8 and 9 are AgK infected cellular extracts immunoprecipated with anti -gB, -gC, -gD and anti-gH antibodies, respectively.


76

Overall, viral protein synthesis in AgK-infected cells was reduced by approximately 5-

fold in comparison to KOS-infected cells. Therefore, five-fold more AgK-infected cell

extract was used for each glycoprotein immunopredpitation. Glycoproteins gB, gC, gD,

gH were detected in AgK-infected cell extracts (Figure 2.9). These results were in

agreement with indirect immunofluorescence assays that detected glycoproteins gB, gC,

gD and gH in AgK-infected cells. Transport of glycoproteins to the cell surface was

examined by fluorescent cytometry measurements using monoclonal antibodies to

glycoproteins gB, gC, gD and gH. All four glycoproteins were transported to the cell

surface.

DISCUSSION

A novel genetic approach was designed to produce the mutant virus AgK with a

deletion of the entire gK gene coding sequences specifying the mature gK. This report

demonstrates that gK is not essential for virus replication in HEp-2 and in

exponentially growing Vero cells, however, gK is required for virus replication in

stationary Vero cells. These results are consistent with a role for gK in virus

envelopment and translocation of infectious virions to extracellular spaces. The

collective findings and their interpretations are substantially different to those

obtained with the FgK-P mutant virus.

Construction of the AgK Virus

The isolation of the AgK mutant was accomplished by rescuing a deletion in the

ICP27 gene which is immediately adjacent to the gK gene, while simultaneously

transferring the engineered deletion in the gK gene. The absence of any intervening DNA

sequence between the gK and ICP27 deletions ensured that the d27-l defect could not

be rescued without the simultaneous transfer of the gK gene deletion (Figure 2.2).

Consistent with this, all putative recombinant viruses, tested after multiple plaque


77

purification steps, were genotypically AgK. Construction of the gK gene deletion was

complicated by the presence of two absolutely essential genes (UL52, UL54-ICP27)

that flanked the gK gene. Furthermore, the UL52 ORF overlaps with UL53 ORF by 45

bp. A recombinant virus that preserved the UL52 gene, while deleting the entire gK

coding sequences that specified the mature gK was constructed successfully. The AgK

defect was rescued by a 1.6 kbp DNA fragment that encompassed part of the UL52

coding sequence and 250 bps of the upstream region of the ICP27 gene. It is unlikely,

that additional mutations were present in the carboxy-terminus of the UL52 gene that

may have contributed to the observed AgK mutant phenotype Furthermore, diagnostic

PCR failed to detect any additional gK deletions, and partial DNA sequencing of the

region immediately proximal to the 5' end of the gK gene deletion did not reveal any

point mutations (Figure 2.4).

The AgK virus is predicted to specify only the first 25 amino adds of the 30 amino

add signal sequence of gK The small polypeptide probably is directed to the

endoplasmic reticulum where it is eventually degraded as would be expected with the

normal gK signal peptide of 30 amino adds that is deaved off the translated gK

protein (Ramaswamy and Holland, 1992).

Role of gK in Virus Envelopment

Herpes virus capsids containing DNA are assembled in the nudeus and acquire

their envelopes by budding through the inner nudear membrane (Darlington and Moss,

1968; Roizman and Sears, 1996). Electron micrographs of AgK-infected Vero cells

revealed an unusually high accumulation of type "B" capsids juxtaposed to the inner

nuclear membrane. Visualization of predominantly "B" capsids in the nudei of AgK-

infected Vero cells may indicate that viral DNA is not effidently packaged into

capsids. Additional experiments are needed to confirm any possible role of gK in DNA

packaging. A few fully enveloped and some partially enveloped viral partides were


78

visualized in the cytoplasm of AgK-infected Vero cells at 72 h.p.L Approximately 10

enveloped virion particles were observed in the cytoplasms of each AgK-infected Vero

cell, and they were mostly found in membrane bound vesicles scattered in the

cytoplasm. By contrast to Vero cells, nudeocapsids did not accumulate in the nuclei of

HEp-2 cells, and approximately 100 enveloped virion particles were found in the

cytoplasm of HEp-2 cells. Viral yields (PFU/cell) were in agreement with the number

of enveloped virions found in the cytoplasm of each cell indicating that most of the

visualized enveloped viruses were infectious. These results suggest that the absence of

gK results in an inefficient capsid envelopment and reducing of the specific infectivity

of enveloped viruses.

Exponentially growing Vero cells released substantially more infectious virions in

their cytoplasms than stationary Vero cells, but 10-fold less than HEp-2 cells. This

indicated that actively replicating Vero cells, and HEp-2 cells complemented the gK-

null defect for virus envelopment. HEp-2 cells are transformed cells originally isolated

from a human epidermoid larynx carcinoma (Moore et al, 1955). In cell culture, HEp-2

cells remain metabolically active even at nearly 100% confluency as evidenced by the

fact that they incorporated ^m ethionine into their proteins, and ̂ H-Thymidine into

their DNA. Since glycoprotein K has been detected exclusively on the surface of nuclei

and ER membranes using anti-gK specific antibodies (Hutchinson and Johnson, 1995),

one can hypothesize that nuclear membranes of dividing cells may complement the

absence of gK because they are primed for mitotic division. In this hypothesis, gK may

cause similar nuclear membrane reorganization events to increase the efficiency of virus

envelopment Similar hypothesis to explain cellular complementation by exponentially

growing cells was reported for the HSV-1 strain 17 syn* ICP34.5 gene which codes for

a viral tegument protein, y 34.5. Deletion of the ICP34.5 (y34.5) viral gene in HSV-1

strain 17 syn* resulted in virions that failed to envelope, while capsids accumulated in

the nuclei of stationary phase 3T6 infected cells.


79

However, the ICP34.5 null virus replicated efficiently in log-phase 3T6 cells (Brown et

al., 1994).

Role of gK in die Translocation of Infectious Virions from the Cytoplasm to the Extracellular Space

Infectious AgK virions accumulated within the cytoplasm of Vero and HEp-2 cells

indicating that gK plays an important role in release of cytoplasmic virions to the

extracellular spaces. A similar retention of infectious virions within cells has been

noted when Golgi-spedfied enzymatic functions were inhibited either by Golgi-spedfic

inhibitors or in mutant cells deficient in specific Golgi-localized enzymes. These

observations suggest that translocation of infectious virions to the extracellular, free

spaces is Golgi-dependent. Our data are consistent with the view that transport

vesicles are formed by the outer nuclear lamellae, since AgK enveloped virions were

observed within cytoplasmic vacuoles. The defect in AgK egress, may be due to the

inability of these transport vesicles to carry their virion particle payload to the Golgi

apparatus. A corollary of this model is that gK-null virions in the cytoplasm of

infected cells should contain underglycosylated glycoproteins, since these precursor

glycoproteins are originating from the RER and the nuclear membrane. Such virions

were previously found to be infectious (Kousoulas et al., 1983). An alternative

explanation is that the absence of gK results in a general compromise of the structural

integrity of the virion particles that somehow alters their specific transport through the

Golgi-dependent secretory pathway. In support of this hypothesis, it has been

observed that the specific infectivity, and kinetics of entry of AgK virions are

substantially reduced in comparison to KOS virions (Baghian, et al personal

communication).

UL20 and gH are known to be involved in the transport of infectious virions to the

extracellular, free spaces (see literature review). UL20-null virions acquire envelopes in

Vero cells, but these get trapped within the perinuclear space (Baines et al., 1991).


80

There are obvious similarities in virion transport defects caused by mutations in UL20,

gH and gK which imply that these proteins share some common functions in

intracellular virion transport. However, AgK virus is not complemented in many cell

lines including the 143 TK~ cell line that complements the UL20-null virus implying that

there are important differences between gK and UL20 functions. Mutant viruses that

carry a ts mutation in gH, synthesize at the non-permissive temperature immature gH

which is not transported to the cell-surface, and infectious virions accumulate in the

cytoplasm (Desai et a l, 1988). In contrast, gH is synthesized and expressed at the cell-

surface of AgK-infected cells implying that gK's role in virion transport is different than

that of gH. As we have discussed earlier, available evidence indicates that glycoprotein

synthesis and cell-surface expression is associated with virion transport, since when

Golgi-dependent glycoprotein processing and transport was inhibited, it resulted in

inhibition of virion transport to extracellular spaces. Apparently the reverse situation

is not true, because glycoproteins gB, gC, gD, and gH are efficiently synthesized and

transported to the cell surface of AgK-infected cells. The AgK phenotype indicates for

the first time that viral glycoprotein transport and cell-surface expression can occur in

the absence of virion transport

Molecular Basis for the Genotypic and Phenotypic Differences Between AgK and F-gKP Viruses

The genotypes of F-gKp and AgK mutant viruses are distinct F-gKp was created

by insertional inactivation of the gK gene using an ICP6::lacZ cassette (Goldstein and

Weller, 1988; Hutchinson and Johnson, 1995). The insertion allowed for the expression

of first 112 amino adds of gK fused to 26 amino adds originating from the 5'-most

DNA sequence of the ICP6 promoter of the ICP6::lacZ gene cassette The extra 26

amino adds that are fused to the amino terminal 112 amino adds of gK are: DPRARRF

CTWYSLT ASAGTR SCWCT (L. Hutchinson, personal communication).


81

Hydrophllldty Window Scale - Kyte-Doolltlle

2 4 6 8 10 12 14 16 18 20 22 24 26P R A R R F C T W Y S L T A S A G T R S C V V C T

Figure 2.10: Hydrophilic and Surface Probability Profiles of the Extra 26 Amino Acids Specified by F-gK[3.

Analysis were performed using the Protein Toolbox application of the MacVector Software (IBI-Kodak).


82

The amino add sequence was also independently predicted in our laboratory from the

complete ICP6:dacZ DMA sequence (S. Weller, personal communication). It was

hypothesized that this fusion protein is degraded because it does not contain the

hydrophobic portion of gK that is thought to anchor gK to membranes. Furthermore, it

was not possible to detect this truncated gK in FgK-p-infiected cells (Hutchinson and

Johnson, 1995). Interestingly, the 112 amino add sequence contains the synl locus that

demarcates the putative fusion domain of gK The 26 amino add segment that is fused

to the amino terminus of gK contains hydrophobic amino add segments which may

transiently anchor the gK-truncated protein to membranes (Figure 2.10). We

hypothesize that the different phenotypic properties of FgK-p and AgK viruses may be

due to expression of the truncated gK in FgK-p-infected cells. The specific phenotypic

differences between FgK-P and AgK mutant viruses are discussed in detail below:

Side-by-side comparisons of the replication and plaquing effidendes of AgK and

F-gKp mutant viruses on different cell lines showed substantial differences. F-gKp

failed to replicate as effidently as AgK virions, and did not form plaques as effidently

as AgK in Vero cell lines. F-gKp is derived from the HSV-1 (F) strain via a homologous

recombination with a plasmid derived from KOS genomic sequences in which a P~gal

cassette was inserted. HSV-1 (F) specifies gB that contains the fast-entry genotype that

causes faster kinetics of entry than KOS (Bzik et al., 1984b; Pellett et al, 1985), and the

kinetics of entry of F-gKp is faster than that of AgK virus on Vero cells (unpublished

observations). In this regard, F-gKp has an advantage for virus growth, since it enters

faster into cells than AgK virus, yet it replicated much less effidently than AgK virus.

