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This is to certify that the
dissertation entitled

THE ROLE OF POLYOMAVIRUS MIDDLE T TYROSINES
IN VIRAL INFECTION

presented by

XIAOYU WANG

has been accepted towards fulfillment
of the requirements for the

PhD. degree in Microbiology and Molecular
Genetics

 

 

 

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6/01 cJCIRC/DateDuopss-p. 15

THE ROLE OF POLYOMAVIRUS MIDDLE T TYROSINES IN VIRAL INFECTION

BY

XIAOYU WANG

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2004

ABSTRACT

THE ROLE OF POLYOMAVIRUS MIDDLE T TYROSINES IN VIRAL INFECTION

By

Xiaoyu Wang

The polyomavirus middle T oncoprotein (MT) plays important roles in the viral
lytic cycle. It strongly upregulates both viral early and late gene expression as well as
viral DNA replication. MT exerts its functions by binding to and constitutively activating
c-Src family tyrosine kinases and their downstream signaling pathways.

In this thesis, I have characterized the role of the “minor” tyrosines Y258, Y288
and Y297 in Src-mediated functions, on their own and in comparison with that of the
“major” tyrosines Y250, Y315 and Y322. I examined the effects of tyrosine to
phenylalanine MT mutations for their effects on Src binding, Src kinase activity, MT
substrate capacity and on MT capability to bind to p85 subunit of phosphatidylinositol-3
kinase. I found that all mutants have the capacity to bind to and activate c-Src. Mutation
of both major and minor tyrosines has an impact on Src activity. This decrease in Src
activity affects the overall degree of both MT and p85 phosphorylation. Mutation of
minor tyrosines reveals a stronger effect when major tyrosines are also removed. In
addition, there are tyrosine specific effects in MT substrate capacity and in p85 binding.

Y250, Y315, and Y322 have relatively equal substrate capacities, and MT containing a

single active tyrosine Y315, and to a lesser extent, Y250, have the capacity to bind to p85
and induce its phosphorylation.

I have also examined the role of MT C-temiinal tyrosines on viral genome
accumulation and gene expression in cell culture. Infection of NIH3T3 cells with
polyomaviruses expressing different tyrosine mutant MTs demonstrates that a tyrosine
sextuple mutant (Y6F) has a severe defect in both viral genome accumulation and gene
expression. The major tyrosine triple mutant (Y3FM) has a moderate defect, whereas the
minor tyrosine mutant (Y3Fm) shows only a minor defect. The capability of different
mutant MT to complement the MT'/ST' defective virus A185 in genome accumulation
and gene expression is also consistent with viral infection data. The MT complementation
study also shows that the effect of the major tyrosines is augmenting. Furthermore, the
ability of MTs to promote viral DNA replication is reduced but not abolished in the
presence of abundant LT. All mutant MTs are also shown to activate polyoma enhancer-
origin dependent gene transcription to different degrees.

Finally, I have examined the function of middle T C-temiinal tyrosines in viral
replication in mice. A tyrosine sextuple mutant (Y6F) shows a 7-10 fold defect in viral
genome accumulation in the acute infection of both neonatal and 4—week old mice. This
defect becomes more severe in the 4-week persistent infection of neonatal mice. The
major tyrosine triple mutant (Y3FM) shows a 2.4 fold defect, whereas the minor tyrosine
triple mutant (Y3Fm) shows no defect in neonatal mice infection. Furthermore, a mild

maturation defect is found in Y6F.

To my wife, Lianjie

iv

ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Michele Fluck for her guidance, understanding,
support and friendship in the course of my graduate studies. I would like to thank the
members of my guidance committee, Dr. Susan Conrad, Dr. Jerry Dodgson, Dr. Sandra
Haslam, and Dr. Richard Schwartz for their collective thoughts and invaluable expertise.
Finally, although too numerous to mention, I would like to thank all of my friends in the
Department of Microbiology and Molecular Genetics, especially in Dr. Fluck’s lab, who
have offered me enormous friendship and support. Finally, I would like to thank Lianjie,

my wife, for her understanding and support through these years.

TABLE OF CONTENTS

Page
LIST OF TABLES ........................................................................................................... viii
LIST OF FIGURES ........................................................................................................... ix
CHAPTER 1: LITERATURE REVIEW ........................................................................... 1
INTRODUCTION ...................................................................................... 1
PROPERTIES OF POLYOMAVIRUS EARLY PROTEINS .................... 2
STRUCTURE AND FUNCTION OF POLYOMA ENHANCER ........... 12
THE POLYOMAVIRUS LYTlC CYCLE, CELLULAR
TRANSFORMATION AND TUMOR INDUCTION ............................. 14
SUMMARY .............................................................................................. 20
APPENDIX 1: TABLES AND FIGURES FOR CHAPTER 1 ................ 21
REFERENCES ......................................................................................... 32
CHAPTER 2: ROLE OF POLYOMAVIRUS MIDDLE T C-TERMINAL
TYROSINES TN MT-MEDIATED SIGNALING ................................... 50
ABSTRACT .............................................................................................. 50
INTRODUCTION .................................................................................... 51
MATERIALS AND METHODS .............................................................. 52
RESULTS ................................................................................................. 58
DISCUSSION ........................................................................................... 65
SUMMARY .............................................................................................. 70
APPENDIX 2: FIGURES AND TABLES FOR CHAPTER 2... ............. 71
REFERENCES ......................................................................................... 92

