THE ROLE OF THE E1B GENE PRODUCTS OF ADENOVIRUS SEROTYPE 12

IN LYTIC INFECTION AND TRANSFORMATION

by

MICHAEL DANIEL SCHALLER, B. Sc.

A Thesis

Submitted to the School of Graduate Studies

in Partial FUlfi1ment of the Requirements

for the Degree

Doctor of Philosophy (Biology)

McMaster University

copyright by Michael D. Schaller

(September) 1990

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FUNCTIONS OF THE E1B GENE PRODUCTS OF ADENOVIRUS TYPE 12

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DOCTOR OF PHILOSOPHY (1990)

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McMASTER UNIVERSITY

(Biology) Hamilton, ontario

TITLE: The Role of the E1b Gene Products of Adenovirus

serotype 12 in Lytic Infection and Transformation

AUTHOR: Michael Daniel Schaller, B. Sc. (McMaster

University)

SUPERVISOR: Dr. S. Mak

NUMBER OF PAGES: XV, 296

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ABSTRACT

It is well documented that two regions of the human

adenoviruses, each encoding multiple polypeptides, are

respo~sible for the transforming activity of these viruses

and that these genes are essential for the efficient

production of virus in permissively infected cells. This

study was undertaken to examine the importance of the

individual proteins of one of these regions from adenovirus

serotype 12 (Ad12), the E1b region, in lytic infection ,and

transformation. Molecular defects have been identified in

the smaller protein, the 19K, in two of the cytocidal (cyt)

mutants of Ad12. Direct evidence has been obtained

demonstrating that the point mutation in the 19K of one;of

the mutants is sufficient to cause the degradation of DNA in

infected KB cells and reduce the transforming activity of

the mutant virus and perhaps its tumourigenic potential,

which are characteristic phenotypes of the cyt mutants. A

mutation was also engineered in the larger, 55K Elb protein

and was found to impair viral DNA replication, reduce the

expression of the late, structural proteins of the virus and

block the inhibition of cellular protein synthesis which is

normally observed upon infection of KB cells with the wild

type virus. The 55K was also found to be necessary for the

efficient expression of the early genes of the virus,

particularly the E2b gene, which encodes essential proteins

iii

for viral DNA replication. The multiplicity dependent

leakiness of the DNA replication defect of this mutant was

exploited to separate the DNA replication defect from the

defects in late protein expression and shut off of host

protein synthesis. The observation that this mutant

exhibited 1% of the transforming activity of the wild type

virus but that transformants were fully tumourigenic also

separated a transformation function from a tumourigenic

function of this protein.

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ACKNOWLEDGEMENTS

I wish to express my thanks to my supervisor, Dr. S.

Mak for providing the opportunity and the guidance required

during most of this wo=k and Dr. F. L. Graham for taking

over as my supervisor during the preparation of this thesis.

Discussions with my colleagues John Gysbers, Harry Caussy

and Shi Yun Zhang as well as comments and suggestions from

the actively participating members of the Adenovirus Work in

Progress group helped shape the direction in which this

research progressed. I would also like to thank Drs. J.

Smiley, L. Prevec and S. Bacchetti for their comments on

this manuscript. I am a£~o indebted to I. Mak and U. Sankar

for their excellent technical assistance and to the National

Science and Engineering Research Council of Canada and the

National Cancer Institute of Canada for financial support.[I

I would also like to express my gratitude to my wife,. Kathy,~:

for her encouragement and tolerance and Geoff and Kelly who

unknowingly sacrificed their time with me so that I could

pursue my career.

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TABLE OF CONTENTS

INTRODUCTION

A. Overview of Lytic Infection and Transformation

B. E1a of the Nononcogenic Serotypes Ad2 and AdS

1. Transcripts and Translation Products

2. Functions of the E1a Proteins

C. E1b of the Nononcogenic Serotypes Ad2 and AdS

1. Transcripts and Translation Products

2. Functions of 58K

3. Functions of 19K

D. E1a of the Highly oncogenic Serotype Ad12

1. Transcripts and Translation Products

2. Functions of the Ela Proteins

E. E1b of the Highly Oncogenic Serotype Ad12

1. Transcripts and Translation Products;.

2. Functions of 55K

3. Functions of 19K:::;:.::3;,'

F. The cytocidal Mutants of Ad12

MATERIALS AND METHODS

A. Recombinant DNA Techniques

1. Materials

a. Enzym~s

b. Cloning vectors (j..