These results suggest that the truncated gK oligopeptide-specified by F-gKp may

interfere with virus replication resulting in a substantial decrease of virus yields.

Comparison of F-gKP and AgK plaque morphologies revealed that F-gKP produced

partially syncytial plaques at late times post infection including the complementing cell

line VK302, while AgK did not produce syncytial plaques. The syncytial plaque


83

phenotype difference between F-gKp and AgK viruses was particularly striking on gK-9

(gK-transformed Vero cell line) and 143 TK' (data not shown). These results suggest

that the putative F-gKp-spedfied gK polypeptide may associate transiently with

membranes causing cell fusion.

Electron microscopic examination of Vero cells infected with F-gKp virus showed

that F-gKp enveloped virions were predominantly found in the perinuclear space, and

a high number of capsids were also detected in the cytoplasm (Hutchinson and

Johnson, 1995). On the basis of these findings, it was suggested that gK may prevent

fusion between viral and cellular membranes, so that the virion particles do not loose

their viral envelopes during intracellular transport Our results with the AgK virus are

markedly different to these findings, since we did not observe neither enveloped virions

in the perinuclear spaces nor an unusually high number of capsids in the cytoplasm of

AgK-infected cells. We hypothesize that a partially functioning gK polypeptide

specified by F-gKp may result in the loss of viral envelopes via nonspecific fusion

events of viral envelopes with the outer lamellae of the nuclear membrane releasing

nudeocapsids in the cytoplasm.

Original descriptions of the syncytial phenotype attributed to the gK gene

induded the hypothesis that gK played the role of membrane fusion inhibitor or

modulator of virus-induced cell fusion. Thus, a gK-null virus would be expected to

cause syncytial plaques (Lee and Spear, 1980; Manservigi et al., 1977; Read el al., 1980;

Ruhlig and Person, 1977; Spear, 1993). Apparently, this hypothesis is not correct, since

AgK virions do not form syncytial plaques. The observation that the gK gene is the

favorite target for mutations that cause virus-induced cell-to-cell fusion, and the feet

that gK is involved in virus envelopment and intracellular virion partide transport

support a role for gK in multiple membrane fusion events. Eluddation of gK's role in

these membrane fusion events remains a crucial target in our quest to understand

herpesvirus maturation, virion transport, translocation of infectious virions to the


84

extracellular space as well as in the ever elusive virus-induced cell to cell fusion

mechanisms, hi this regard, the availability of the AgK mutant virus in conjunction with

already developed methods for gK gene site-directed mutagenesis (Chouljenko et al.,

1996) will facilitate the delineation of the functional domains of gK.


CHAPTER HI

VIRAL INTERFERENCE MEDIATED BY gK-TRANSFORMED CELL LINES TO HSV-1 (KOS) EGRESS AND TO HSV-1 (AgK) REPLICATION

INTRODUCTION

HSV-1 Nudeocapsid Envelopment and Virion Egress

The final step in the morphogenesis of infectious HSV-1 virions is the acquisition

of the viral envelope by budding of DNA filled viral capsids through the inner lamella

of the nudear membrane (Roizman and Sears, 1993). The process is poorly

understood, but available data indicate that capsids and nudear membranes need to

be appropriately primed for effident virus envelopment Thus, defects in capsid

assembly or DNA encapsidation do not effidently produce enveloped virions

(Roizman and Sears, 1993; Spear, 1993). Similarly, defects in certain tegument proteins

that reside between the capsid and the viral envelope can also inhibit virion

envelopment (Roizman and Sears, 1993). Herpesvirus capsids acquire their envelope

by budding through the inner lamellae of the nudear membrane (Compton and

Courtney, 1984; Morgan et al., 1959; Schwartz and Roizman, 1969). The release of

infectious virions from infected cell requires the transport of infectious virions from the

perinuclear to the extracellular space. The translocation most likely occurs within

transport vesides derived from the outer lamellae of the nudear membrane and the

contiguous ER (Campadelli-Fiume et al., 1991; Compton and Courtney, 1984;

Darlington and Moss, 1968; Morgan et al., 1959; Roizman and Sears, 1996; Schwartz

and Roizman, 1969). The transport is modulated by both viral and cellular factors that

have been reviewed in Chapter I and Chapter IL One of the viral proteins that has been

directly implicated in transport is the UL20 protein. In the absence of UL20, virions

envelope, but remain in the perinudear spaces of Vero cells. However, most other cells

induding 143 TIC complement the UL20 defect indicating that Vero cells may lack

certain cellular factors that are involved in transport (Baines et al., 1991).

85


86

Interference of HSV-1 Induced Cell Fusion by gB and gK-Transformed Cell Lines

Virus induced syncytium formation is mediated by intercellular recognition by the

virus-specified glycoproteins present on the plasma membrane by the viral receptors of

the adjacent cells. Wild-type infections in cell culture caused limited amounts of cell

fusion; however, mutant viruses were isolated that caused extensive cell fusion. The

syncytial phenotype was mapped to at least five different loci of the viral genome. The

predominant syn loci were present in genes that code for either glycoproteins or

membrane bound proteins. The syn 1 loci was mapped to the UL53 ORF that encodes

gK (Bond and Person, 1984; Debroy et al., 1985; Hutchinson et al., 1992b; Manservigi et

al., 1977; Pogue-Geile et al., 1984; Ryechan et al., 1979), syn 3 to the carboxyl terminal

of UL27 encoding gB (Bzik et al., 1984a), syn 4 was located in UL20 ORF (Baines et al.,

1991), syn 5 was present in the UL24 ORF (Jacobson et al., 1989; Tognon et al., 1991)

and syn 6 was mapped to the terminal repeat regions that flank the Ul, nestled

between the ICPO and y 34.5 genes (Romanelli et al., 1991). Cell to cell fusion induced

by mutant HSV-1 with gB or gK syn mutation were shown to be inhibited by cells

expressing the corresponding wild-type gB or gK proteins, respectively (Hutchinson et

al., 1993). Expression of wild-type HSV-1 gK suppressed membrane fusion induced by

syncytial mutants with mutations in gK but did not suppress fusion caused by

syncytial mutant with mutations in gB or vice versa. Also, inhibition of fusion was

shown to be dependent on the gK gen e-copy number. gK-transformed cell lines

decreased the viral yields and reduced the average plaque size in comparison to Vero

cells. These results indicate that both gB and gK play significant but separate roles in

virus-induced cell fusion.

To characterize the role of gK in virus replication and membrane fusion, we

previously constructed and characterized HSV-1 mutant virus AgK that lacks the entire

UL53(gK) gene (Jayachandra, S. et aL, submitted). AgK replicated in actively dividing

Vero and HEp-2 cells, and predominantly enveloped capsids were observed in the


87

cytoplasm of infected cells. In stationary phase Vero cells, AgK accumulated capsids in

the nucleus, and there was a concomitant decrease in the average number of enveloped

capsids in the cytoplasm. In the process of generating the AgK viruses it was found

that the AgK virus could not be propagated efficiently on certain gK-transformed cells.

In particular, the AgK virus did not form plaques on BL-1 cells. The inability of BL-1

cells to complement the gK-null defect was further analyzed by examining the

replication properties of AgK and KOS virus on BL-1 and other gK-transformed cell

lines. This study shows that gK-transformed cells restrict virion egress of wild-type

HSV-1. Furthermore, AgK infectious virion production was restricted in gK-

transformed cells.


Cells and Viruses

Vero cells were propagated and maintained in DMEM supplemented with 10%

FBS (see chapter II materials and methods). Cell lines gB-gK and SV-gK, constructed in

our laboratory were maintained in DMEM supplemented in 10% FBS and 300 ug per

mL of G418 (Geneticin; GIBCO Laboratories, Grand Island, NY). BL-1 cells are Vero

cells transformed with HSV-1 (KOS) BamHI-L fragment was obtained from Dr. S. K

Weller (University of Connecticut Health Center) and was described previously

(Goldstein and Weller, 1988). These cells were grown in DMEM supplemented with

10% heat inactivated FBS and 250 ug per mL of G418. VK302 and gK-9 cell lines, are

two gK transformed Vero cell lines obtained from Dr. D. C Johnson, Oregon Health

Sciences University and were maintained in DMEM histidine minus medium (GIBCO

Laboratories, Grand Island, NY) supplemented with 10% FBS and 03 mM histidinol

(Hutchinson and Johnson, 1995). All transformed cells were passaged once in DMEM

containing 7% FBS without selection prior to infection with virus. The HSV-1 (KOS), the

parental wild-type strain used in this study was originally obtained from Dr. P. A


88

Schaffer (Dana-Faber Cancer Institute, Boston, MA). AgK virus, a gK null virus, where

the entire gene coding for gK deleted was previously described (Figure 3.1)

Reagents

Restriction enzymes and DNA modification enzymes were obtained from New

England BioLabs (Beverly, MA).

Plasmids

Plasmid pSG28 contains the EcoRI E-K fragments of HSV-1 (KOS) inserted into

pBR325 was a gift from Dr. R. M. Sandri-Goldin (University of California, Irvine, CA)

and was described previously (Goldin et al., 1981). The 3.2 kbp EcoRI-BamHI

fragment, which contains the carboxy-terminal two-thirds of the UL52 ORF and the

complete UL53 ORF, was removed from pSG28 and was inserted into the multiple

cloning site (MCS) of plasmid pUC19 to generate plasmid pSJ1710. The 13 kbp Kpn I-

BamH I fragment from pSJ1710 was transferred to pUC19 to generate plasmid

pSJ1683. Plasmid pSJ1680 contains the 700 bp promoter region of HSV-1 gB derived

by PCR amplification of HSV-1 (KOS) viral DNA. The 700 bp fragment was PCR

amplified with a synthetic EcoR I at the 5' terminal and a BamH I site at the 3' terminal

to facilitate cloning into MCS of pUC19 to generate plasmid pSJ1680. The PCR primers

used to generate the 700 bp gB promoter were gBPl(5'-TAT CAA GAA TTC TCG

AGA AGA TGC TGC-3') and gBP2(5'-AAC TAA GGA TCC AGG GGG CCC GTC

GT-3'), respectively. The 1.3 kbp Kpn I-BamH I from pSJ1683 was excised, blunt-

ended with T4 polymerase and cloned into the blunt-ended Pst I site in the MCS of

pSJ1680 to generate the plasmid pSJ1684. Thus, plasmid pSJ1684 contains the gene

coding for gK under the control elements of the gB promoter.


