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CHARACTERIZATION OF HUMAN ADENOVIRUS TYPE 5
EARLY REGION 1 PROTEIN
USING ANTI-PEPTIDE ANTIBODIES
by
Siu-Pok Yee
A Thesis
submitted to the School of Graduate Studies
in Partial Fulfillment of the Requirements
for the Degree
Doctor of Philosophy
~cMaster university
October, 1985. ©
----,
...,
Characte=izatioh~of Hu~an Adenovirus
ty?e 5 Early Region 1 ?roteins Using
Anti-peptide Antibodies
--"
DOCTOR OF PHILOSOPH~ (1985)(Medical Science) ,
MCMASTER UNIVERSITYHamilton, Ontario
TITLE:
AUTHOR:
SUPERVISOR:
"--Characterization of Human Adenovirusty?e 5 Early Region 1 Proteins UsingAnti-?e?tide Antibodies
Siu-pok Yee
Dr. Philip E. Branton
c
NU~BER OF PAGES: 290
•
.'
ii
,
ABSTRACT~
HQman adenovirQses are known to transform rodent
cells in cQltQre and these cells are tQmorgenic when
injected into new born animals. It has been well
established that the early region 1 (El) of human
adenovirQs type 5 is necessary and sQfficient for
oncogenic transformation •. The El region is comprised of
two transcription Q~~S known as E1A (0 to 4.5~ of the
genOm;).~13 (4.5 to 11.2%), each of which prodQces
mQltiple species of mRNAs and polypeptides. E1A is also
required-to activate the transcription of other viral
early regions. In the present stQdy anti-peptide sera
were Qsed to identify and characterize these viral
proteins.
Anti-peptide sera specific for the amino- and
carboxy-ter~ini of ElA were raised ana these two sera
precipitated an identical set of fo~r major
polypeptides of 52, SO, 48.5, and 45K and two minor
species of 37.5 and 35K. Studies using EIA mutant
virQses also revealed
~OlypePtides are derived
that
from
52, 48.5, and 37.5K
the 1.1 kb mRNA, and the
. SO, 45, and 35K species from the 0.9 kb mRNA of E1A.
These sera were also used to identify ?olypeptides that
are associated with El proteins. A set of five cellular
iii
,polypeptides consisting of >250K, 1051 (doublet), 68K,
and 651 species were found to co-precipitate with ElA
-
various conoitionsproteins under
this,
association was
~nd the
investigated
nature
using
of
the
anti-nentide sera as well as an EIA-specific monoclonalI 0 0
.. ' • 40-ac ... lvl ...y.
An~isera against synthetic peptides corresponding
to the both termini of EIB 58K were also raised and
used to identfy 58K from wild-type and mutant-infectec, .
cells. It had previously been shown that protein kinase
activity was associatec wit~
~inase activity was intrinsic
58K. To as~ if protein,
to this 'vlral protein::...,..
seve~al conventional met~oas were used to purifvo "
58"
and the -results suggested that such activity may be
intrinsic to this viral protein.
The anti-peptide sera were used to purify °El
proteins. A simple purification procedure using these
sera and their corresponding synthetic peptides was
developed and highly purifieJ 58K and ElA proteins were
obtained. Attempts were mace to study protein kinase
activity using these purified El proteins, however, the
results were inconclusive ana it was not possible- to
unequivocally determine
intrinsic to the~.
iv
.~1 _
kinase activity was
•
;ACKNOW4EDGEMENTS
It is difflcult for me to adequately express my
appreciation to my supervisor, Dr. Philip Branton, for his
patience and guidance during the ups and downs of the study.
Throughout these years he has not only taught me the
excitment of science, but also provided encourgement in the,
tough time when nothing seemed to work. With his support I
made -this thesis a reality.
I would also like to thank the members of my
supervisory committee Dr. James smiley and Dr. Frank "Graham
for their advice throughout this work and thorough review of
my thesis and the latter for also providing the various AdS
mutants during these studies. My appreciation is also
extended to Dr. Mark McDermott and Dr. N. Balachandran for
their valuable suggestions on the immunological techniques.
