CHARACTERIZATION. OF THE HOMAN ADENOVIRUS

TYPE 5 EARLY REGION IB 1.76R PROTEIN

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By

CATHERINE JANE MCGLADE

A Thesis

Submitted to the School of Graduate Studies

in Partial Fulfilment of the Requirements

for the Degree

Doctor of Philosophy

McMaster University

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c CHARACTERIZATION OF THE HUMAN ADENOVIRUSTYPE 5 EARLY REGION 18 176R PROTEIN

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DOCTOR OF PHiLOSOPHY (1990)(Medical Science)

McMaster universityHamilton, ontario

TITLE:

AUTHOR:

Characterization of Human AdenovirusType 5 Early Region lB 176R Protein

Catherine Jane McGlade

SUPERVISOR: Dr. Philip E. Branton

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NUMBER OF PAGES: 238

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ABSTRACT

Cellular t~ansformation by human adenoviruses requires the

expression of two early transcription units, E~ (0 to 4.5% of

the genome) and EIB (4.5 to 11.2%). The products of both genes

are required to disrupt normal cellular growth control and

morphology. Early region 1A products alone can induce cellular

immortalization, but establishment of fully transformed cells

requires cooperation with EIB. Early region IB produces two

unrelated proteins, 496R and 176R, each of which can act

independently with E1A to induce cellular transformation.

176R is a membrane associated protein whose molecular role

in transformation is not known. Anti-peptide sera specific for. ~---

the amino- and carboxy-termini of 176R were produced. These two

sera were used for further purification and biochemical

characterization of the protein. The 176R migrated as two species

termed 18.5 and 19K on 5DS-PAGE • The "generatio~\ of the two

species was not due to proteolysis, nor any post-translational

modification investigated.

Two post-translational modifications of 176R were

identified: phosphorylation and acylation. The phosphorylation

site was mapped to serine residue 164, and a mutant virus in

which this amino acid was altered, ~2204, was produced by

oligonucleotide-directed mutagenesis. Characterization of ~2204

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as well as an El plasmid carrying this mutation suggested that

phosphorylation is not important for the biological activity of

176R. 176R was also found to contain covalently bound palmitate

and myristate. Both tryptic and chymotryptic peptide mapping as

well as CNBr cleavage were used to localize the acylation site.

These experiments suggested that multiple acylation sites exist,

and at least one site was localized to the amino terminal region"-by tryptic peptide mapping. The linkage of fatty acid to 176R was

o found to be via an amide, or an unusually stable ester bond to an

internal amino acid. Acylation of 176R likely facilitates its,~

membrane association, but it was not possible to test\this"

hypothesis without precise mapping of the acylation site(s) and

construction of specific mutants.

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ACKNOWLEDGEMENTS

I am deeply indebted to my supervisor Dr. Philip Branton,

not only for his constant support and encouragement throughout

the course of my Ph.D. work, but for making the experience one

which I will look back on with fond memories. I am especially

grateful for his advice and friendship which helped me through

some frustrating times and made completion of this work possible.

Thanks also to Dr. Stan Bayley and Dr. David Johnson for

their advice over the years and for their careful ~~ading of this

thesis.

To Siu-Pok Yee, a special thank you for being so generous~

with his time and knowledge while getting me started in the lab.

I am also indebted to Michel Temblay and Bruce Rowley for

their friendship and for many helpful discussions. Their

suggestions and advice contributed greatly to this work •

.r am also grateful to the other·-members of the lab, past and

present, with whom I spent many enjoyable hours having a few.~;

beers and talking science.""

It was a pleasure to work in the cooperative environment of

the Cancer Research Group and I thank all its members, both

faculty and students, f~r many helpful discussions.

Finally, I would like to thank my parents, who gave me the

confidence to pursue this work, and Jim who had to put up with me

while I completed it.

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This thesis is dedicated to

my parents,

and to Jim.;~".

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TABLE OF CONTENTS

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531.3 Thesis proposal •••••••••••••.•••••••••••••••••

1.2 Cellular transformation by adenoviruses.1.2.1 General features •••••••••••••••••••••••1.2.2 Transforming genes •••••••••••••••••••••1.2.3 Role of E1 products

a) structure and function of E1Apolypeptides••••••••••••••••••.••.•••

b) structure and function of E1Bpolypeptides •••••••••••.••••.••••••.•

Chapter 1: Introduction.1.1 Adenovirology.

1.1.1 Classification of human adenoviruses •••1.1.2 Structure of the virion••••••••••••.••.1.1.3 Organization of the genome•••••••••••••1.1.4 Lytic infection by adenoviruses

a} adsorption and uncoating••••.••••••••b) early gene expression••••••••••••••••c) replication and the early to late

switch••••••••....••.•.•••...•.•••••.d) asseJDbly•••••••••••••••••••••••••••••e) cytopathology .