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gK-Transformed Cell-Lines U l ___________________________ Us

KOS

BL-1

UL52 "

- • — — *UL53-gK

. -

UL54-ICP27

BamHI EcoRIUL52

KpnlEZZZZZ-jH

BamHI

VK302

gK9 (200 copies)

SV-gK

gB-gK

Hpal BamHIUL53-gK

Kpnl Hpal BamHIrD promoter UL53-gK

Kpnl Hpal BamHI

EcoRI

r D promoter UL53-gK .IT|~ * * * * * * # 1 j

Kpnl Hpal BamHIadenovirus major late promoter UL53-gK

[ Hpal B ar13*3

Kpnl

gB promoter

Ipal

UL53-gK

BamHI

Kpnl Hpal BamHI

Figure 3.1: Schematic Representation of HSV-1 gK-Transformed Cell Lines Used in this Study.

The top line represents the HSV-1 genome and the relevant UL52, UL53 and UL54 ORFs are expanded below, showing the restriction endonuclease cleavage sites. Relevant HSV-1 genomic regions which were stably expressed in Vero cells are shown.

00o

90

p91023 is a eukaryotic expression plasmid that contains the SV40 origin of replication,

the adenovirus major late promoter coupled to the cDNA copy of the adenovirus

tripartite leader and the SV40 early polyadenylation signal (Wong et al., 1985). The 13

kbp Kpn I-BamH I fragment from pSJ1710 was blunt-end cloned into the blunt-ended

EcoR I site of p91023 to generate plasmid pSJ1720, thus placing the gK gene under the

control elements of the adenovirus major late promoter. pSV2rteo is a plasmid carrying

the dominant selectable neomycin resistance (NeoR) gene under the controls elements of

the SV40 origin of replication, SV40 promoter and the SV40 polyadenylation signals

(Qontech Laboratories, Inc. Palo Alto, CA).

Construction of gK-Transfonned Cell Lines, gB-gK and SV-gK

Cell lines gB-gK was constructed by cotransfecting subconfluent Vero cell

monolayers with 2 ug of plasmids pSJ1684 and pSV2neo and cell line SV-gK was

constructed by cotransfecting 2 ug of plasmids pSJ1720 and pSV2neo, respectively.

Transfections were carried out using the calcium phosphate precipitation method

described previously (Baghian et al., 1993; Chen and Okayama, 1987). Two days post

transfection cells were trypsinized and reseeded in the presence of selection media

(DMEM supplemented with 10% FBS and 150 ug/m L G418). Cells were selected for

neomycin resistance using increasing amounts of G418. Individual colonies of cells were

picked, grown and subcloned three times, checked for the presence of gK by PCR,

before establishing these cell lines for further experiments.

Genomic DNA

Genomic DNA was extracted from the cell lines by lysing the cells with 1% NP40,

0.5% deoxycholate in 10 mM Tris, 1 mM EDTA (TE). Lysates were treated with RNase

(lOmg/mL) for 10 min. at 37°C, followed by addition of 1% SDS and proteinase K

(lOmg/mL) at 55°C overnight DNA was purified through two phenol ether extractions


91

and precipitated with 3M sodium acetate and ice cold ethanol. The precipitates were

resuspended in distilled water and was used in PCR and dot blot assays.

Cellular RNA

Total RNA from BL-1 and Vero cells was extracted using the Tri-Reagent kit

according to manufactures instructions supplied by (Molecular Research Center, Inc.

Cincinnati. OH). RNAs were suspended in 10 mM Tris, 1 mM EDTA pH 8.0 buffer

and used in dot blot assays.

PCR Detection of the gK Gene in gK-Transformed Cell Lines

PCR was performed using the GeneAmp Reagent kit supplied by Perkin

Elmer/Applied Biosystems (Foster City, CA). Genomic DNA was prepared as

described above. Approximately 100 ng of cellular DNA was used in a total reaction

volume of 100 uL. PCR primers FF1 (5'-TACCCTGCAGCAGCA AAGCCT-3') and FF2

(5'-TGCGTAGTTCAAGAGGTAGGT-3'). All the primers were synthesized in our

laboratory with a Perkin Elmer/Applied Biosystems DNA Synthesizer Model 394

using the phosphoramidite chemistry. The PCR reaction conditions were as follows:

98°C for 1 sec, 58° C for 1 min., 72° C for 2 mins for 30 cycles, followed by a 5 min.

extension at 72° C.

Dot Blot Hybridization of Cellular DNA and RNA

Total cellular DNA from Vero, gB-gK, SV-gK and BL-1 cells were extracted

as described previously. The purified DNA was treated with 0.4 mM sodium

hydroxide, lOmM EDTA for 30 mins. at 37°C. The denatured DNA solution was

applied to the positively charged nylon membrane present in the dot blot manifold

(Bio-Rad Laboratories, Hercules, CA) according to manufactures instructions. For

northern dot blot hybridization, total cellular RNA from Vero and BL-1 cells were


92

denatured with lOmM sodium phosphate, dimethyl sulfoxide (DMSO), 6M glyoxaL

After blotting the membranes were rinsed gently with 2X SSC (sodium chloride and

sodium citrate buffer), air dried, and hybridized with a biotin labeled probe. The probe

was a 13 kbp Kpn I-BamH I fragment derived from plasmid pl683 which contains

portions of the UL52 ORF and the complete UL53 ORF. The 13 kbp fragment was

purified using Prep-A-Gene DNA purification systems according to manufactures

instructions and labeled with biotin-dUTP using the NEBlot phototope kit (New

England Biolabs, Beverly, MA.) which incorporates biotin into the probe through a

random primer reaction. Prehybridization, hybridization and detection of the

biotinylated probe was achieved using the Southern-Light"^ chemiluminescent

detection system according to manufactures instructions (Tropix, Inc. Bedford, MA).

Kinetics of Infectious Virus Production

Sub-confluent monolayers of Vero, BL-1 VK302 cells were infected with either AgK

and KOS at an m. o. L of 10. After a 60 min. adsorption at 4° C viruses were removed

and cultures were washed with media. Fresh pre-warmed media was added to the

cells and cultures were incubated at 37°C for the duration of the study. At selected

times, the supemates and the cell pellets were harvested to separate tubes. The

samples were frozen and thawed three times, sonicated and plaque assay were carried

out on Vero cells.

Visualization of Intracellular Virus Particles in Vero, BL-1 and VK302 Cells Using Electron Microscopy

Vero, BL-1 and VK302 cells were infected with AgK or wild type KOS virus at an

m. o. L of 10, incubated at 37°C for 48 hours and were prepared for electron

microscopy as described in material and methods in Chapter II.


93

RESULTS

Glycoprotein K (gK) - Transformed Cell Lines

Initial attempts to inactivate the gK gene failed indicating that gK may be essential

for virus replication (MacLean et al, 1991). To assess the role of gK in HSV-1

replication a number of gK-transformed cells were used. BL-1 cells were transformed

with the HSV-1 (KOS) BamHI-L DNA fragment containing the entire gK gene and

portions of the adjacent DNA sequences representing the portions of the UL51 and the

entire UL52 ORF (Goldstein and Weller, 1988). The UL52 ORF specifies a subunit of

the helicase-primase complex that is essential for viral DNA replication. BL-1 cells

efficiently complemented the UL52 defective virus, h rll4 and the copy number was

established 5-10 copies per cell (Goldstein and Weller, 1988). In addition, two stably

transformed gK cell lines, gB-gK and SV-gK were constructed (Figure 3.1). The

presence of the gK gene in the gB-gK and SV-gK cell lines were detected by PCR gK

gene specific PCR primers were used to amplify the gK gene from the transformed cell

lines (Figure 3.2). Results show that a 1.1 kbp was amplified, similar to the control

BL-1 cells. The copy number of the gK gene in both cell lines were established by dot

blot hybridization. The copy number of BL-1 was previously established to be 10-15

copies per celL By comparison to BL-1 cells the copy number of gB-gK and SV-gK cells

was established to be 15 copies and 8 copies per celL

Two additional gK transformed cells, VK302 and gK-9 were later obtained from

Dr. D. C. Johnson. In both VK302 and gK-9 the cellular copy of gK is under the control

elements of the HSV-1 gD promoter and the bovine growth hormone polyadenylation

signals. The exact copy number of gK in VK302 cells is not known, however, the VK302

cells partially complements gK-null virus, AgK and FgK-p. The expression of gK in

VK302 was estimated to be similar to the levels of wild-type HSV-1 infections in Vero

cells (Hutchinson and Johnson, 1995). In gK-9 cells the copy number of the gK-gene was

estimated to be over 200 copies per cell (Hutchinson et a l, 1993).


94

1 2 3 4 5 6 7 8 9 10 tl

Figure 3. 2 : PCR Detection of the gK Gene in Transformed Cell Lines.

Agarose gel electrophoresis of ds DNA PCR products to detect the gK genotype.Lane 1 through 5: cellular DNA from gB-gK cells; Lanes 6 through 9: cellular DNA from SV-gK cells; Lane 10: Viral DNA of KOS. All DNA were amplified with gK gene specific PCR primer pair PI and P2, respectively. Lane 11: lambda phage DNA digested with HindO (marker).


95

A) 1 4 5 6t t •• # •

Vero BL-1

B)BamHI

UL52

EcoRI Kpnl

UL53-gK« / / / / / / / >

J , »Bbsl Hpal BamHI

UL54-ICP27

Biotinylated probe (5.6 kb)

Figure 3.3: Northern Dot Blot Analysis of Vero and BL-1 cellular RNAs.

A) Duplicate samples of RNA from Vero cells (Lane 1) and BL-1 cells (Lanes 4, 5 and 6) were blotted onto nylon membranes and probed with the biontylated probe.B) Location of the 5.6 kbp biotinylated probe relative to the viral genome.


96

mRNA Synthesis in BL-1 cells

mRNA was extracted from non-infected BL-1 and Vero cells and Northern dot

analysis was performed as described in materials and methods. Results showed that

biotinylated gK DNA probe hybridized to mRNA from BL-1 cells whereas the probe

did not bind to Vero cell RNA extracts (Figure 33)

Plaque Morphology

In the process of generating a gK-null virus, the recombinant AgK stocks were

plated on Vero and BL-1 (Figure 3.4). AgK produced numerous small plaques on

actively dividing Vero cells (5-10 infected cells/plaque) as previously reported

(Jayachandra, S. et aL, submitted). The viral recombinants did not produce plaques

when propagated on confluent BL-1 cell monolayers. At high m. o. L individual

rounded up cells infected cells were visualized at late hours post infection in AgK

infected BL-1 cells. However, from 72 hour post infection (h. p. L)., small numbers of

microscopic plaques (5-10 cell infected cells/ plaque) were visualized when AgK was

propagated on actively dividing BL-1 cells. Wild-type KOS virus plaques were reduced

in size when propagated on confluent or actively dividing BL-1 cells when compared to

the plaque size on Vero cells (Figure 3.4). The plaque morphology of AgK and KOS

was examined on SV-gK, gB-gK and gK-9 cell lines and in these gK-transformed cells

AgK produced microscopic plaques, however, the KOS plaques were similar to those

on BL-1 cell monolayers. Furthermore, the plaque morphology in AgK infected VK302

was larger with 50-100 infected cells/plaque thus indicating to some partial

complementation of the gK-null defect However, the wild-type KOS plaque size was

reduced when compared to Vero cells (Figure 2.6).