CHAPTER 3

CHAPTER 4

THE ROLE OF MT C-TERMINAL TYROSINES IN VIRAL DNA
REPLICATION AND GENE EXPRESSION IN CELL CULTURE 95
ABSTRACT .............................................................................................. 95
INTRODUCTION .................................................................................... 95
MATERIALS AND METHODS .............................................................. 96
RESULTS ................................................................................................. 99
DISCUSSION ......................................................................................... 104
SUMMARY ............................................................................................ 107
APPENDIX 3: FIGURES AND TABLES FOR CHAPTER 3 .............. 108
REFERENCES ....................................................................................... 123
THE ROLE OF MT C-TERMINAL TYROSINES TN GENOME
ACCUMULATION AND PERSISTENCE IN MICE ........................... 127
ABSTRACT ............................................................................................ 127
INTRODUCTION .................................................................................. 127
MATERIALS AND METHODS ............................................................ 128
RESULTS ............................................................................................... 130
DISCUSSION ......................................................................................... 136

vi

SUMMARY ............................................................................................ 139
APPENDIX 4: FIGURES AND TABLES FOR CHAPTER 4 .............. 141
REFERENCES ....................................................................................... 162

vii

Table 2—1

Table 2-2

Table 3-1

Table 4-1

Table 4-2

LIST OF TABLES

Page
Tyrosine mutant middle T ........................................................ 75
Summary ofthe levels of phosphorylated p85. . . . . . .. ................................ 91
in the in vitro kinase assays
Properties of polyomaviruses used in cell culture studies .................. l 10
Properties of polyomaviruses used in mouse studies ........................ 143
A summary of viral genome accumulation in organs ...................... 147

from neonatal mice following a 6-day acute infection

viii

Figure 1-1
Figure 1-2
Figure 1-3
Figure 1-4
Figure 1-5
Figure 2-1
Figure 2-2
Figure 2-3
Figure 2-4

Figure 2-5

Figure 2-6

Figure 2-7

Figure 2-8

Figure 3-1

Figure 3-2
Figure 3-3

Figure 3-4

Figure 3-5

LIST OF FIGURES

Page
Physical map of polyomavirus genome ........................................ 23
Lytic cycle of polyomavirus ...................................................... 25
A schematic representation of middle T ....................................... 27
Diagram ofthe signaling pathways mediated by middle T .................. 29
Physical map of the enhancer region of polyomavirus A2 genome ........ 31
A schematic representation of middle T ....................................... 73
Analysis oftyrosine mutant middle T expression ............................. 77
Pulse—chase analysis ofthe stability of Y6F middle T ........................ 79
Analysis of c-Src activation in cells expressing mutant MTs ............... 81
MT-associated in vitro kinase assay and the effect of
Src inhibitor SU6656 ............................................................. 83
Analysis of Src activity and MT phosphorylation by 85
MT-associated in vitro kinase assay
MT phosphorylation in cells infected with polyomaviruses ................. 87
by in vitro kinase assay
Requirement of MT tyrosines for p85 binding ................................ 89
Comparison of viral genome levels in wild type and mutant ............. 1 12
polyomavirus infected cells
Analysis ofthe early viral proteins ............................................. 1 14
C omplementation of MTs in A185 genome accumulation .................. 1 l6
Complementation of MTs on A185 viral early and late ..................... I 18
protein expression
Comparisons of MT complementation in NHI3T3 and ..................... 120

NIH-LT cell lines

Figure 3-6 MT activation of gene expression through polyoma enhancer ............ 122

Figure 4-1 Polyomavirus genome accumulation in the skin close to the site ......... 145
of injection in neonatal mice following a 6-day acute infection

Figure 4-2 Genome accumulation of polyomaviruses A2-RE and Y6F in ............ 149
neonatal mice following a 6-day acute infection

Figure 4-3A Comparison ofdifferent mutants in neonatal mice following ............. 151

a 6-day acute infection

Figure 4-3B A summary of viral genome accumulation in skin (close to the ........... 153
site ofinjection) from neonatal mice following a mixed infection

Figure 4-4 Genome accumulation of polyomaviruses A2-RE and Y6F in ............ 155
neonatal mice following a 4-week persistent infection

Figure 4-5 Determination of adult mouse infection period .............................. 157

Figure 4-6 Genome accumulation of polyomaviruses A2-RE and Y6F in ............ 159
4-week old mice following a 5-day acute infection

Figure 4-7 Genome analysis of live virus production of polyomavirus ............... 161
A2-RE and Y6F

CHAPTER 1

LITERATURE REVIEW

INTRODUCTION

Mouse polyomavirus is a small non-enveloped double-stranded DNA tumor virus.
The viral genome consists of 5297 base pairs of closed circular DNA organized as
minichromosome and packaged in an icosahedral capsid. The viral genome is divided
into early and late regions, separated by a non-coding regulatory region (Figure 1-1,
[154]). The viral regulatory region contains two promoters for early and late gene
transcription, an enhancer and an origin of replication (Ori). The early gene encodes three
viral regulatory proteins - large T (LT), middle T (MT) and small T (ST), which are
translated from three differentially spliced transcripts. Large T is a 100-kDa
multifunctional nuclear protein that is vital for viral replication, regulation of early
transcription [41] and immortalization of primary cells in culture [137, 138]. Middle T is
a 56-kDa oncoprotein localized at the plasma membrane. MT is required for cellular
transformation [97] and plays an important role in the viral lytic cycle [32] and hence in
viral replication and persistence in mice [72]. MT functions are mediated by a series of
interactions between MT and cellular signaling molecules involving several tyrosines at
the C-terminal half of MT. Small T is a 22-kDa protein located in both the nucleus and
the cytoplasm. ST can enhance the function of LT in DNA synthesis [18, 128],

complement the function of MT in transformation [14, 126] and tumor induction [12, 13].

The late region encodes three viral capsid proteins — VPl (45 kDa), VP2 (35 kDa) and
VP3 (23 kDa), which are also synthesized from three differentially spliced transcripts.
This thesis will focus on the role of MT C -terminal tyrosines in the activation of
MT signaling, viral replication and gene expression during viral lytic infection using cell
culture and mouse model. Therefore, this literature review will address the following
topics: (1) properties of viral early and late proteins, especially the role of MT in signal
transduction and viral lytic cycle; (2) polyoma enhancer and its role in viral replication
and gene expression; and (3) polyomavirus lytic cycle, cellular transformation and tumor

induction.
PROPERTIES OF POLYOMAVIRUS EARLY PROTEINS

Large T protein;

Large T (LT) is a lOO-kDa multifunctional nuclear protein. It contains 785 amino
acids, with the first 79 amino acids shared by MT and ST. LT plays important roles in
initiation of viral DNA replication [49, 187], regulation of early and late gene
transcription [68, 100], genome integration [56, 69] and cellular immortalization [58, 66,
74].

