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

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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

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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

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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