97

Figure 3. 4: contd.

Figure 3. 4: Plaque Morphology of KOS and AgK Viruses on Vero and BL-1 Cells.

Panels A and B represent KOS and AgK virus infected Vero cells and panels C and D represent KOS and AgKvirus infected BL-1 cells, respectively. Cells were infected at an m. o. i. of 0. 2 PFU/cell and photographed through a phase contrast microscope at 72 h. p. i.


Figure 3.4


99

Table 3.1: Flaqtiing Efficiency on gK-Tranformed Cells

Virus* Cells Titered On

Genotype Phenotype TiterPFU/mL

KOS Vero gK+ gK+syn+ 2x 10l°

VK302 gK+ gK+syn+ 5 x 10*

gB-gK gK+ gK+syn+g

1 x 10*

BL-1 gK+ K+syn+ 5.3x 10*

SV-gK gK+ gK+syn+ 4 x 10*

gK-9 gK+ gK+syn+ 3 x 106

AgK Vero gK- gK syn+ 4x 10*

VK302 gK- gK syn+ 6x 107

SV-gK gK- gK syn+ 1.2 x 10s

gB-gK gK- gK syn+ 3x 10*

gK-9 gK- gK syn+ 4x 10*

BL-1 gK- gK syn+ 2.6x 101

“Virus titers represent an average of three independent experiments with individual titers varying less than 2-fold. KOS and AgK viral stocks were prepared in Vero and titered on the indicated cell lines.


100

Plaquing Efficiency of AgK and KOS on Different gK -Transformed Cell Lines

The replication properties of AgK and KOS in the gK-transformed cells was

determined in a series of titration experiments. AgK and KOS viral stocks obtained by

propagation on the permissive actively dividing Vero cells, were used to infect

subconfluent Vero, BL-1, SV-gK, gB-gK, gK-9 and VK302 cells. The results of these

experiments are shown in Table 3.1. KOS titers were reduced by 10-fold (1 log) in

VK302 and gB-gK cells; 100-fold (2 log) in BL-1 and SV-gK cells and 10,000 fold (4

logs) in gK-9 cells. The AgK viral titers in Vero cells were reduced by 100 fold(2 logs)

when compared to KOS titers in Vero cells. However, all the gK-transformed cells

showed varying decrements in titer when infected with AgK virus. In VK302 cells, AgK

titers were reduced 10 fold (1 log); in SV-gK the titer reduced by 1000 fold (3 log);

10,000 fold (4 log) reduction was observed in gB-gK and gK-9 cells and in BL-1 cells

the reduction in titer was 100,000 (5 log) when compared to the AgK in Vero cells

(Table 3.1).

Replication Kinetics of AgK and KOS in Vero, BL-1, and VK302 Cells

To further elucidate the replicative characteristics of AgK and KOS, one-step

replication curves were established on Vero, BL-1 and VK302 cells (Figure 2.7; 3 .5A

and 3 .5B). Previously, we have reported that the infectious virus production kinetics

of AgK was markedly different from that of wild-type KOS virus, and that over time

there was an accumulation of virions in the cytoplasm of cells (Figure 2.7). In VK302

cells the replication kinetics of AgK revealed an increase in the accumulation of

infectious virions in the cytoplasm over time, with a 2 log difference in the titer between

extracellular and intracellular virions (Figure 3 .5B). In BL-1 cells, KOS infectious

virions accumulated inside the cells with a 6-log drop between the extracellular virus

and the amount of cell associated virions.


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11 - -11

10 - b-10

9 -

3l/J60Od5PH

2 -

0 4 8 12 16 20 24 28 32 36 40 44 48 0 6 12 18 24 3036 42 48 54 60 66 72 78 84 90 96HOURS POST INFECTION HOURS POST INFECTION

Figure 3.5: Kinetics of Infectious Virus Production in BL-1 and VK302 Cells.

Subconfluent BL-1 and VK302 cells containing approximately 8 x 10 ^ cells per 9.62 cm^ at the time of infection(panels A and B) were infected at an m. o. i. of 10 w ith either KOS or AgK viruses. At various times after infection, virus stocks were prepared from the supernates and cell extracts and were titered on Vero cells. Symbols: □ , extracellular KOS virus; O , cell associated KOS virus; A, extracellular AgK virus; O , cell associated AgK virus; extracellular F-gKp virus; SJ, cell associated F-gKp virus.

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102

The AgK replication was greatly reduced compared to KOS, and the titers of both the

extracellular and intracellular virus remained constant at approximately 1000

PFU/mL (Figure 3 .5A).

Electron Microscopic Examination of KOS and Agk on Vero, BL-1 and VK302 Cells

AgK infected Vero cells revealed an accumulation of capsids in the nucleus, a few

enveloped particles in the cytoplasm and the extracellular spaces devoid of virions as

previous reported (Figure 2.8). Wild-type KOS virus replicated efficiently in BL-1 cells

(Table 3.1). However, electron microscopic analysis of KOS infected BL-1 cells

revealed an accumulation of enveloped virus particles in the perinuclear space, while

a few enveloped virions were found in the cytoplasm and intercellular spaces (Figure 3.

6A). In contrast, examination of more than 100 different AgK-infected BL-1 cells

revealed that most cells did not contain any virion particles (Figure 3. 63), while 2% to

4% of the cells contained a small number of capsids within their nuclei and some

aberrant enveloped virions in the cytoplasm (data not shown). In VK302 cells both

AgK and KOS capsids were seen in the nucleus (Figure 3 .7A and 3 .7B). Enveloped

AgK and KOS virions were visualized in the perinuclear spaces, cytoplasmic transport

vesicles and in the extracellular spaces (Figure 3 .7A and 3 .7B).

D I S C U S S I O N

Partial Complementation of AgK in VK302 Cells

Preliminary evidence demonstrated that the gene encoding gK, the UL53 ORF

could not be deleted and it was hypothesized that this gene product was essential for

viral replication (MacLean et al., 1991). Also, F-gKp, a partial gK-null virus (the gK

gene was insertional inactivated with a p-galactosidase gene) could not be propagated

on Vero cells but replicated efficiently, on VK302 cells.


103

Figure 3. 6: Electron Micrographs of BL-1 cells Infected with AgK and KOS Viruses.

Subconfluent BL-1 cells were infected with KOS virus (panel A) and withAgK virus (panel B) at an m. o. L of 10 PFU/cell, incubated at 37°C for 72 hours and prepared for electron microscopy. The arrows in panel A point to multi-laminar structures formed by the nuclear membranes. n=nudeus, c=cytoplasm, scale bar = 0.5 pm.



105

Figure 3.7: Electron Micrographs of VK302 Cells Infected with AgK and KOS Viruses.

Subconfluent VK302 cells were infected with KOS virus (panel A) and with AgK virus (panel B) at an m. o. L of 10 PFU/cell, incubated at 37°C for 72 hours and prepared for electron microscopy. The arrows in panel A and B points to extracellular virus. n=nucleus, c=cytoplasm/ scale bar = 0.5 pm.


106


107

However, the gK-null virus, AgK (with the entire gene coding for gK deleted) was

isolated in the absence of a complementing cell line and gK was shown to be

dispensable for viral replication in actively dividing cells (Jayachandra, S et aL,

submitted). Interestingly, certain gK-transformed cell lines, BL-1, gK-9, gB-gK and SV-

gK, restricted the replication of AgK virus. The possibility that gK-transformed cells

can neither complement nor inhibit gK-null viruses was examined in detail previously

(Hutchinson et al, 1993; Hutchinson and Johnson, 1995). A rather complicated picture

emerged from those studies. F-gKp failed to form plaques on Vero cells, formed

syncytial plaques in cell lines that expressed low amounts of gK, and its replication

was inhibited in cell lines expressing high amounts of gK in comparison to wild-type

levels. However, F-gKp was able to efficiently replicate in the VK302 cell line that

produced intermediate amounts of gK similar to the levels produced by wild-type

infections (Hutchinson and Johnson, 1995). Enveloped KOS virus was found in the

perinuclear spaces and in the cytoplasm of infected VK302 cells (Figure 3.7), however,

there was a 10-fold decrease in infectious virions between extracellular and

intracellular virions (Figure 3. 5B). This aspect is similar to the reported phenotype of

wild-type KOS virus infections in VK302 cells (Hutchinson and Johnson, 1995).

Suprisingly, VK302 efficiently complemented both the gK mutant viruses, F-gKp and

AgK virus, although the viral yields were reduced in comparison to wild-type

(Hutchinson and Johnson, 1995); Jayachandra, S., submitted), while BL-1 and the other

gK-transformed cells did not efficiently support the replication of AgK virus. One

possible explanation of these differences between different cell lines is that in VK302

and gK-9 cells the gK gene is under the control of the HSV-1 gD promoter whereas in

BL-1 cells the gK gene is under its own promoter, in SV-gK and gB-gK cells, gK is under

the control elements of adenovirus major late promoter and gB promoter, respectively.

The difference between VK302 and gK-9 cells was that the latter had over 200 copies

of the gK gene per cell whereas VK302 cells expressed similar to wild-type levels of gK


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(Hutchinson and Johnson, 1995). Therefore, differential expression due to different

promoter controls may not be responsible for the observed phenotypic differences.

An alternate explanation is that the gK gene that is expressed in VK302 cells

has secondary mutations that overcome the gK-mediated interference. This kind of

restriction has been previously reported for gD-transformed cells. HSV-1 gD

transformed Vero cells inhibits wild-type HSV-1 and HSV-2 infection in several cells

lines (Campadelli-Fiume et al., 1988; Dean et al., 1994; Johnson and Spear, 1989). Most

likely the presence of the expressed wild-type gD precludes penetration of virus into

cells by interacting directly with the virion or by interfering with virus binding to the cell

surface receptors (Dean et al., 1994). Mutant viruses that overcome the gD-mediated

interference have mutations mapping to gD and/or to the gK and gC genes. These

HSV-1 gD mutants that overcome gD mediated interference de-envelope in gD

expressing cells, and there was a 50% reduction in infectious virus in the extracellular

fluids compared to wild-type virus indicating inefficient cellular transport and egress

(Campadelli-Fiume et al., 1990). Furthermore, cells transformed with mutated form of

gD do not restrict wild-type or the gD mutant replication, indicating that mutated form

of gD expressed by these transformed cells does not block the relevant entry. Similarly,

VK302 cells can be postulated to posses second site mutations in gK that help

overcome the restriction to both wild-type virus and AgK virus replication. Currently,

we are in the process of sequencing the gK gene from VK302 cells.