The excellent technical help of Sylvia Cers, Joceline
Otis and John Rudy are grateful appreciated. The helpful
discussions of Michel Tremblay during the lunches, coffee
r
•
breaks and ping-pong games, and as well the people in the
lab, past and present, are greatly acknowledged, I would
also like to thank Ernest Chan and his family for providing
unlimited access to their Apple IIe,
and Applework for the
preparation of this manuscript.
The work leading to this thesis would have never been
completed without the encouragement and support of the
persons to whom this thesis is dedicated.
v
•
)
This thesis is dedicated to
:nom and dad
and•
~'!a i -$ i n
"
vi
..
TABLE OF CONTENTS
Chapter 1 Introduction
1.1 Human adenoviruses'.
1.1.1 Classification of humanadenovi ruse-s
1.1.2 Structure and composition -ofadenovi ruses ~.
1.1~3 The adenovirus genome
1.1.4 Functional organization of theviral genome
Page1
1
1
2
7
9
1.1.5 productive infection by adenovirus 14
(a) Absortion anc uncoating
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•
•
L2
1.3
1.4
2.2
(b) Early gene expression
(c) viral DNA replication
(d) Transition from ~rly to iategene expression
(el Assembly of adenovirion
Transfor~ation by adenoviruses
1.2.1 Transforming genes
1.2.2 Proteins encoded in ~El
1.2.2 Functions of El
Antisynthetic peptide 'antibodies
proposal of this thesis
Materials and ~ethocs
Cells and viruses
Infection and radioactive labellingof cells
vii
16
, 20
23
26
28
30
34
38
46
51
54
54
55
2.16 Estimates of protein ab~ndance
2.11 Glycerol gradient centrif~gation
2.3 preparation of anti-peptide sera
2.3.1 Conj~gation of peptide tobovine perum albumin
,55
55
56
58
59
59
61
62
62,
63
64
65
66
67
68
68
70
70
Imm~noaffinitv p~rification ~sing
anti-synthetic peptide antibodies
Immunofluorescence
Cleveland Mapping
One-dimensional polyacrylamide gelgel electrophoresis
Two-di~ens~onal oolvactvlamide gelgel elect~o?horesis· .
2.14
2.15 Meas~remen~ of incorporation.ofradioactivity by TeA precipitation
2.13 P~rification of 58K by DEAE-sephacelchromatography
3.1 Analysis of El polypeptid~s ~sing
antisera to synthetic peptidecorresponding to carboxy termini
2.3.2 Immunization of rabbits andpreparation of antiserum
2.12 P~rificatidn of viral polypeptide bya~~onium sulfate precipitation
2.9
2.10 Association of Ad EIA polypeptides withcell~lar proteins
2.8
2.6
2.4 Preparation of cell extracts andirnmunoprecipitation,-
2.5 Protein kinase assay
Chapter 3 Analysis of AdS El proteins ~sing
anti-peptide antisera
viii
3.1.1
3.1. 2
preparation of antisera specificfor the carboxy termini of AdS Elproteins
Immunoorecioitation .of AdSpolypeptide"with antisera raisedagainst the synthetic peptidescorresponding to the carboxytermini of Er-proteins
70
73
3.1.3 Specificity of antipeptide sera 85
3.1.4 Comparsion of viral proteins 90precipitatec by anti tumour andantipeptide sera by Clevelandpeptide map;:>ing
3.1.5 Localization of AdS proteins by 95immunofluorescence
3.2 Analysis of El poly;:>e;:>tides usingantise~a to synthetic peptidescorresponding to the amino-termini
99
3.2.1 preparation of .antisera against" 99the amino-termini of Elpoly;:>e;:>tides
3.2.2 Immunoprecipitation of AdS 102polyppeptides with antiserum"raised against the amino terminalsynthetic peptides
3.2.3 Analysis of EIA polypeptides 108synthesized by the AdS mutantsma;:>ping in EIA
3.2.4 Failure to detect oroducts from 121the 0.6 kb EIA mRNA using ElA-Nlserum
342.5 Immunoprecipitation of EIB 58K 125synthesized in cells infected with
. wild-type or group II :,ost-rangemutants 0.£ AdS
•
Chapter 4 Association of AdS EIA polypeptideswith cellular proteins in AdS-infectedcells
ix
130I
J
I 4.