LT binds to the polyoma origin as double hexamers. ATP-dependent DNA
helicase activity of LT unwinds DNA in the 3’ to 5’ direction on the DNA strand to
which it is bound. LT then forms complexes with cellular proteins, e. g. DNA polymerase
oz, single strand-specific DNA binding (SSB) proteins, replication protein A (RPA) and
topoisomerase I, to initiate DNA replication [67].

LT can also interact with the product of pr tumor suppressor gene, p105Rb and

related proteins, p107 and pl 30 [55, 65, 89]. The domain responsible for pr binding and

immortalization is located at the N—terminal of LT between amino acids 141 and 146
[104]. Further study reveals that LT binds to the pr, p107 and p130 through a common
region located between amino acids 141 to 158 [135]. This region contains a consensus
pr binding sequence D/N-L-X-C-X-E. Substitution of any amino acid within this core
pr-binding sequence abolished pr and p107 binding in vitro and immortalization
activity in vivo. The binding of LT to pr releases transcription factor E2F [188],
resulting in the transcriptional activation of E2F controlled cellular genes, including DNA
polymerase oz, dihydrofolate reductase (DHFR), thymidylate synthetase and thymidine
kinase and the subsequent S-phase entry of host cells. The interaction between LT and
pr is also involved in the ability of LT to immortalize primary rodent cells [104].
Mutants containing a single amino acid substitution (Asp-141+ Glu or Glu-146—> Asp)
in LT failed to bind pr and were unable to immortalize primary rat embryo fibroblasts.
However in some cases, immortalization function of LT in cell culture is not required for
tumor induction in mice [70]. Mutant viruses encoding altered large T proteins that fail to
bind pr, are defective in immortalization of primary rat embryo fibroblasts by a variety
of tests, but are capable of transforming both primary and established fibroblasts in
culture as well as induce tumors in mice. The DnaJ domain at the N-terminus of large T
is required for the regulation of pr function. Mutations of large T sequences conserved
with the DnaJ family affect large T binding to heat shock protein 70 (Hsc 70) [150]. The
same mutations abolish large T activation of ’E2F-containing promoters and pr binding-
dependent large T activation of cell cycle progression. These functions of large T, which
cause cells to enter the S-phase are also required for viral DNA replication, presumably

as they provide the cellular environment required for DNA synthesis.

p300 and C BP [cAMP-responsive-element-binding factor (CREB)-binding
protein] are large nuclear proteins that are involved in transcriptional regulation,
chromatin modification and cell cycle control. LT binds to CBP/p300 through its C-
terminal region [39, 124]. The QLFP sequence between amino acid 668 and 671 is
homologous to the CBP/p300 binding region in adenovirus El A and SV40 LT. Mutation
at residue 671 results in a loss of CBP/p300 binding and ofthe repressive activity oflarge
T on CREE-mediated transactivation by CBP/p300. These mutants transform rat cells
normally but fail to induce a broad spectrum of tumors in mice. Proline 671 substitution
mutants grow poorly in nonnal NIH 3T3 cells but can be partially complemented for
growth in mouse cells expressing wild type large T. These findings indicate that
C BP/p300 binding by Py large T is essential for virus growth.

Other functions of the C-terminal half of LT comprising approximately residues
264-785 are also studied [78]. This region is sufficient for driving viral DNA replication
in vivo in dividing mouse cells using an origin-containing plasmid as a reporter, but is
strikingly deficient for replication in serum-starved cells. However, this deficiency can be
complemented by coexpression of N-terminal half of LT due to its ability to inducing S-

phase entry.

Middle T grotein

Middle T’s (MT) activities can be observed in both viral growth and
transformation. MT is required for high level ofviral replication during viral lytic cycle
[32, 33]. MT activates polyoma enhancer and increases viral early and late gene

expression [40, 51, l 17, 190]. The expression of MT is sufficient to transfomi established

cell lines [97, 138, 167] and to induce multifocal metastatic mammary adenocarcinomas
in transgenic mice [87].

Middle T is a 421 amino acid protein with a molecular weight of 56 kDa. MT
shares a common N-terminal region (aa. l-79) with LT and ST, and a further common
region with ST (aa. 80-191). There is a stretch of 22 hydrophobic amino acids near its C-
terminus that serves as membrane-binding domain. MT lacking this sequence can not
bind to cell membrane and does not transform [27]. However, replacement of this
hydrophobic region with membrane targeting sequences from vesicular stomatitis virus
glycoprotein G [166] fails to maintain transformation while preserves membrane
association. In addition, point mutations within this region can also eliminate the
transformation ability of MT without affecting membrane binding [1 12], suggesting that
membrane binding is not the only function of this hydrophobic region.

A highly conserved sequence HPDKGG between residues 42 and 47 which is
common to all T antigens shares functional homology with J-domain of DnaJ/hsp40
molecular chaperones [99]. J-domain can stimulate ATPase activity of hsc70 and may be
involved in the assembly of multi-subunit complexes [156]. Deletion of J-domain in MT

does not affect MT transformation [24, 80].