Chapter 2: Materials and Methods. ,2.1 Cells•••••• of 552 • 2 Viruses ~ '. •• 552.3 Infection. • • • • • • • • • • • • • • .. • • . • • • • • • • • • • • • •• 562.4 Synthetic peptide antisera••••••••••••••••••• 562.5 Radioactive labeling••.•••••••••••••••••••••• 572.6 Immunoprecipitation•••••••••••••••••••••.•••• 592.7 Polyacrylamide gel electrophoresis••••••••••• 602.8 western immunoblot••••••••••••••••••••••••••• 612.9 Immunofluorescence••••••••••••••••••••••••••• 622.10 Cleveland peptide mapping••••••••••••••••••• 632.11 Protein extraction from acrylamide••••••••••• 642.12 Phosphoamino acid analysis •••••••••••••••••••.,652.13 Chemical cleavage of proteins using CNBr•••••· 652.14 Immunoprecipitation of v-a digests ••••••••••• 66

2.15 DNA cloning and maniplation.2.15.1 bacterial strains and culture••••••••• 662.15.2 restriction digests, ligation reactions,

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agarose and acrylamide gels for DNA,transformation of bacteria and DNA. .m~n~-pre~s. • • • • • • • • • • • • • • • • • • • • • • • • • • •• 67

2.15.3 large scale preparation of plasmidDNA by CsCl Banding•••••••••••••••••••• 68

2.15.4 isolation of DNA fragments fromacrylamide gels•..•......•..•.....•.... 69

2.16 site directed mutagenesis ••••••••••••••••••••. 702.17 Rescue of mutations into pXC38 •••••••••••••••• 722.18 Mutant virus rescue.

2.18.1 transfection of 293 cells •••••••••••••• 732.18.2 i~olation and screening of recombinant

v~ruses. . • • . . . . • . . • • . . . • • . . . . • . . . . . . . •. 742.19 Transformation assay.

2.19.1 preparation of BRK cells••••••••••••••• 752.19.2 transfection of BRK cells and scoring

.transformants. • • • • • • • • • • • • • • • • • • • • • • • •• 762.20 Biochemical characterization fatty acid­

protein interaction.2.20.1 hydroxylamine treatment of gels •••••••• 772.20.2 methanol-KOH treatment of gels ••••••••• 77

2.21 Methanol hydrolysis and analysis of fattyacids by HPLC 78

2.22 Peptide mapping by HPLC.2.22.1 Performic acid oxidation and protease

digestion. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 792.22.2 Separation of peptide by reverse-

phase HPLC.............................. 792.22.3 ~solation ~f.T1 ~sing 19-N1

1mmunoprec~p1tat1on•••••••••••••••••••• 802.23 Ion exchange chromatography of CNBr cleavage

products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. 81

Chapter 3: Analysis of AdS r/6R using antipeptide sera.3.1 preparation of antipeptide sera •••••••••••••••• 823.2 Immunoprecipitation of 176R polypeptides••.••••• 853.3 Cleveland peptide mapping of the 18.5 and' '::.

19 kDa species of 176R••••••••••••••••••••••••• 883.4 western blot analysis of 176R polypeptides

produced in bacteria••••••••••••••••••••••••••• 913.5 Localization of 176R by indirect

immunofluorescent antibody staining•••••••••••• 943.6 Glycosylation as a possible post-

translational modification••••••••••••••••••••• 94

Chapter 4: Phosphorylation of AdS 176R.4.1 Detection of phosphorylated forms of 176R•••••• I004.2 Identification of the phosphorylated amino

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acid in 176R l034.3 Estimation of the level of 176R

phosphorylation••••..•••.•••..•.••........••... l034.4 Mapping of the phosphorylation site.

4.4.1 chemical cleavage with CNBr••.•••••••••• 1064.4.2 digestion with V-8 protease and

immunoprecipitation••••••••••••••••••••• 1124.5 confirmation of ser-164 phosphorylation

site by site-directed mutagenesis.4.5.1 oligonucleotide directed

mutagenesis•...•..•..••...•.••......••.• 1184.5.2 rescue of mutation into pXC38 ••••••••••• 1234.5.3 rescue of mutation into infectious

adenovirus type 5•••••..•••••..••••.•••• 1264.6 Biological role of phosphorylation of 176R.