..
Association of AdS E1A polypeptideswith cellular proteins in AdS-infectedcells
130
Chapter 5 Analysis of S8K and its associatedprotein kinase activity usingconventional methods o~ proteinpuri fi'cation
162
5.1 Ammonium sulfate fractionation of AdS 162E1B S~K
5.2 Fractionation·of AdS E1B S8K using 166ion-exchange chromatography
5.3 Glycerol gradient centrifugation 172
Chapter 6 Purification of AdS El proteins by 176immunoaffinity using antipeptide sera
6.1 Releasing of El proteins fromimmunoprecipitates using correspondingsynthetic peptide
177
6.2 Purification of AdS El proteins usingimmunoaffinity chromatography
192
Chapter 7 Discussion
REFERENCES
x
202
228
LIST OF TABLES
Table NO. Title
1 Immunoorecipitation of AdS E1Aproteins coded for bv wt andvirus by E1A-Nl and E1A-Cl sera
Page
117
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2 Analysis of pro-t~'ins associatedwith AdS E1A products
xi
142
Figure NO.
1
2
3
4
5
6
LIST OF FIGURES
Title
virion structure of adenovirus
Transcription maps of Ad2
The predicated carboxy-terminalsequence and synthetic peptidesof Ad5 58K and E1A proteins.
!m~unoprecipitation of AdS- Elpolypeptides by anti tumour anaC-ter~inal an~ioectide sera- . .32IDunoprecipitation of
?-labeled AdS El polypeptidesbv antitumour and C-terminal
....... .... • ..::lan~lpep~lue sera.
comparsion of phosphoproteinsprecipitatec by combined hamsterantitumour ana EIA-CI sera.
page
5
12
72
76
79
82
7
8
9
10
11
T~o-dimensional gel electrophoresis.of AdS 81'),,· proteins precipitatec byE1A-C1 serum.
Effect of synthetic peptides onprecipitation of viral proteins byantitumour and C-ierminalantipeptide sera.
Effect of heterologous peptide onprecipitation of viral p~oteins byC-terminal antipeptice sera.
Partial hvdrolvsis of the 58Kpolypeptide pr~cipitated byanti tumour or 58-Cl serum withStaphyloccal V-8 protease.
Partial hvdrolvsis of the 52Kpolypeptide pr~cipitatec byanti tumour or EIA-Cl serum withStaphyloccal V-8 protease.
xii
84
- 87
89
92
95
12
13
15
Fluorescent-antibody staining with 98C-terminal antipeptiae sera.
The predicted amino-terminal 101sequences and synthetic peptidesof AdS 58K and EIA proteins.
Immuhoprecipitation of 58K and ·104effect of synthetic peptide onprecipitation of 58K by 58-Nlserum.
Analysis on one- and two-dimension 107gels of EIA proteins immunoprecipitated by EIA-Nl and EIA-Clantipeptide sera.
Two-dimensional gel electrophoresis 115- of EIA proteins synthesized in
mutant-infected cells anaim~unoprecipitated by EIA-Nl serum.
16
,- I
Map locations· of AdS EIA mutants. 110
18
19
Analysis on two-dimensional gels of 120EIA proteins from host-rangegroup I mutants immunoprecipitatedby EIA-Nl serum".
Analysis of EIA proteins synthesized 124late during infection
20
21
22
23 •
Analysis of 58K-related viralproteins ·from host-range group IImutants by anti tumour aDdantipeptide sera.
SDS-PAGE analysis of polypeptidescoprecipitated with EIA proteinsusing EIA-Cl serum.
Analysis of polypeptidescoprecipitated with EIA proteinsusing EIA-Cl serum.
SDS-PAGE analysis of polypeptidescoprecipitated with EIA proteinsusing EIA-Cl and EIA-Nl sera.
xiii
128
134
137
139
?'-~
25
26
27
28
29
30
31
32
SDS-PAG~ analysis of polypeptides 144coprecipitated with EIA-Cl serumand mouse monoclonal antibody M73.