Protein Phosghatase 2A

MT stoichiometrically associates with protein phosphatase 2A (PP2A), a
serine/threonine phosphatase. The PP2A core structure is a dimer, consisting of a 36—kDa
catalytic C subunit and a 65-kDa regulatory A subunit. A third regulatory B subunit

associates with this core enzyme, conferring substrate specificity and subcellular

distribution. Both MT and ST bind to the core dimmer PP2A A+C through their N-
terminal region (amino acid 90-120 in MT) [79, 80, 84, 85, 1.32, 169, 177]. Mutants that
do not bind PP2A fail to bind c-Src and to transfomi. It is not clear at present what other

functions are carried out by MT-PP2A association.

c-Src Tyrosine Kinase

 

MT has no intrinsic enzymatic activity by itself. However, following the binding
of MT to PP2A, MT binds to c-Src family tyrosine kinases, including pp60c'src [47, 48],
pp59°ryn [38] and pp62c’ycs [102]. Src protein tyrosine kinases are 52-62 kDa proteins
composed of 6 distinct functional regions: (1) a short N-terminal Src homology (SH) 4
domain for membrane localization; (2) a unique region for each family member; (3) an
SH3 domain, which can bind to proline-rich ligands containing a core consensus
sequence of PXXP; (4) an SH2 domain can bind to short continuous amino acid
sequences containing phosphotyrosine, and the specificity of individual SH2 domains lies
in the 3-5 residues following the phosphotyrosine (+1, +2, +3, etc); (5) a catalytic domain
possesses tyrosine-specific protein kinase activity; (6) a short C-terminal negative
regulatory tail containing a conserved tyrosine residue.

pp60c'Src was the first protein identified to be associated with MT [47]. Residues
185 to 210 of MT sequence are required for c-Src binding, but have no role in PP2A
binding [22, 81]. The activation of pp60”rc by MT is accompanied both by the
dephosphorylation of Tyr 527 [44], a site which negatively regulates pp6OC'SrC kinase
activity and by the autophosphorylation of Tyr 416 [30, 41, 45]. Once activated, MT-

associated Src phosphorylates at least three tyrosine residues in the C -terminal region of

MT, Tyr 250 [90], Tyr 315 [96, 130, 145] and Tyr 322 [146]. These phosphorylated
tyrosines then provide docking sites for several cellular molecules involved in signal

transduction, including ShcA, the p85 subunit of Phosphatidylinositol-3’ kinase and PLC-

71.

Phosphatidylinositol -3 kinase

Phosphatidylinositol-3 kinase (PI3K) was the first signaling intermediate found to
bind to MT [46, 184]. PI3K is a heterodimer, consisting of an 85-kDa regulatory subunit
(p85) and a 110-kDa catalytic subunit (p110) [28]. The SH2 domain of p85 interacts with
phosphorylated tyrosine 315 on MT which contains the preferred PI3K binding motif,
YMXM [164, 189]; in turn, p85 phosphorylation stimulates kinase activity of the p110
subunu.

Recently, other studies have reported that p85 can also bind to Y250, although
through different mechanisms. The amino acid sequence surrounding tyrosine 250 is
NPTXSVM. NPTY is the binding motif for ShcA PTB domain (see below), while
YXXM forms a potential binding site for p85. Hong, et. al. reported that Y250 provides a
secondary binding site for p85 based on genetic and biochemical analysis [95]. On the
other hand, Ong, et al. [129] showed that Y250F shows decreased PI3K activity, which
can be restored by insertion of growth-factor-receptor-bound protein 2 (Grb2) binding
sites from EGF receptor (YVNQ) or from ShcA (YYND). Since Grb2-associated-
binding protein (Gabl) forms a complex with ShcA, Grb2, Ras guanine nucleotide

exchange factor son of sevenless l (8051), and MT [125] as well as other SH2 domain-

containing proteins, e.g. PI3K [94] and PLC-‘yl [86], they suggested that Y250 can
indirectly recruit p85 through ShcA-Grb2-Gab1 interaction.

Binding of MT translocates PI3K from cytoplasm to a membrane location where
its substrates are present [42]. PI3K phosphorylates the D-3 position of the inositol ring
of phosphatidylinositol (4,5)-biphosphate [Pl(4,5)P3] to generate inositol 3,4,5-
trisphosphate [PI(3,4,5)P3], which can be dephosphorylated by a 5’ phosphatase to
generate Pl(3,4)P2 [149]. These PI3K products bind to the pleckstrin homology (PH)
domain of Akt/PKB/RAC, a serine/threonine protein kinase and recruit it to the plasma
membrane. PI(3,4,5)P3 and PI(3,4)P2 can also bind to the PH domain of the protein
kinase 3-phosphoinositide-dependent protein kinase-1 (PDK-l). Following activation by
PI3K, PDK-l and a less well-defined kinase PDK-Z then phosphorylate Akt at threonine
308 and serine 473, respectively, thus activate Akt [4, 175]. Akt regulate many biological
processes, such as proliferation, apoptosis, and growth. Some of the substrates of Akt
include the glycogen synthase kinase 3 (GSK3), the ribosomal protein S6 kinase

(pp70‘w’K), the transcription factor E2F, and the apoptosis regulating protein Bad.

ShcA [Src homology 2 (SH2) domain-containing transfomiing protein A]

Phosphorylated tyrosine Y250 of MT was discovered as a binding site for ShcA
when a proline 248 to leucine mutation in the NPTY motif of MT was found to cause loss
oftransfonnation phenotype, while still able to associate with c-Src and PI3K [25, 59-61].
ShcA is expressed as three isoforms of approximately 46, 52 and 66 kDa. Each ShcA
peptide contains an SH2 domain at its C-temiinus, a collagen homology region (CH1)

and an N-tenninal phosphotyrosine binding (PTB) domain. The 66 kDa isofonn contains

an additional N-tenninal CH-like region (CH2) [139]. ShcA binds to phosphorylated
Y250 of MT through its PTB domain and is phosphorylated by MT-c-Src complex at
Y239, Y240 and Y317 in the CH1 region. When phosphorylated, these tyrosines can
interact with the SH2 domain of Grb2. Grb2 associates through its SH3 domain with
$051. Relocation of SOS] to a membrane site activates p21ms [10], which further activates

the mitogen-activated-protein kinase (MAPK) signaling cascade.