Virus-Induced Cell Fusion in gK-Transformed Cells

Previously it was reported that gB-transformed cells inhibited the fusion caused

by the syncytial mutants with mutations in gB and that gK-transformed cells inhibited

fusion caused by syncytial viruses with mutations in gK (Hutchinson et al., 1993).

Interestingly, viral induced cell fusion was not drastically inhibited in BL-1 infected

with syncytial mutants HSV-1 (MP) or HSV-1 (fsB5). However, infectious viral titers


109

of HSV-1 (MP) were reduced by 100-fold when compared to KOS infected BL-1 cells

and HSV-1 (tsB5) viral titers were similar to KOS titers (Jayachandra et al., 1995). The

inhibition to fusion was at low m. o. L, at high m. o. L viral fusion induced by these

syncytial mutants were not inhibited. Moreover, like wild-type virus, BL-1 cells

infected with syncytial mutants accumulated virions in the nucleus and perinuclear

membranes and no virus was visualized in the cytoplasm or the extracellular spaces of

infected cells implicating defects in post-perinuclear egress defects (Jayachandra et al.,

1995). Furthermore, the synthesis of viral glycoproteins in KOS infected BL-1 cells were

reduced by 3-fold compared to KOS-infected Vero cells.

Restriction of AgK Virus Replication in BL-1 cells and Other gK-Transformed Cell Lines

BL-1 cells that are transformed with the HSV-1 (KOS) BamHI-L fragment

containing the entire UL52 and UL53-gK genes did not support the replication of AgK

(Figure 3 .5A). Wild-type KOS virus replicated on BL-1 cells, however, KOS viral titers

were reduced by two logs when compared to Vero cells (Table 3.1). Electron

microscopic examination of KOS infected BL-1 cells revealed an unusual accumulation

of enveloped virions in the perinuclear space, while there were few enveloped virions in

the cytoplasm and on the surface of infected cells (Figure 3.6). In agreement with the

electron microscopic results, the growth kinetics on BL-1 showed an accumulation of

infectious virions in the cells, with no accumulation of virus in the extracellular fluids.

The phenotypic property is reminiscent of the UL20-null phenotype that caused

accumulation of enveloped virions in the perinuclear space in Vero cells and HEp-2

cells but not in 143TK\ (Baines et al., 1991). A possible interference by UL52

expression in BL-1 cells can be ruled out since BL-1 cells efficiently complement the

UL52-null virus, h rll4 (Goldstein and Weller, 1988). Furthermore, BL-1 cells are

transformed with 10-15 copies of the gK gene which is under the control elements of its

own promoter. Also, other gK transformed cells with the exception to VK302 behaved


110

like BL-1 cells. It is unlikely that BL-1 cells express high amounts of gK, yet it severely

restricts the replication of AgK virus. Also electron microscopic visualization of AgK

infected BL-1 cells did not detect an accumulation of virions in the nucleus, perinuclear

spaces or in the extracellular spaces (Figure 3.6). The level of cellular host-range

restriction provided by BL-1 cells on AgK virus replication is unusually severe, since

viral titers were reduced by 5 logs when compared to KOS titers (Table 3.1)

The mechanism of gK-mediated restriction was further investigated by analyzing

various gK-transformed cells with the gK gene under different transcriptional

promoters. KOS infectious virus production in the gB-gK and SV-gK cells were

marginally reduced, with a 2-fold and 10-fold decrease, respectively (Table 3.1).

However, in gK-9 cells (a gK transformed cell line with 200 copies of the gK gene/cell)

the KOS viral titers were decreased by 1000-fold. Electron microscopic examination of

wild-type HSV-1 strain F, infected gK-9 cells showed an accumulation of enveloped

virions in the perinuclear spaces with no virus detected in the cytoplasm (Hutchinson

and Johnson, 1995). It was postulated that the over expression of gK in gK-9 cells was

detrimental to wild-type HSV-1 egress which resulted in the reduction in infectious

virion production. Interestingly, preliminary evidence shows the wild-type entry

kinetics in gK-transformed cells is delayed when compared to KOS entry in Vero cells,

however, immunofluorescent studies of gB expression revealed that there was a 5-fold

reduction either in the transport of the virions to the nucleus or a decrease in overall

viral protein synthesis (Baghian et al., 1997).

Interference to AgK virus replication in BL-1 and the gK-transformed cell lines

could be explained by the fact that these gK-transformed cells consdtutively express

low amounts of gK which are expressed in nuclear membranes and possibly other

intracellular membranes in the absence of viral infection. The idea is supported by

northern blot studies that indicated low amounts of gK transcripts in uninfected BL-1

cells (Figure 33). Presumably, the KOS virus is able to overcome the fnms-dominant


I l l

inhibitory effect of low amounts of gK expressed in BL-1 cells. The early presence of gK

in BL-1 cells inhibits the translocation of virion particles from the perinuclear spaces to

the cytoplasm, thus effectively trapping enveloped virions in the perinuclear space. In

the case of AgK virus, the absence of viral gK gene expression, causes maximum

inhibition of AgK virus in BL-1 cells. It is not clear at the present time what step of the

AgK virus replication cycle in BL-1 cells is affected by this trans-dominant inhibition of

virus replication.

Wild-type virus, syncytial mutants and AgK infectious virion production was

reduced in gK-transformed cells, however, AgK infectious virion yield was significantly

reduced by 1000-10,000 fold. Moreover, virtually no capsids accumulated in the nuclei

of BL-1 cells infected with AgK virus which may implicate that this resistance by gK-

transformed cells to AgK infection may be due to a multitude of cellular and viral

specified factors. Available data with anti-peptide gK serum, revealed that gK was not

detected in the plasma membrane or in the virion particle and that gK localized to the

RER, Golgi apparatus and the nuclear membranes (Hutchinson et al., 1995). One can

postulate that gK may be functioning in virus entry, and the gK-specified by the gK-

transformed cells may be interacting with host cellular factors like the recently

described accessory protein, HVEM (Montgomery et al., 1996). Herpes virus entry

mediator (HVEM), a HeLa cell cDNA when stably expressed transfers susceptibility

to HSV-1 entry to the normally resistant Chinese hamster ovary (CHO) cells. HVEM-

transformed cells were shown to enhance entry of a variety of HSV-1 strains and is

implicated to mediate post-attachment events (Montgomery et al., 1996). Interestingly,

the syncytial mutant virus, HSV-1 (MP) that has the syn 1 mutation in gK and is gC

negative, enters and replicates albeit inefficiently in CHO cells that were normally

resistant to HSV-1 wild-type. However, in HVEM transformed-CHO cells, the entry of

HSV-1 (MP) was significantly enhanced which raises the possibility that some other

cell-specified accessory factors may be mediating the entry of syncytial mutants. In gK-


112

transformed cells this interaction could be at an earlier event occurring during the

glycoprotein processing and maturation of both the cellular HVEM-like mediator and

the cell-specified gK. The interaction could render the HVEM-like mediator inactive/

precluding infection by AgK mutants. However, the binding of the accessory entry

mediator may not be absolutely essential for entry of wild-type strains, which have

been shown to enter gK-transformed cells, although with slower entry kinetics (Baghian

et a l, 1997)

An alternate explanation is that the gK antipeptide serum could not efficiently

detect the mature form of gK that may be present in the virion particle or in the plasma

membrane of infected cells. The gK specified by these transformed cells may interfere

with HVEM or other unidentified gK-spedfic plasma membrane receptor resulting in

the inaccessibility of this portal of entry by AgK virions. The low amounts of gK that

are expressed in gK-transformed cells can partially block receptors, and incoming wild-

type virus overcomes this block or uses alternate entry pathways due to presence of

functional gK

A second possible explanation is that transport of virions from the plasma

membrane to the nucleus may be significantly delayed in gK-transformed cells.

Preliminary evidence shows that wild-type KOS has a 2 hr delay in the synthesis of

glycoproteins in gK-transformed cells (Baghian et al., 1997). The transport of AgK

virions in gK-transformed cells may be delayed even further, and the viral capsids may

not form the appropriate post-entry intermediates required for progress of virion

particles to the nucleus which could result in the AgK virions being directed to

endosomes and lead to their eventual degradation as evidenced by wild-type KOS

infections of gD transformed cells.

The third possibility is that AgK viral capsids are blocked from docking at the

nuclear membrane, while KOS viral capsids enter the nucleus and viral replication

ensues. The absence of gK in AgK virions may alter the tertiary structure of the virion


113

particle that enables the recognition of nuclear receptors. The possibility that the viral

DNA encoded by AgK is defective is unlikely, because the AgK virions are able to enter

Vero and HEp-2 cells normally and establish efficient replication.

Lastly and more importantly, gK plays a crudal role in virion egress and may

modulate this pathway. The intracellular vesicular pathway comprises a series of

membrane bound compartments between which the vesicles are shuttled during the

process of transport of macromolecules. One important aspect of this pathway is that

macromolecular synthesis and transport originating in the ER must be transported,

sorted and distributed by transport vesicles to their correct destinations. The process

thus requires a machinery that is responsible for the formation, targeting (pilots and

sorters) and fusion of transport vesicles. HSV-1 may utilize this vesicular pathway to

egress from cells, indicating that certain viral factors either mimic or act in coordinance

with cell factors to facilitate virion intracellular transport In wild-type infected gK-

transformed cells virions accumulate in the perinuclear spaces, reminiscent of the

UL20-null virus. In the previous chapter it was reported that AgK virions are most

likely transported in vesicles derived from the outer nuclear lamella, but in the absence

of gK these vesicles failed to correctly transport the virion particles to extracellular free

spaces. Interestingly, in gK-transformed cells these outer lamella derived transport

vesicles are not formed, resulting in accumulation of wild-type virions in the

perinuclear spaces. These results are consistent with the role of gK in formation of

transport vesicles from the outer lamella and the subsequent piloting and or/sorting of

such vesicles to the Golgi and eventually to the plasma membrane. Further, it points to

a possible role for nucleus-bound factors that are needed for transport vesicle

formation, and in gK-transformed cells such cellular factors are inhibited by the early

expression of cellular gK


CHAPTER IV

SITE-DIRECTED MUTAGENESIS OF THE AMINO TERMINAL OF HSV-1 GK

INTRODUCTION

HSV-1 glycoprotein K is involved in viral envelopment and virion egress, as

reported in the previous chapters. gK is also involved in modulation of viral induced

membrane fusion events (reviewed in detail in literature review). The syn 1 loci maps to

gK and this locus can be further separated into two syncytial domains, one domain

located at the amino terminal a t/o r around amino add 40 and the other located

towards the carboxy terminal of gK (Dolter et al., 1994) (Figure 1.5 and Figure 4.1). A

number of syncytial mutants have mutations mapping to the N-terminal of gK (Bond

and Person, 1984; Bzik et al., 1984b; Debroy et al., 1985; Hutchinson et al., 1992b;

Manservigi et al., 1977; Pogue-Geile et al., 1984; Ryechan et al., 1979). Five of eight such

mutants were N-terminal domain mutants (Dolter et al., 1994). Four of the five HSV-1

(MP), HSV-1 (KOS) syn 20, syn 102 and syn 105 have mutations at amino add 40 of

the primary sequence of gK (Figure 4.1). In all mutants except syn 20, the change was

from Ala 40 to Val 40. In syn 20 the mutation was from Ala 40 to Thr 40. The single

point missense mutations that resulted in these amino add substitutions rendered the

virus to cause extensive cell-cell fusion or syncytia. In cell culture, infections with MP,

syn 102, and syn 105 were considered to be dominant as they caused extensive

(strong) fusion, whereas infections with syn 20 were considered to be weakly

codominant because the plaques were only partially fused (Dolter et al., 1994).