4.6.1 protein stability••••••••••••••••••••••• 1354.6.2 virus growth, and cyt/deg phenotype .•••• 1354.6.3 cellular transformation••••••••••••••••• 138

4.7 Rescue of mutation into a 496R- background.4.7.1 subcloning of mutations into pXC38 •••••• 1384.7.2 cellular transformation•••• ~ •••••••••••• 143

Chapter 5: Acylation of Ad5 176R.5.1 Detection of acylated forms of 176R•••••••••••• 1495.2 Identification of the fatty acid bound

to 176R••••••••.••••••••••••••••••••.••••.•••.• 1525.3 Analysis of the protein-fatty acid linkage

5.3.1 hydroxylamine treatment of 176R••••••••• 1555.3.2 chloroform:methanol extraction and

treatment with KOH•••••••••••••••••••••• 1565.4 Localization of the acylation site.

5.4.1 CNBr cleavage••••••••••••••••••••••••••• 1615.4.2 analysis of 176R tryptic p~ptides••••••• 164 ~5.4.3 ion-exchange chromatography of

176R CNBr cleavage products ••••••••••••• 178~> 5". 4 ~ 4 analysis of 176R chymotryptic

peptides 184

.' Chapter 6:' DisCussion••••••••••••••••••••••••••••••••••• 192·

REFEREtICES •••••••••••••••••••••••••••••••••••••••••••••• 213

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Figure No.

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LIST OF FIGURES

Title

Virion structure of human adenovirus.

strllcture of the adenovirus genome.

Transcription map of the E1 region.

Functional domains of the E1a poly­peptides.

Transcription map of the E1B region.

Predicted amino and carboXY,terminalsequences of AdS E1B-176R andsynthetic peptides.

Immunoprecipitation of 176R with19-N1 and 19-Cl antipeptide sera.

Partial proteolysis peptide mappingof the 18.5 and 19 kDa.176R species.

Western blot "analysis of 176Rproteins produced in bacteria.

Indirect immunofluorescent stainingusing 19-C1 serum.

Effect of tunicamycin treatment on176R species.

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SDS-PAGE of 176R time course labelingwith [35s]methionine and [32p]ortho­phosphate.

Two-dimensional phosphoamino acidanalysis.

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Amino Acid sequence of Ad5 E1B-176Rshowing CNBr and V-8 proteasecleavage sites.

Cleavage pattern of 176R by CNBr.

Cleavage pattern of 176R with s.aureus V-8 protease.

v-a protease cleavage pattern of3H-amino acid labeled 176R.

Generation of mutant pm2204.

Single lane sequencing of M13 phage.

BanII digest of recombinant pXC38.

southern blot of recombinant Ad5plaques.

Restriction digests of recombinantAd5 DNA.

Phosphorylation of 176R in cellsinfected by wt Ad5 or mutant pm2204.

Pulse-chase labeling of 176R fromwt Ad5 or pm2204 infected cells.

Analysis of Hirt extracted viral DNAto detect the deg+ phenotype.

Generation of pXC2204(!j4).

Restriction analysis of pXC2204(lj4).

Labeling of 176R with (3H]fatty acids.

Analysis of the fatty acid presentin 176R.

Sensitivity of fatty acid.linkage toliydroxylamine (HA) treatment.

Sensitivity of fatty acid linkage tomethanol-KOH treatment.

CNBr cleavage of (3H]myristate labeled176R.

Trypsin cleavage sites of Ad5 176R.

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Separation of t~tic peptides of 176Rlabelled with [3 ]methionine [35]_cysteine, [3H]arginine and [~H]lysine.

Identification of T1 by immuno­precipitation.

Tryptic peptides of the 176R mutant \)pm2072.

Separation of [3H]myristate labelled176R tryptic peptides.

Ion-exchange chromatography of 176RCNBr cleavage products.

Chymotryptic cleavage sites of Ad5­176R.

separation of 176R chymotrypticpeptides.

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174

176

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182

185

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Table No.

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LIST OF TABLES

Title

Estimation of the level ofphosphorylation of ~76R.

Plaguing efficiency on HeLa and293 cells.

Transformation of. baby rat kidneycells.

Tr}~tic peptides of AdS-E1B 176R.

Chymotryptic peptides of AdS-E1B 176R.

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AdS

Ad2--.

Ad12

BSA

dATP,\\

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dCTP

CNBr

CPE

DMSO

DNA

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LI:ST OF ABBREVIATIONS

adenovirus type 5

adenovirus type 2

adenovirus type 12

bovine st:rum albumin -

deoxyadenosine S' triphosphate

complimentary deoxyribonucleic acid

deoxycytosine S' triphosphate

cyanogen bromide

cytopathic effect

dimethylsulfoxide

deoxyribonucleic acid

ethylenediaminetetraacetic acid

ethidium bromide

hydroxyl amine

high performance liquid chromatography

potassium hydroxide

mess~nger ribonucleic acid

minute.,','

map unit

micro-CUrie

microgram

microlitre

xiv