M

Phosphorylation of Y322 in MT creates a binding site for the SH2 domain of
phospholipase C-yl (PLC-yl) [163]. PLC-yl is phosphorylated on tyrosines upon
association of MT. On activation, PLC-yl catalyzes hydrolysis of PI(4,5)P2 to produce
the second messengers inositol 1,4,5-trisphosphate (1P3) and diacylglycerol (DAG). 1P3
provokes a transient increase in intracellular free Ca2+, while DAG serves as a direct

activator of protein kinase C (PKC).

14-3-3

14-3-3 denotes a large family of ~30-kDa acidic proteins that exist primarily as
homo- and heterodimers within all eukaryotic cells. 14-3-3 proteins interact with
phosphoserine in the RSXSXP motif [123]. MT contains this consensus sequence around
serine 257 and two 14-3-3 family proteins (27-kDa mixture of several subspecies, and 29-
kDa epsilon subspecies) bind to MT through phosphorylated Ser 257 [50, 131], though
the kinase responsible for the phosphorylation of Ser-257 is unknown. 14-3-3 has been

shown to bind PI3K [21], cdc25 [43] and Raf-l [108]. 14-3-3 is also involved in the Ras-

dependent activation of Raf-l [98, 143]. Ser 257 is also involved in MT multimerization
[148]. Removal of Ser 257 does not affect transformation of fibroblasts in vitro, since
8257A and 8257C mutant MTs retained the ability to form foci or colonies in agar, but a
virus expressing S257C MT failed to induce salivary gland tumors in mice [50]. The

basis for this defect is unknown.

Downstream transcription factors

 

The downstream targets of MT-induced signaling pathways include transcription
factors, including PEAl/AP-l [147, 181, 182], PEA3/c-Ets [151] [180] and Myc [136].

PEAl is a mouse homolog of human AP-l. AP-l family of transcription factors
consists of homo- and heterodimers of Jun (v-Jun, c-Jun, JunB, JunD), Fos (v-Fos, c-Fos,
FosB, Fral, Fra2) or activating transcription factor (ATF2, ATF3/LRF1, B-ATF) bZIP
(basic region leucine zipper) group DNA binding proteins [9, I76]. Jun-Jun and Jun-Fos
dimers preferentially bind to the phorbol l2-O-tetradecanoate-l3-acetate (TPA)-
responsive element (TRE; TGACTC A). Three different types of MAPKs, the ERK, the
JNK and the p38 MAPK, contribute to the induction of AP-l activity in response to a
variety of extracellular stimuli. c-fos is activated by ERK [35] or ribosomal S6 kinase
(RSK) [36] and c-jun is activated by .INK [93].

PEA3 is a mouse homolog of human c-Ets. Ets family transcription factors share
an approximately 85-amino acid structurally conserved Ets DNA binding domain.
Thirteen subfamilies have been identified [105]. Ets binds to a purine-rich GGAA/T core
sequence. Sequences flanking this motifdetennine the specificity of binding by particular

Ets proteins [82].

10

In the polyoma enhancer, there are PEAl/AP-l and PEA3/c-Ets binding sites.
When activated by MT through signal pathways, PEAl/AP-l and PEA3/c-Ets can bind to

the polyoma enhancer and activate viral DNA replication and gene expression.

Small T protein

Small T (ST) is a 195 amino acid cysteine-rich protein located in both cytoplasm
and nucleus. ST binds PP2A [I32]. Mullane et. al. showed that although still able to bind
to PP2A, a Y250/315F mutant MT did not promote cell cycle progression in serum-
starved NIH3T3 cells like ST did. It also failed to activate c-fos whereas ST could.
However, this mutant MT was able to activate c-jun, whereas ST was not [119].
Therefore ST-PP2A interaction may have a different role than MT-PP2A interaction.

ST can enhance the function of LT in DNA synthesis [18, 128]. Berger et. al.
showed co-expression of ST could partially overcome the replication defect of a mutant
polyomavirus genome encoding only LT. Using a dexamethasone-inducible system,
Ogris, et. al. further demonstrated that expression of both LT and ST could induce a high
percentage of cells to enter S phase, whereas expression of LT or ST alone failed. ST can

also complement the function of MT in transfomiation [14, 126] and tumor induction [12,

13].

Lady-mm

The late gene of polyomavirus encodes three capsid proteins, VPl (45 kDa), VP2
(35 kDa) and VP3 (23 kDa). VPl is the major capsid protein. The icosahedral capsid
contains 72 pentameric capsomers, which each contains five molecules of VPl. VPl is

the only capsid protein exposed on the outside of the virion. The sequence of the minor

11

capsid proteins, VP2 and VP3 are overlapping. VP2 has 115 amino acids at its N-
terminus and contains the entire VP3 sequence at its C-terminus. The highly conserved
C-termini of VP2 and VP3 interact with the N-temtinus of VP] [15, 37].

VPl consists of about six isoelectric species designated A through F, which is
caused by post-translational modification [7, 20]. The minor species D, E, and F are
phosphorylated, mainly on threonine residues, and to a less extent, on serine residues.
Mapping of the phosphorylation sites on VPl identified in vivo phosphorylation of
threonine residues Thr 63 and Thr 156 [106] and in vitro phosphorylation of serine
residue Ser 66 [107]. Mutation of Thr 63 does not cause growth defect of the virus,
whereas mutation of Thr 156 affect virion assembly. Mutation of Ser 66 does not produce
viable viral particles. Casein kinase II can mediate the phosphorylation of Ser 66 on VPl,
and is often activated by serum, epidermal growth factor, and insulin-like growth factor
[2, 29, 101, 155]. Polyomavirus host range transforming (hr-t) mutants [16] encoding
defective MT and ST have been shown to have defect in VPl threonine phosphorylation
at Thr 63 and Thr 156, causing defect in virion assembly [75, 76]. These data suggest a
role of MT and/or ST in regulating VPl phosphorylation.