Another syncytial mutant virus HSV-1 (KOS) syn 8 has amino add substitution at

position 33, that results in a Pro 33 to Ser 33 change. This mutant has two additional

mutations in gK that may or may not be related to the fusion phenotype. However, the

Pro 33 to Ser 33 is a drastic change from small nonpolar, helix breaker to a more

hydrophilic polar amino add.

114


115

S Y N C Y T I A L M U T A T I O N S I N G L Y C O P R O T E I N K

1 * * 50HSV-1 (KOS) MLAVRSLQHL STWLITAYG 1VLVWYTVFG ASPLHRCIYA VRPTGTHNDTHSV-1 (MP). Syn 1 V ........HSV-1 (KOS). Syn 8 S..............HSV-1 (KOS). Syn 20 T .........HSV-1(KOS). Syn 30 .............................................HSV-1(KOS). Syn 31 .............................................HSV-1 (KOS). Syn 102 V ........HSV-1(KOS). Syn 103 .............................................HSV-1 (KOS). Syn 105 V ........

HSV-1 (KOS)HSV-1 (MP). Syn 1 HSV-1(KOS). Syn 8 HSV-1(KOS). Syn 20 HSV-1(KOS). Syn 30 HSV-1(KOS). Syn 31 HSV-1(KOS). Syn 102 HSV-1(KOS). Syn 103 HSV-1(KOS). Syn 105

51 100* *

ALVWMKMNQT 1LFLGAPTHP PNGGWRNHAH ICYANLIAGR WPFQVPPDA

N.

HSV-1 (KOS)HSV-1 (MP). Syn 1 HSV-1(KOS). Syn 8 HSV-1(KOS). Syn 20 HSV-1(KOS). Syn 30 HSV-1(KOS). Syn 31 HSV-1(KOS). Syn 102 HSV-1(KOS). Syn 103 HSV-1(KOS). Syn 105

101 * 150TNRRIMNVHE AVNCLETLWY TRVRLVWGW FLY1AFVALH QRRCMFGWS M............................................

Figure 4.1: contd.

Figure 4.1: Alignments of Protein Sequences of gK Specified By Different HSV-1 Syn Mutants.

Sequences were aligned using the University of Wisconsin Genetic Computer Group software package. Dots represent amino adds identical to the parental wild-type HSV-1 KOS sequence. Uppercase letters denoted below the wild-type HSV-1 KOS sequence represent mismatch amino adds. " * " depicts the mutated amino adds that result in the syncytial phenotype.


116

151 200HSV-1 (KOS) PAHKMVAPAT YLLNYAGRIV SSVFLQYPYT KITRLLCELS VQRQNLVQLFHSV-1(MP). Syn 1 .............................................HSV-1(KOS). Syn 8 .............................................HSV-1(KOS). Syn 20 .............................................HSV-1(KOS). Syn 30 .............................................HSV-1(KOS). Syn 31 .............................................HSV-1 (KOS). Syn 102 .............................................HSV-1(KOS). Syn 103 .............................................HSV-1(KOS). Syn 105 .............................................

201 250HSV-1 (KOS) ETDPVTFLYH RPAIGVIVGC ELIVRFVAVG LIVGTAFISR GACAITYPLFHSV-1 (MP). Syn 1 ML.......................HSV-1(KOS). Syn 8 .............................................HSV-1(KOS). Syn 20 .............................................HSV-1(KOS). Syn 30 .............................................HSV-1(KOS). Syn 31 .............................................HSV-1(KOS). Syn 102 .............................................HSV-1(KOS). Syn 103 .............................................HSV-1(KOS). Syn 105 .............................................

251 300HSV-1(KOS) LTITTWCFVS TIGLTELYCI LRRGPAPKNA DKAAAPGRSK GLSGVC®CCHSV-1(MP). Syn 1 .............................................HSV-1(KOS). Syn 8.................................................HSV-1(KOS). Syn 20 .............................................HSV-1(KOS). Syn 30 .............................................HSV-1(KOS). Syn 31 .............................................HSV-1(KOS). Syn 102 .............................................HSV-1(KOS). Syn 103 .............................................HSV-1(KOS). Syn 105...............................................

301 338* *

HSV-1 (KOS) SIILSGLAMR LCYIAWAGV VLVALHYEQE IQRRLFDVHSV-1 (MP) . Syn 1.... V..........................HSV-1(KOS). Syn 8......................................HSV-1(KOS). Syn 20 ..................................HSV-1 (KOS). Syn 30 .. .P...............................HSV-1(KOS). Syn 31 ..................................HSV-1(KOS). Syn 102 ..................................HSV-1 (KOS) . Syn 103 ...... L .........................HSV-1(KOS). Syn 105 ..................................

Figure 4.1


117

In vitro transcription, translational and truncation experiments have also shown

that gK has a signal peptide that is cleaved at amino add 30, has two N-linked

glycosylation sites at amino add position 48 and 58, respectively, and the first

hydrophobic domain anchors the amino one-third of the protein to membranes

(Ramaswamy and Holland, 1992) (Figure 1.5). Considering all these facts it was

suggested that residues 31 to 124 form the ectodomain of gK and is probably involved

in viral induced cell fusion.

In an attempt to rapidly delineate and map the functional domains that are

involved in virus-cell fusion and in viral egress, a long-PCR site-directed mutagenesis

method was developed that results in nearly 100% mutagenesis of the final DNA

products (Chouljenko et al., 1996). The method utilizes long-PCR enzymes and

conditions to efficiently amplify DNA fragments. The long PCR fragments carrying

specific mutations could efficiently recombine with intact viral DNA in the novel

recombinant system that was developed in chapter 2. This procedure of creating site-

specific mutations was demonstrated by generating a new restriction endonuclease site

and a missense mutation in the glycoprotein K(gK) gene specified by herpes simplex

virus (HSV-1). A recombinant virus that carried the mutated gK gene caused extensive

cell fusion of infected cells (Chouljenko et a l, 1996). Using this long PCR site-directed

mutagenesis procedure three different mutations were introduced into the gK gene, such

that the translated gK proteins would have amino add substitutions at Ala 31 to Val

31, Thr 50 to Val 50 and Thr 60 to Val 60.


Cells and Viruses

Vero, rabbit skin cells and V27 cells were obtained and maintained as

described previously (Chapter 2 materials and methods). HSV-1 (KOS), the parental


118

wild-type virus strain and HSV-1 (KOS) d27-l were propagated in Vero and V27 cells

respectively (Chapter 2 materials and methods).

Equipment and Reagents for PCR

All experiments were performed using the GeneAmp PCR System 9600 according

to manufacturers instructions (Perkin Elmer Corporation, Norwalk, CD. All PCR

reagents were provided as part of the XL PCR Kit (Part No. N808-0182; Perkin

Elmer/Applied Biosystems, Foster City, CA). Hot Start PCR was performed with

Ampli Wax PCR Gem 50 (Part No. N808-0150; Perkin Elmer/Applied Biosystems,

Foster City, CA).

Restriction Enzymes and Plasmid

Restriction enzymes Bam HI, Bbs I and Kpn I were obtained from New England

BioLabs (Beverly, MA). Plasmid pSJ1710 described previously (Chapter 2 materials

and methods) contains the 3.2 kbp EcoR I-BamH I fragment, which contains the

carboxy terminal two-thirds of the UL52 ORF and the complete UL53 ORF. The

plasmid was digested with Kpn I-BamHI to generate a 1340 bp DNA fragment that

was used as a 'cast7 DNA.

Primers for Long PCR and Mutagenesis

Primers LN5 (5’- GGC AAC GAC GAC GAC GGG GAC TGG T-3’), C3 (5’-GGC

CTT GGA ACG GGG ACG GAT TTA T-3’), Mutagenic primer-MUTl (5’-CCG TCT

TTG GTG TCA GTC CGC TGC A-3% Mutagenic Primer-MUT2 (5’-TGC AGC GGA

CTG ACA CCA AAC ACG G-3’), P2 (5'-TGG GTC CTC CTA CAG CTA-3') and P3

(5' -GGC ACA GAC AAG GAC CAA TCA GAC ACC A-3') were synthesized in our

laboratory with a Perkin Elmer/ Applied BioSystems DNA Synthesizer Model 394

using phosphoramidite chemistry. The approximate location of primers LN5, C3,


119

MUT1 and MUT2 and the gK gene on the viral genomic map is shown in Figure 4.2.

The PCR fragment produced by the LN5 and C3 primer pair was predicted to be 4314

bps-long with an average GC content of 65.2%. The DNA fragment amplified by

LN5/MUT2 primer pair was predicted to be 1156 bp with a GC content of 64.4%, and

the PCR primer pair MUT1 /C3 produced a product of 3183 bps with a GC content of

65.6%. Primers P5 and P6 bracket the point mutation and were used for detection of

the engineered mutation in mutant viruses (Figure 4.2).

Extension of the LN5/MUT2 PCR Product Using the Cloned gK DNA Fragment or "cast*

The template wild-type KOS viral DNA was prepared as described in materials

and methods of chapter 2. The 1130 bp PCR fragment derived from the primer pair

LN5/MUT2 (500 ng) was mixed with the cloned Kpn I-Bam HI DNA fragment (125

ng), denatured and reannealed. The total volume of DNA mixture was brought up to

55.6 mL and the parameters for extension were: Extension reaction mix (100 mL):

DNA solution (55.6 ml), Mg(OAc)2 (4.4 ml), 3.3X XL buffer (30.0 ml), dNTPs, (8.0 mL

of 2.5 mM-stock), rTth, XL (2.0 ml). Thermal profile: 3' at 95°C; 1' at 94°C, and 15' at

72°C for 7 times.

Final Overlap-Extension

The LN5/MUT2 PCR extended product (50 ng) was gel-purified vising the Prep-

A Gene purification method as detailed by the manufacturer (Bio-Rad, Inc, Richmond,

CA), and was used for the next overlap-extension with the MUT1/C3 product (50 ng).

Extension reaction mix (100 ml): DNA solution (55.6 ml), Mg(OAc)2 (4.4 ml), 3.3X XL

buffer (30.0 ml), dNTPs (8.0 mL of 2.5 mM stock), rTth, XL (2.0 ml). Thermal Profile:

3’ at 95°C; 1' at 94°C, and 15' at 72°C repeated 3 times.