Analvsis of the association of the 150>250~' protein with ElK'polypeptides.
Complexing of cellular proteins 153with Elri polypeptides in vitro.
Phospho=~lation of EIA-associated 157protei ns".
Analysis of immunoprecipitates from 160om975- and hrl-infected cells.~
Purification of 58K by ammonium 165sulphate precipitation.
Two-dimensional 'gel electrophoresis 168of im~unoprecipitates EIB 58Kprotein
Analysis of AdS EIB 58K and protein 171~kinase activitv by DEAE-sephacelchromatography.
. -.Analysis of AdS EIB 58K and protein 17'4kinase activity by glycerolgradient centrifugation.
33
34
Purification scheme of E1 proteinsusing' antipeptide sera.
9isplacement of EIA proteins fromEIA-Cl immunoprecipitates by EIA-Cpeptide.
179
181
35 Displacement of 58K from 58-Cl 183immunoprecipitates by 58-C peptide.
36 DisplaceMent of EIA proteins fromEl~-~l immunoprecipitates by EIA-Npeptide.
"186
,
,
37 Displacement of 58K from 58-Nl 189immunoprecipitates by 58-N peptide.
xiv
u ___
(
38
39
40
,
Effect of ionic detergents in thedisplacement ~f El oroteins fromimmunoprecipitates.
Purification of EIA oroteins and58K using C-terminal~antipeptidesera.
Two-step purification of E1Aproteins and 58K using antipeptidesera.
,
xv
191
--194
198
-List of Abbreviations
SDTA
deT?
dCMP
MR
DNA
nRNA
BS.;
.I eDNA
4'rCA
PPO
D'130
sv~D
RSV
TE:-tED
PBS
SDS
ethylenediamine~raacetic acid
oeoxycytosine ~riphosphate
deoxycytosine monophosphat~
molecular weight
deoxyribonucleic acid
~essenager ribonucleic acid
bovine se~urn al~umin
com91ementary aeoxy~i~onucleic acid
trichloroacetic acid
2,S-diphenyloxazole
dimethvlsulfoxide. ..
simia:"! virus 40
-ROllS sacroma virus
N1N,~I/N'-tetrametfiylethylenedia~ine
phosphate buffered saline
sodium dodecyl sulfate
, .., '.~ .,
SDS-?AGE
/'
sodium dodecyl sulfate-polyacrylamide gel
elec~ro?horesis,
•
AdS
Ad2
uCi
AT?
ug
ml
ul
mM
:nin
vol
T:-is
T::is-HCl
m. u •
--
adenovirus type 5
adenovirus type 2
micro-Currie
acenosine triphosphate
rnicrogra~
millilitre
microlitre
millimolar
ffiioLJte
volume
Tris hycroxy~ethyl aminomethane
Tris-nyc=ochoric acid
map unit
}.."Vii
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I
Chapter 1
Introduction
'-
1
1.1 Human Adenoviruses
1.1.1. Classification of Human Adenoviruses
•To date at least thirty-one recognized serotypes
of human adenoviruses have been classified into several
g=Oups that share common properties (Flint, 1980a;
;ladell et al"~ 1980). They have been classified into
subgroups according to the base composition (G+C
content) of their D~~ (?ina and Green, 19.65), the
degree of their nucleic acid homology as measured by
DNA:DNA and DNA:RNA hybridization (Lacy "and Green 1964,
1965, 196i; BartoK et al 18i4; Garon et al 19i3), and
also their ability to aggulutinate erythrocytes of
human, monKey, and rat in vitro (Rosen 1958, 1960;
Kasel et al 1960, ZuscheK 1961). They have also been./ "
gathered into four groups according to their ability to
induce tumours in newborn hamsters. Serotypes in
subgroup A which are highly oncogenic are able to
induce tumours rapidly in the majority of inoculated
animals. The weaKly oncogenic subgroup B only has a
limited capacity to induce tumours and members of
subgroup C and D which are weakly oncogenic have a---------- -limited capacity to induce tumours --(Hllebn-er, 196i;
~cAllister et al., 1969). A fifth and sixth group
top related