The N-terminal glycine of the VP2 is myristylated [103, 161]. The removal of the
myristyl group by mutating the N-temiinal glycine to glutamic acid does not result in a

total loss of infectivity, but the efficiency of viral replication is severely diminished.

STRUCTURE AND FUNCTION OF POLYOMA ENHANCER

Polyomavirus enhancer is a 244-bp sequence (nt. 5023-5267) between Bell and
PvuII, located between replication origin and the beginning of late protein coding

sequences [53, 54, 168]. It contains DNAse I hypersensitive sites [31] and is devoid of

12

nucleosomes in 30% of the viral chromatin in infected cells [92, 170], and thus bears
characteristics of active chromatin. The polyoma enhancer can be divided into two
distinct elements: A domain (BclI-Pvull, nt. 5023-5130) and B domain (PvuII-Pvull, nt.
5130-5267). The A domain comprises an auxiliary subelement (nt. 5073-5102) and an 01-
core subelement (nt. 5108-5126). Similarly, B domain comprises two auxiliary
subelements, (nt. 5130-5172 and nt. 5202-5218) and a B—core subelement (nt. 5172-5202)
[118, 120]. Several binding sites for cellular proteins have been identified, including
PEAl/AP-l[113], PEA2 [134, 178] and PEA3/c-Ets [179, 180]. In particular, the oz-core
of A domain contains juxtaposed binding site for PEA3, PEA] and PEA2. Many genes
activated by signaling through the Ras—Raf—ERK pathway utilize transcriptional control
elements containing similar tandem AP-l and Ets binding sites, e.g. Cyclin D1 [3], the
urokinase-type plasminogen activator [170], and interstitial collagenase/matrix
metalloproteinase 1 (MMPl) [19]. PEAl/AP-l and PEA3/c-Ets has been shown to bind
to the enhancer [l 13, 122, 127, 134, 180, 190], leading to the activation of early and late
gene transcription [40, 51, 54, 117, 165, 168, 190]. PEAl/AP-l and PEA3/c-Ets can also
bind to polyoma enhancer and directly stimulate DNA replication [11, 122, 172].
PEAl/AP-l and PEA3/c-Ets are also shown to stimulate DNA replication directly
through the bipartite PEAl-PEA3 site in vitro [179-181]. Therefore, polyoma enhancer
can regulate viral DNA replication by two mechanisms. (1) The activation of early gene
transcription by PEAl/AP-l and PEA3/c-Ets increases the expression of LT, therefore
increases viral DNA replication. (2) PEAl/AP-l and PEA3/c-Ets can also bind to

polyoma enhancer and directly stimulate DNA replication.

13

The two enhancer domains play different roles in controlling cell-specific
transcriptional activation in cell culture [92]. In mouse fibroblasts, element A provides a
3-fold higher enhancement of the alpha 2-collagen promoter than element B. In mouse
embryonal carcinoma cells, element B shows the same efficiency as in fibroblasts,
whereas that of element A decreases by a factor of 3.5. They also play different role in

controlling the age- and organ-specific pattern of viral replication in mice [6].

THE POLYOMAVIRUS LYTIC CYCLE, CELLULAR TRANSFORMATION AND

TUMOR INDUCTION

Lytic cycle of polyoma infection

Polyomavirus was first discovered in 1953 by Ludwig Gross as an infectious
agent in cell free filtrates from mouse leukemic extracts which caused salivary gland
carcinomas when inoculated back into mice [83]. Later this virus was shown to cause a
variety of tumors in newborn mice [159], and was subsequently named mouse
polyomavirus. It soon became a popular model for studying viral lytic infection, cell
transformation and tumorigenesis.

A productive infection of polyomavirus starts with the adsorption of the virion to
the surface of permissive cells that support a full lytic infection. The major capsids
protein VPl binds to specific sialyloligosaccharide receptors on the surface of cell
membrane [23, 34, 73, 158]. The myristylation of VP2 has also been shown to be
important for the infectivity of the virus through an unknown mechanism [103, 144, 161]
since VP2 is entirely internal to the virion outer shell. The internalization of virions after
the initial adsorption is through monopinocytosis. However, the exact route of

polyomavirus entry is still unknown [77], and is unlike SV40 which enters via caveola-

l4

derived vesicles [8, 157], or human JC virus which uses a clathrin-coated pit endocytic
pathway [133]. Once uncoated inside the nucleus [1 10], the viral minichromosome serves
as a template for early gene transcription. Pre-mRNA is transcribed from the early region
and is spliced into three early transcripts and the early proteins (large T, middle T and
small T) are produced. Large T binds to the origin of replication and initiates viral DNA
replication. As DNA replication starts, the late promoter is activated and produces the
late transcripts. The late mRNA precursor is spliced into three late mRNA, which is
translated into capsid proteins, VPl, VP2 and VP3. New virions are assembled and
accumulated in the nucleus. When the cells are filled with progeny virions, they lyse and

the mature viral particles are released [1].

Virus replication in! mice

The course of polyomavirus infection in vivo consists of two distinct phases:
acute phase and persistent phase [64]. Following infection of neonatal mice, the level of
live virus and viral genome peaks at 7-10 days postinfection [141, 142]. This coincides
with the onset of anti-viral immune response [63, 142]. The immune response clears most
of the viruses and viral genomes from infected organs within a week, opening the way for
the persistent phase. The level of viral genomes gradually decreases, but persists over a
long period of time in the kidney, the salivary gland, the bone, the skin and the mammary
gland [64, 114, 115, 142, 186]. The overall de novo viral replication potential also
progressively decreases with the age of the mouse, thus mice infected as neonates
produces more viruses than those infected after 3 weeks of age [63, 141, 186]. An organ-
and age-specific pattern of viral replication has been observed [57, 63, 185]. In organs

such as bone, skin and mammary gland. virus replicates at very high levels in neonates

15

and at moderate levels in adults, whereas in organs such as kidney, liver and lung, virus
replicates at high levels in neonates but at very low levels or not at all in adults. Further
study demonstrated that polyoma enhancer plays an important role in determining this
age- and organ-specific viral replication pattern [6]. The B domain mediates
polyomavirus replication in most organs, in particular in kidney, at neonatal stage but is
not required in those organs which can sustain replication in the adult, i.e. mammary
gland, skin and bone. The A domain mediates polyomavirus replication at adult stage.