120

Long PCR Mutagenesis Procedure

( a ) Location of primers on the HSV-1 viral genome™ » M g lU L S ^ K „ „

3* •// ------- ■ '----- — — — — ------— 5*M U R P3 C3

^ LN5:MUT2 PCR product

3’ „ MUT1C3 PCR ProductS’.*____________________ •»©^ _ Cloned gK gene "CAST1

'ssssssss»

@ Stepl: Extension of LN5:MUT2 PCR product using cloned gK gene. Predicted frequency for mutation is 50%

S’ - * x -3’ --------------- 1

No extension3: •"S' No extension

/p \ s te p i Extension of product from step 1 using the MUT1:C3PCR '■*' Product shown in (c)

S' _____________ 6.__ _________3. -------------------------------S'*-2------------

No extension ** ■ *■y No extension

(g) Step3: PCR with LN5 andC3 to produce final productPredicted frequency far mutation is 100%

5’- J ^ _______ __ __________3'

R tH 5'

r 5'

P3

Figure 4.2: Schematic D iagram of the M utagenesis Procedure.

A) The HSV-1 genome showing the location of all the primers and the gK gene. The location of the engm eeredm utaion is denoted(A). (B and C) The LN 5/M ut2 PCR product (B) and the M u tl/C 3 PCR product (C). (D) The cloned DNA gK fragment used for the first extension reaction. (E) The first extension reaction (— )of the LN5/M ut2PCR product using the doned gK DNA fragment. (F) The extensionof the PCR product from step E using the PCR DNA fragm ent represented in step (C). (G) The final PCR product using primers LN5 and C3. This DNA fragm ent contains the desired m utation (A).


121

Final PCR Conditions

The primer pair LN5/C3 was used to amplify the 4279 bp DNA fragm ent PCR

parameters were: 3' at 95°C; 30' at 94°C, 8' at 68°C repeated 30 times, and final

extension for 15' at 72°C. Hot Start conditions were applied according to the

Manufacturer (Perkin-Elmer/Applied Biosystems) and the composition of lower and

upper reaction mixtures were as follows: Lower Reaction Mixture (40 ml-total): water

(12.4 ml), 3.3X XL Buffer (12.0 ml) dNTPs (8.0 mL of 2.5 mM stock)), Primer LN5 (1.6

mL of 20 mM stock), Primer C3 (1.6 mL of 20 mM stock), Mg(OAc)2, (4.4 mL of 25

mM stock). Upper Reaction Mix (60 ml): water (38 ml), 33X XL Buffer (18 ml), viral

DNA (1 mL of 7 mg/ml-stock), rTth DNA PoL XL (2 mL of 2 U/ml-stock).

Generation of Recombinant Virus and Detection of Site-Specific M utation

Rabbit skin cells were transfected with the mutagenized long PCR fragment

using the calcium phosphate technique (Chen and Okayama, 1987) and twenty four

hours post transfection cells were superinfected with d27-l viruses. Forty eight hours

post infection, recombinant viral stocks were made and titered on Vero cells. For PCR

analysis, viral DNA mini-preparations were prepared from plaque-purified virus

stocks as described previously (Chapter 2 materials and methods). Viral DNAs from

each of the viral plaques were subjected to PCR analysis using internal gK primers P2

and P3 and the subsequent PCR products were digested with restriction endonuclease

Bbs I.

RESULTS

PCR Mutagenesis

A pair of mutagenesis primers MUT1 and MUT2 were designed such that a Ala

to Val change was created at position 30 of the gK primary sequence and also the

existing cleavage site for the restriction enzyme Bbs I was obliterated.


122

1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 13 14

Figure 4.3: Agarose Cel Electrophoresis of Viral PCR Products Restricted with Bbs L

P5/P6 PCR products from putative mutagenized SJVal31 virus (lane 2 thro 9), laboratory virus strain KOS (lane 10 thro 13). PCR DNA fragments were digested with restriction endonuclease Bbs I (lanes, 3,5,7,9,11 and 13 respectively). Molecular weight marker, X phage DNA digested with HindEH (lane 1 and 14).


123

The mutagenesis procedure is outlined in Figure 4.2 and was composed of three main

steps, hi step 1, the cloned gK gene was used to extend the LN5/MUT2 PCR product

Fifty per-cent of the extension reaction products are predicted to contain the

engineered mutation. The size of the first extended product was 2190 bps. The

extended product was used in step 2, with the MUT1 / C3 PCR product this second

extension was predicted to raise the frequency of the mutation to 100%. Final

amplification was performed with primer pair LN5/C3 (Figure 4.2). Final PCR

products were tested for the absence of Bbs I restriction endonuclease site by digestion

with Bbs I (Figure 43).

Markex-rescue and Transfer of the Mutation (Ala 31 to Val 31 change) and Elimination of the Bbs I Restriction Site from the KOS Genome

Viral DNA from individual plaque purified recombinant viral plaques obtained

from the transfection of full length mutagenized DNA fragments (4279 bps) and

infection with d27-l virus were subjected to PCR using primers P2 and P3 that were

designed to bracket the site of the mutation (Figure 4. 2, A). P2 was located 281 bases

5' to the Bbs I site, and P3 was located 1323 bases 3' to the Bbs I site. Therefore, in

wild-type fragment, digestion by Bbs I of the P2/P3 product is expected to produce

two DNA species of approximately 281 and 1323 bps. The PCR fragments obtained

from purified viral DNAs from the recombinant viral plaques were restricted with Bbs

I. The loss of the Bbs I site was confirmed as both the undigested and digested PCR

fragments were of the same size (1604 bps). (Figure 4.3). Four viral isolates SJVal31

that contained the mutations that change Ala to Val 30 and which do have the Bbs I

restriction endonuclease site were obtained (Figure 4.3 lanes 2 through 9). Control viral

strain HSV-1 (KOS) that has the Bbs I restriction site (Figure 4.3 lane 10 through 13).


124

DISCUSSION

An efficient long PCR site-mutagenesis procedure was developed to produce

mutations in the gK gene (Chouljenko et al., 1996). Making use of the strategy to insert

site specific mutations into long DNA fragments in conjunction with the newly

developed recombinant system (discussed in chapter 2), additional mutations were

generated to delineate the 5' boundary of the N-terminal syncytial domain in gK.

The Ala to Val substitution at amino add 31 is a conservative change, in that a

small polar Ala residue was substituted with a larger polar Val residue. A sim ilar

substitution at residue 40 causes extensive cell fusion (Chouljenko et a l, 1996; Dolter et

al, 1994). The recombinant viruses with the Ala 31 substitution were designated

SJVal31, did not cause extensive cell fusion and the plaque morphology was

indistinguishable from the parental wild-type strain KOS.

Mac Vector protein analysis software package was utilized to analyze the

predicted primary structure of proteins. The first 50 amino adds specified by the wild-

type gK and by the SJVal31 gK are shown in Figure 4.4. These results show the amino

add substitution at residue 31 did not result in any drastic changes in the

hydrophilidty, surface probability and secondary structure when compared to the

parental wild-type gK. However, there was a slight reduction in the flexibility and

antigenic index in this region (Figure 4.4). Also, the Ala 31 to Val 31 amino add

substitution did not eliminate the existing signal peptidase deavage site.

These results along with the previously published data fin e-maps the 5'

boundary of the N-terminal syncytial domain. Ala 31 is the first amino add after

deavage of the 30 amino add signal peptide, does not play a role in the viral induced

cell fusion. The amino add residues from 31 thro 36 are not conserved among the other

alphaherpesviruses, however, Ala 31 is part of the first antigenic portion of gK that is

predided to form a (5 turn (Figure 4.4 Secondary Structure).


125

Figure 4.4: Secondary Structure Predictions and Comparison of KOS-Ala31 and SJ-Val31.

Analysis of the engineered amino add in SJ-Val31 compared to the parent KOS-Ala31. The First 51 amino adds were analyzed using the "Protein Toolbox" application of the MacVector software package (IBI-Kodak). Hydrophilidty, Antigenic indices, Flexibility, and secondary structure profiles were compared. CF=Chou-Fasman secondary structure algorithm; RG= Robson-Gamier secondary structure alogrithm


126

K0S-Ala31

Hydrophiticity Window Size = 7 Scale = Kyte-Doolittle

4 .00

2 . 0 0

0 . 0 01 . 00

t r5 10 15 20 25 30 35 40 45 50MLAVRS LQHLS TVVLITAYGLVLVWYTVFGAS P LHRCIYAVRP TG TNND TA

SJ-Val31

Hydrophilicity Window Size

o.oo- 1 . 00- 2 . 0 0

-4 .0 0-5 .0 0

Scale = Kyte-Doolittle

I r

5 10 15 20 25 30 35 40 45 50MLAVRSLQHLSTVVLITAYGLVLVWYTVFGVS PLHRCIYAVRPTGTNNDTAK O S-A la31

X<D■o

c<U

1.0.0.0.0.0. 1

-o.;-o . -- 0.1- 0 . )- i . <

SJ-Val31

f j.....T" ' .......... f I j

f 1 1 1 tE E J j1 ______

5 10 15 20 25 30 35 40 45 50MLAVRSLQHLSTVVLITAYGLVLVWYTVFGASPLHRCIYAVRPTGTNNDTA

1.00

S 0 .60

c<0o>"c< -0 .8 0

5 10 15 20 25 30 35 40 45 50MLAVRS LQHLSTVVLITAYGLVLVWYTVFGVSPLHRCIYAVRPTGTNNDTA

Figure 4. 4: contd.


97

127

K 0S-A la31

1 .2 51 .201 .153k 1 .101 .0 5

A 1 .00X 0 .9 5iZ 0 .9 0

0 .8 50 .8 00 .7 5

SJ-Val31

t ------ 1------- 1------ 1------ r5 10 15 20 25 30 35 40 45 50ML AVRS LQHLS TVVLITAY GLVLVWYTVF GASPLHRCIYAVRP TGTNND TA

1 .251 .201 .151 .101 .051 .00

X 0 .9 50 .9 00 .8 50 .8 00 .7 5 i ------ 1------- r

5 10 15 20 25 30 35 40 45 50ML AVRSLQHLSTVVLITAYGLVLVWYTVFGVSPLHRCIYAVRPTGTNNDTAKOS-Ala31

CF H e lix ' CF S h e e t CF T urns RG H e lix RG Sheet- RG T urns CfRg Hlx CfRg S h t CfRg Tmcr>

5 10 15 20 25 30 35 40 45 50MLAVRSLQHLSTVVLITAYGLVLVWYTVFGASPLHRCIYAVRPTGTNNDTA

SJ-Val31

h CF H e lix CF S h e e t

= CF T urns ~ RG H e lix % RG S h e e t »- RG T urns ■§ CfRg Hlx- § CfRg S h t a CfRg Trn

T5 10 15 20 25 30 35 40 45MLAVRSLQHLSTVVLITAYGLVLVWYTVFGVS PLHRCIYAVRPTGTNNDTA

Figure 4. 4


128

Previously, a syncytial mutation HSV-1 (KOS) syn 8 was reported to have a mutation

at Pro 33 and other mutations elsewhere in the gK gene. Whether the Pro 33 to Ser 33

substitution in the absence of other mutations results in a similar cell fusion phenotype

is yet to be determined.