vi

1

2

6

6

11

14

14

17

19

22y

22

25

27

27

29

31

33

35

35,

35

35

36

c. Recombinant Plasmids containing Ad12 or Mutant

DNA Sequences 38

d. Bacteria 42

e. Broth and Plates 43

2. Transformation ofE. coli 45('"' ~""3. DNA Extraction from E. coli 47

4. Protocols for the Preparation of Single stranded DNA

48

a. Growth of M13K07 48

b. Preparation of Single stranded pUC118/1.19 49..

c. Preparation of single Stranded M1.3 DNA 50

B. Tissue Culture Techniques 50"

1. Cells and. Mediar~

2. Viruses"~,

3. Infection Procedures

4. Purification of Virus

5. Plaque Assay f'::)

C. construction of Recombinant Viruses::":'. • . • .1":-

1. Construction ol Recomb1nant V1ral DNA Molecules

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2. Transfection of MH12-C2 Cells

3. Growth of Recombinant Viruses and Plaque

Purification

D. Transformation @ndTumourigenicity Assays

1. Preparation of Primary BRK Cultures

vii

50

51

52

53

54

55

55

55

58

59

59

2. Selection and staining of Foci 60

3. Tumourigenicity Assay 61

E. Techniques for the Analysis of DNA 62

1. Extraction of DNA from Cells in Tissue Culture 62

2. Gel Electrophoresis of DNA 63

a. Agarose Gel Electrophoresis 63

b. Polyacrylamide Gel Electrophoresis 64

c. Visualization of DNA on Gels 64

d. Fragment Isolation 65

i} from Agarose Gels

ii} from polyacrylamide Gels

65

66

o . 3. Transfer to and Immobilization of DNA on Membranes:~:'

·67

a. Southern Blotting 67I:~

b. Electrophoretic Blotting 67

., c • Slot Blotting 68

4. Preparation and Use of Radiolabelled Probes 69

a • Nick Translation 69.-0~

b. Primer Extension l~ 69

c. Probe Purification 70

d. Hybridization and Washing of Filters 70

5. DNA sequencing '.71

0

a. Maxam and Gilbert ,Technique 71

i) Radiolabelling DNA " 71 ..

ii) sequencing ~ea~tions 7i

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b. Chain Termination Technique

c. sequencing Gels

F. Tec~niques for the Analysis of RNA

72

75

75

1. Extraction and Purification of RNA from Cells in

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CUlture

2. Northern Analysis

a. Gel Electrophoresis and Blotting

b. Hybridization and Washing Filters

c. staining with Methylene Blue

G. Techniques for the Analysis of Proteins

1. Materials

a. Sera

b. Protein A Beads

2. Preparation of Radiolabelled Cell Lysates

a. Labelling Cells in Culture

b. Preparation of Cell Lysates

3. Immunoprecipitation

4. Analysis by Gel Electrophoresis

a. SDS-PAGE

b. Fluorography

75

76

76

77

78

78

78

78

79

79

79

81

82

82

82

RESULTS 84

I. IDENTIFICATION OF AN E1B 19K DELETION IN cyt~·68 84

A. Restriction Enzyme Analysis of Cloned Viral DNA 85

B. Restriction Enzyme Analysis of Viral Genomic DNA 97

C. preliminary Sequencing Analysis 101

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D. Identification of a 107 bp Deletion in cyt 68 104

E. Sequencing Analysis of the E1 Region 114

1. Analysis of Strain P 114

2. Analysis of cyt 68 119

F. Expression of the E1 Proteins in cyt 68 Infected Cells

127

II. IDENTIFICATION OF AN E1B 19K MUTATION IN cyt 62 132

A. Construction of wild Type/cyt 62 Chimeric Viruses 133

B. AdC22 Induces DNA Degradation in Infected KB Cells 154

C. AdC22 is Transformation Defective 158

D. Tumourigenic Potential of pP7-2 Transformed Cells 162

DELETION MUTANT

E. Nucleotide Sequence of E1 of cyt 62

F. AdC22 Encodes an Unstable E1b 19K Protein

"III. CONSTRUCTION AND ANALYSIS OF AN EIB 55K;

