michael daniel schaller, b. sc
TRANSCRIPT
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
/;
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
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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
x
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
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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
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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
219'.::-:-;.,
6-1. E1b 19K Mutants of Ad2 And Ad12
c::
xiv
242
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