The two other amino add substitutions that were engineered by site directed

mutagenesis were Thr 50 to Val 50, and Thr 60 to Val 60 that are conservative amino

add substitutions. The plaque phenotype of the recombinant viruses were similar to

wild-type KOS. PCR analysis showed that the d27-l deletion was repaired, however,

restriction enzyme analysis foiled to detect the engineered restriction endonudease

sites. This indicates that the recombinant viruses did not have the site-specific

substitutions. The obvious explanation for these results is that the deletion in d27-l

virus was rescued by the DNA fragment, however the recombination sites were

probably to the right of the mutations as there is approximately 1 kbps of intervening

sequence between the d27-l deletion and the engineered mutations. Also the low

frequency of recombination with these fragments could point to the fact that the amino

adds substitutions disrupt the N-glycosylation sites in gK that could render this

protein non-functional which would be lethal for viral replication. To further analyze

these mutations, a more stringent recombination system is required. A better virus that

could serve this purpose would be the AgK virus, which has the entire gene coding for

gK deleted. The mutagenized DNA fragments could be used in conjunction with AgK

virus such that the deletion in gK is repaired by the DNA fragments with simultaneous

transfer of the site-specific gK mutations. However, a potential drawback to this

strategy lies in the inability to phenotypically differentiate between the AgK virus

plaques and the recombinant mutant virus plaques which makes the screening process

more labor intensive.


C H A P T E R V

C O N C L U D I N G R E M A R K S

S U M M A R Y

Glycoprotein K (gK) was hypothesized to be involved in virus induced cell to cell

fusion mechanisms which involves fusion of adjacent cellular membranes resulting in

polykaryotes or syncytia, because a number of HSV-1 mutants that cause extensive cell

to cell fusion were mapped to the gK gene. Hutchinson, et al., (1995), constructed a

partially deleted FgK-p virus and demonstrated that gK is essential for viral replication

and egress, because FgK-P virus could be propagated only on a complementing gK-

transformed cell line. In this research, a novel recombination method for transferring

mutations and deletions into the gK gene was developed and a gK-null virus, AgK, with

a complete deletion of the gK gene was constructed. In contrast to previous findings

(Hutchinson, et al., 1995), characterization of the phenotypic properties of AgK virus

revealed that gK was dispensable for viral replication in exponentially dividing cells,

however gK was essential for viral replication in stationary phase cells. Furthermore,

gK was required for efficient viral envelopment and in the translocation of infectious

virions from the perinuclear spaces to the cytoplasm and from the cytoplasm to the

extracellular spaces. Based on the findings from this research, a unique role for gK in

the formation and translocation of virion transport vesicles has emerged.

It has been hypothesized that single amino add mutations in gK result in the

syncytial phenotype by decreasing the ability of gK to inhibit cell to cell fusion and in

this regard the gK truncation in FgK-p behaves in a similar manner. Apparently the

hypothesis that gK is an inhibitor of cell to cell fusion is incorrect, because deletion of

the entire gK does not cause cell fusion. It remains unclear how gK partidpates in the

cell fusion phenomenon. Available data indicate that the HSV-induced cell fusion

involves a complex mechanism which involves multiple viral and cellular proteins. It is

worth pointing out that in the carboxy-region of gK, a conserved double Cys motive

129


130

was found, CXXC where X is any amino add, which has sequence similarities to the

mammalian Mss4 protein that is required for vesicular transport Thus it is conceivable

that gK may possess two functional domains where the amino-terminal one-third of

the glycoprotein functions in membrane fusion, probably in the triggering of budding

and/or the formation, of virion transport vesides, whereas the carboxy one-third of

the protein is required for targeting and transport of the vesides. Interestingly, the

presence of unenveloped FgK-(3 capsids in the cytoplasm of infected cells indicate that

the amino-terminal of gK when expressed alone causes aberrant fusion of membranes

and probably inhibits the formation and transport of vesides. However, in the total

absence of gK the AgK virions acquires envelopes and are found in the cytoplasm of

actively dividing HEp-2, and Vero cells, indicating that certain cellular factors can

partially compensate for loss of gK function in actively dividing cells. It is tempting to

speculate that gK could mimic or alter the function of certain cellular GTP binding

proteins (such as ARF and SAR) that are involved in the veside budding and

trafficking from the ER to the ds-phase of the Golgi.

Information about the function of gK was also obtained by studying the

replication of wild-type and gK-null viruses in gK-transformed cells. gK-transformed

cell lines are unique in that they restrict the replication of the gK-null viruses and the

plaque production of wild-type viruses, respectively, with the exception of one cell

line. Enveloped wild-type virions were trapped in perinuclear spaces of gK-

transformed cells confirming the role of gK in virion translocation and further alluding

to the role of cellular or other viral factors that may interact with the cellular gK and

modulate the formation of virion transport vesides.

Finally, feasibility experiments were performed to fine-map the amino-terminal

syncytial domains in gK by utilizing a long PCR site-directed mutagenesis method.

Three mutations were constructed and one virus recombinant, SJVaBl containing an

Ala to Val substitution at amino add 31 was isolated.


131

FUTURE RESEARCH CHALLENGES

Infection of cells by enveloped viruses requires that viruses overcome multiple

membrane barriers. In the case of HSV these barriers include the plasma membrane

during virus entry and the outer nuclear lamella, the Golgi membranes and the plasma

membrane during egress. Also, cells possess complex and well regimented networks by

which proteins and macromolecules that are synthesized within the cell are

transported to the exterior. Viruses with their limited coding capacity have evolved

optimal mechanisms by which they can modulate and overcome these physical barriers

and in turn utilize the cellular environment for both entry, maturation and egress. Thus

there are various viral and cellular proteins that either independently or in synergy

regulate the different membrane fusion events and the translocation of virions.

In particular, these investigations elucidated that gK is involved at various levels

at both virus egress and preliminary evidence shows that it may be involved in viral

entry. This research supports the model that virion particles are transported in

specialized vesicles (modules) as it is thermodynamically unsuitable for viruses to de

envelope and re-envelope multiple times as it transits through the vesicular transport

system. The sequential de-envelope/re-envelopment could compromise the structural

integrity of the virus. It is interesting to note that vesicular glycoprotein transport has

been studied in great depth, however, mechanisms by which large macromolecules

transit through cells are unknown. The vesicles that shuttle proteins between the RER

and the ris-phase of the Golgi have diameters of ~70nm, HSV with a particle size of

-130 nm seems to utilize a similar pathway. A possible mechanism for intracellular

virus transport would be to fuse vesicles together to produce a large transport vesicle.

In this regard, gK with membrane fusing and transport properties can be envisaged to

interact with other cellular or viral factors to modulate and regulate this mechanism.

Future investigations should be aimed at clarifying the role of gK and gK-

mediated interference in virus entry and to examine the molecular basis of gK's


132

involvement in the vesicular transport. Some possible questions and research ideas

proposed in this regard are:

1) To clarify the role of the amino-terminal one-third of gK in fusion by constructing a

KOS mutant virus that is similar to FgK-(3, expressing the amino-terminal one-third of

gK and the 26 amino adds specified by the ICP6 promoter region. The mutant virus

would express a truncated form of gK in a syngendc background; it would be possible

to determine if the partial fusion phenotype of FgK-p is due to the presence the amino-

terminal or if amino-terminal interacts with other HSV-1 specified proteins and

regulates the fusion phenomena.

2) To examine the carboxy-terminal region of gK and identify its role in virion vesicular

transport by either site-directed mutagenesis of the Cys residues in the wild-type KOS

virus or by doning the Mss4 gene into the AgK virus to determine enhanced transport of

viral partides in stationary phase Vero cells.

3) To examine the structural integrity of the AgK virion partide by analyzing the

glycoprotein profile of sucrose gradient purified AgK virions and comparing it with the

purified wild-type KOS virions.

4) To analyze the role of gK in viral attachment and entry and to detect if gK is present

in the virion partide by epitope tagging gK and tracing the intracellular localization of

gK in infected cells.

5) To determine the molecular basis for complementation and interference by different

gK transformed cell lines for both wild-type KOS and AgK viral replication by i)

Obtaining revertant KOS and AgK virions that overcome the viral replication

restrictions in the gK-transformed cells and mapping the genes involved by marker

rescue experiments, ii) Obtain the DNA sequence of the cellular gK gene from the gK

transformed VK302 cells that complement both KOS and AgK virus replication, iii) To

examine the level of constitutive transcription and translation of the cellular gK genes

from different gK transformed cell lines in the absence of viral infection.


133

In conclusion this research aimed to improve existing knowledge of HSV-1

glycoprotein K and in particular intracellular infectious virion maturation and egress,

and in this regard, it contributes to the overall understanding of intracellular transport

of viruses and macromolecules not only for herpesviruses but in other enveloped viral

systems. This study along with work from other laboratories would hopefully lead to

advances in understanding of virus-host cell interactions the successful establishment

of infection by viruses and to exploit new targets for antiviral therapy.


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VITA

Sukhanya Jayachandra was bom in Bangalore, India, on September 6,1965, the

middle child of Mr. R. Jayachandra and Mrs. J. Jayakumari. She received her high

school diploma from Sophia High School (Bangalore, India) in April, 1982. The author

obtained a Bachelor of Science degree in Botany, Zoology and Chemistry from M ount

Carmel College (Bangalore, India) in 1987. Sukhanya came to the United States in 1988

to pursue higher education; received a Master of Science degree in Biology from Clarion

University (ClarionPennsylvania) in 1991 for her thesis entitled "Isolation and

Preliminary Characterization of Noncytopathic Mutants of Bluetongue Virus". She

came to Louisiana State University in September of 1990 to study the molecular

biology of herpesviruses with Dr. Konstantin G. Kousoulas in the Department of

Veterinary Microbiology and Parasitology. She earned her Doctor of Philosophy degree

in May, 1997. Her hobbies include reading books on science, novels, and cultural

philosophies. She also enjoys gourmet cooking, gardening, astronomy (star gazing) and

sewing.

154


DOCTORAL EXAMINATION AND DISSERTATION REPORT

C a n d i d a t e : Sukhanya Jayachandra

M a jo r- F i e l d : Veterinary Medical Sciences

T i t l e o f D i s s e r t a t i o n : Genetics and Functions of Herpes Simplex VirusType-1 (HSV-1) Glycoprotein K (gK) in Virus Envelopment and Egress.

■ o v ed :

M a j o r P r o f e s s o r a n d C h a i r m a n

o r t n e G r a d u a t e S c h o o l

EXAMINING COMMITTEE:

D a t e o f E x a m i n a t i o n :

12/ 20/96


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Genetics and Functions of Herpes Simplex Virus Type 1 (HSV