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A. Deletion of 102 bp of the Coding Sequences

166

175

179

of 55K 180

B. The SSK Mutation Impairs Virus Growth in KB Cells 192

C. Identification of the Mutant Protein Product 196

D. dl1201 is Defective for Viral DNA Replication 200

E. dl1201 is Defective for Early Gene Expression 207

F. synthesis of Viral Late and Cellular Proteins 217

G. d11201 has an Altered Oncogenic Potential

DISCUSSION

223

228

A. The Function of 19K in Lytic Infection and Transformation

228

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1. Identification of Mutations in 1wo cyt Mutants of

Ad12 231

2. Role of the 19K Protein in Lytic Infection 232

3. Role of the E1b 19K in Transformation 233

4. Role of the 19K in Tumourigenicity 238

5. comparison of E1b 19K Mutants of Ad2 and Ad12 241

B. The Function of 55K in Lytic Infection and Transformation

244

1. dl1201 Expresses Reduced Levels of the Mutant

Protein

2. Requirement of 55K for viral Growth and DNA

Replication

3. The 55K Regulates Early Gene Expression

245

247

248

4. The 55K and Protein Expression in Infected Cells

249

5. Role of the 55K in Transformation and

Tumourigenicity

C. Concluding Remarks

APPENDIX I

APPENDIX II

REFERENCES

xi

251

253

257

261

263

LIST OF FIGURES

~-1.. Transcription Map of the Adenovirus Genome 3

~-2. Transcription Map of the E~a and E~b Genes of Ad2 8

~-3. Transcription Map of the E~a and Elb Genes of Ad12 23

2-1.. Recombinant Plasmids used in this study 39

- 2 strategy for the Rescue of Chimeric E~ Regions into.l- .Virus 56

2-3. strategy for Sequencing the E~ Region of Ad~2 73

3-~. Restriction Maps of the Left 3496 bp of Ad12 Strain

Huie 87

3-2. Restriction Analysis of DNA Fragments Isolated from

Recombinant Plasmids 89

3-3. Restriction Analysis of Viral DNA 98

3-4. preliminary'Nucleotide Sequencing of cyt 68 102

3-5. Construction of M~3 Recombinants for Sequencing 106

3-6. Autoradiograms of Selected Sequencing Gels 110

3-7. Construction of Recombinants for Sequencing strain P

DNA 116

3-8. Construction of Recombinants for sequencing cyt 68 DNA

Ii 121

3-9. Comparison of the Nucleotide Sequence of Region E1 from

strains Huie and P 123

3-10. El Protein Expression in cyt 68 Infected KB Cells ~29

xii

4-1. strategy for the Construction of wild Type/cyt 62

Recombinants 135

4-2. Construction of the Recombinant Plasmid pP67 137

4-3. construction of Chimeric wild Type P/cyt 62 Plasmids

139

4-4. construction of Plasmids pP7-1, pP7-2 and pP7-3 143

4-5. Analysis of the structure of the El Region of

Recombinant Viruses 147

4-6. Analysis of the structure of the Genomes of Recombinant

Viruses 151

4-7. DNA Degradation Analysis 155

4-8. Transforming Activity of the Recombinant Viruses 160

4-9. Construction of Recombinant Plasmids for sequencing

168

4-10. Autoradiogram of Nucleotide Sequence of cyt 62 173

4-11. E1 Protein Synthesis in AdC22 Infected KB Cells 176

5-1. Construction of Recombinant Plasmid pH6dl-l 181

5-2. Autoradiogram of Nucleotide Sequence of pH6dl-l 185

5-3. Restriction Maps of Ad12 188

5-4. Analysis of the Genome Structure of dl120l 190

5-5. E1 Protein Expression in Mutant Infected KB Cells 198

5-6. Viral DNA Replication in Mutant Infected Cells 202

5-7. MUltiplicity Dependent Leakiness of the dl120l DNA

Replication Defect 205

5-8. Early Gene Expression in dl1201 Infected KB Cells 210

xiii

5-9. Early Gene Expression in KB Cells Infected at High

MUltiplicity with dl1201 215

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5-10. Total Protein Synthesis in Mutant Infected KB Cells

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6-1. E1b 19K Mutants of Ad2 And Ad12

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xiv

242

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