Early studies showed that middle T is essential for virus replication and
persistence in mice. For example, a mutant virus PTA-1387T, which encodes a truncated
MT lacking the C-tenninal membrane insertion region, has a strong detect in viral
replication and persistence and fails to induce tumor [72]. The role of MT in viral
replication is probably mediated by transcription factors PEAl/AP-l, PEA2 and PEA3/c-
ets [1 13, 134, 165, 179, 182] through polyoma enhancer [33].

The route of infection could also affect the site of persistence. For instance, virus
persisted in both lungs and kidneys in an intranasal infection, while only persisting in the

kidneys in an intraperitoneal infection [62-64].

Cellglar transformation and tumor induction

In tissue culture cells that are permissive to polyomavirus infection, virus
replication takes place, followed by the production of progeny virions. It ends with the
completion of viral lytic cycle and the lysis of the cells. In cells that are non-permissive
to polyomavirus infection, the expression of viral early proteins alters the phenotype of
the infected cells, which displays properties of transformation, including loss of contact

inhibition and anchorage independence. Some of the short-temi changes are also seen in

16

lytically infected permissive cells. However, the viral genome replicates poorly in
nonpermissive cells, due to the inability of LT to interact with the host DNA polymerase-
or/primase [121], and, therefore is gradually lost. The transformed phenotype disappears
and the cells return to normal growth. This process is called “abortive transformation”
[160]. At a low frequency, the viral genome integrates into the host cell genomes at a
random site, which results in the continued expression of MT and permanent acquisition
of transformed phenotype. The infected cells are transfomted by the virus. In cell culture,
the expression of MT is sufficient to transform established cell lines [97, 138, 167], but
LT is required to transfomi primary cells [137]. MT Tyr 250 is required for
transformation [59, 1 l 1]. MT Tyr 315 is not essential for transformation, but mutation of
tyrosine 315 to phenylalanine greatly reduces transformation efficiency [26, 116, 130].
Interestingly, expression of MT alone can induce tumors in transgenic mice. In fact, MT
is the most potent oncogene tested in transgenic mice.

Polyomavirus infection induces a variety of tumors in mice [52] and MT is
required for tumor induction [72]. Three major mouse models have been developed to
study tumorigenesis: (1) Neonatal mice infection model; (2) Adult nude mice infection
model; and (3) Transgenic mice model.

Infection of neonatal mice leads to a rapid systemic infection. Most of the viruses
are cleared from infected tissues following immune maturation, but virus can persist in a
lower degree in certain organs throughout the life. Tumor induction occurs later in life.
There is a correlation between high genome replication and high tumor incidence,
because tumors are developed from organs that support high level of genome replication

and persistence in adult mice [186]. The tumor response depends on the genetic

l7

backgrounds of both virus and host. Certain inbred mouse strains are susceptible to tumor
induction by polyomavirus. More than 30 cell types can be infected and tumor arises
from a subset of these tissues, including the mammary gland, the skin, the bone, the
salivary gland and the kidney [52]. Susceptibility in mice is modulated by their MHC
type (H-2k)-related anti-tumor activity and also by an endogenous mouse mammary
tumor provirus, Mtv-7. Mtv-7 encodes a superantigen (sag) that delete V36+ T cells
required for mounting an effective cytotoxic T lymphocyte (CTL) response to
polyomavirus induced tumors [109]. Superantigen-independent susceptibility has also
been reported recently [173, 174]. Two wild-derived inbred mice strains, which carry no
detectable endogenous Mtv-7 are as fully susceptible to tumor induction by polyomavirus.
This superantigen-independent susceptibility is transmitted in a dominant fashion in
crosses with MHC-identical immunologically resistant mice [173, 174]. The genetic
background of viruses also plays important roles in determining the tumor response. The
“high tumor” strain, PTA induces a variety of epithelial and mesenchymal tumors in high
incidence and short latency. The “low tumor” strain, RA induces only two types of
mesenchymal tumors after a long latent period [52]. The key determinant of PTA in the
induction of a high tumor profile is a single amino acid substitution in the major capsid
protein VPl [71].

Infection of adult athymic (mi/mi) nude mice provides a means to study tumor
induction at a specific age and homtonal dependence. In Balb/C mice, tumors appears
with a high frequency and a short latency. Tumor induction is restricted to the bone, the
salivary gland, the uterus, the skin and the mammary gland [5, 17, 91, 162, 171]. The role

of age and ovarian hormone on mammary tumorigenesis has also been demonstrated

18

[140]. In the ovary-intact mice, the tumor incidence is 100% in mice infected at 3, 6 and
10 weeks of age; it decreases to 55% and 10% in those infected at 20 and 30 weeks of
age, respectively. Removal of ovaries 1 week prior to polyomavirus infection reduces the
tumor incidence to 61%, 11% and 0% in mice infected at 6, 10 and 20 weeks of age,
respectively. Removal of ovary 1 week post infection does not have a significant effect
on mammary oncogenesis in 6 week-old mice. Treatment of ovariectomized adult mice
with estrogen + progesterone for 1-3 weeks prior to infection was unable to restore
tumorigenesis to the level observed in intact mice. These different effects suggest the
existence of different status in hormonal responsiveness at these ages, which may
correlate with the transition in hormonal responsiveness and mammary gland morphology
that occur between these ages.

Transgenic mice expressing polyoma MT has been a widely used model to study
mammary oncogenesis [152]. One model system using polyoma MT under the control of
MMTV promoter/enhancer demonstrates that expression of polyomavirus middle T
antigen results in the widespread transformation of the mammary epithelium and the
rapid production of multifocal mammary adenocarcinomas and secondary metastatic
tumors in the lungs [87]. To further study c-Src in middle T antigen-induced mammary
tumorigenesis, a transgenic mouse strain carrying the MMTV/PyV middle T antigen
fusion gene and a disrupted c-src gene was generated [88]. In contrast to the rapid tumor
progression seen in the original MMTV/PyV middle T antigen strains, mice expressing
MT in the absence of functional c-Src rarely develops mammary tumors, suggesting that
c- Src tyrosine kinase activity is required for middle T antigen- induced mammary

tumorigenesis. The requirement of ShcA and phosphatidylinositol 3' kinase signaling

19

pathways for polyomavirus middle T-mediated mammary tumorigenesis has also been
studied [183]. Transgenic mice that express Y250F or Y315/322F MT developed
extensive mammary epithelial hyperplasias. The mammary epithelial hyperplasias
expressing Y315/322F MT were highly apoptotic. A proportion of the metastatic tumors
arising in mice expressing Y250F MT displayed reversion of the mutant ShcA binding
site. These results suggest that polyomavirus MT-mediated tumorigenesis requires
activation of both ShcA and PI-3' kinase, which appear to be required for stimulation of

cell proliferation and survival signaling pathways, respectively.

SUMMARY

This literature review focused on the MT-induced signaling pathways and the
roles of MT in polyomavirus lytic infection and transformation. This study investigates
the contribution of C-terminal tyrosines in MT signaling and establishes a model for MT
function in viral lytic infection in its natural host. In addition, this study demonstrates a
novel role of MT tyrosines in viral replication, providing new insight into the function of

MT in virus life cycle.

20

APPENDIX 1: TABLES AND FIGURES FOR CHAPTER 1

21

Figure 1-1 Physical map of polyomavirus genome

The inner circle represents the restriction endonuclease sites of MspI on the
polyomavirus genome. The early and late coding regions are shown in the outer circles.
The coding regions are represented by boxes and the introns are represented by jagged
line. The early transcripts are differentially spliced from a common precursor mRNA and
translated into the large T, middle T and small T. The late transcripts are processed
similarly to produce capsid proteins, VPl , VP2 and VP3. The non-coding viral regulatory
region contains the origin of replication, an enhancer, and the early and late promoter.

This figure is a modification ofthe original figure from Soeda et. al. [153].

   

3cm:
Eoow

 

Figure 1-2 Lytic cycle of polyomavirus

The lytic infection of cells by polyomaviruses can be divided into early and late stages.
The early stage starts with attachment of the virion to cell surface and continues until the
beginning of viral DNA replication. This stage is marked by the adsorption and
penetration of the virion and its migration to the nucleus, where the viral genome is
uncoated and ready for DNA replication and late gene transcription. The late stage begins
with the onset of viral DNA replication and ends with release of progeny viruses. This
stage includes viral DNA replication, late gene expression, assembly of progeny virions
and finally release of viral particles. Multiple steps of a productive polyomavirus
infection are depicted. This is a modified version of the figure from Fundamental

Virology, 4th ed

24

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25

Figure 1-3 A schematic representation of middle T

Tyrosines on middle T are presented above the line. The position of DnaJ domain, intron
and membrane insertion domain and the binding sites for MT-associated proteins
including PP2A, c-Src, ShcA, 14-3-3, PI3K and PLC-yl are marked below the line. The
signaling pathways activated by these proteins are shown. The common region shared

between MT and ST is shown at the bottom.

26

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27

Figure 1-4 Diagram of the signaling pathways mediated by middle T

After binding to cell membrane, MT binds to PP2A and c-Src, leading to the activation of
c-Src tyrosine kinase activity. c-Src phosphorylated at least three tyrosines at the C-
terminal of MT, including Tyr 250, Tyr 315 and Tyr 322. Binding of ShcA to
phosphorylated Tyr 250 recruits Grb2 and SOS, resulting in the activation of Ras. Ras
activates MAPK signaling pathways. Binding of the p85 subunit of phosphatidylinositol—
3 kinase (PI3K) to phosphorylated Tyr 315 activates PI3K, resulting the activation of
PDKl, which further activates Akt. Binding of PLC-yl to phosphorylated Tyr 322

activates PKC.

28

 

 

    

PI3K PLC-gammal

L\ \.
l \ l

Rac PDKl

R'ar Akt

 

 

 

Nucleus

Figure 1-5 Physical map of the enhancer region of polyomavirus A2 genome

The 244 base pair polyomavirus enhancer (BclI-Pvull, nt. 5023-5267) consists of two
distinct elements, A domain (BclI-Pvull, nt. 5023-5130) and B domain (PvuII-PvuII,
5130-5467). Each domain can be further divided into a core element and one (A domain)
and two (B domain) auxiliary subelernents. Cellular transcription factor biding sites are
found in the enhancer, including one PEAl (mouse homolog of human AP1) and three
PEA3 (mouse homolog of human c-Ets) sites. The location of polyomavirus origin of
replication and the early and late promoters are also indicated. E = Early Promoter; L =

Late promoter; HS = DNase I Hypersensitive site.

30

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it

tiles

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3.5 Sum 5.5 SE? SE?
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31

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Wirth, J .J ., et al., Organ- and age-specific replication of polyomavirus in mice. J
Virol, 1992. 66(6): p. 3278-86.

Wirth, J .J ., L.G. Martin, and M.M. Fluck, Oncogenesis of mammary glands, skin,
and bones by polyomavirus correlates with viral persistence and prolonged
genome replication potential. J Virol, 1997. 71(2): p. 1072-8.

Wold, M.S., J.J. Li, and T.J. Kelly, Initiation of simian virus 40 DNA replication
in vitro: large-tumor-antigen- and origin-dependent unwinding of the template.
Proc Natl Acad Sci U S A, 1987. 84(11): p. 3643-7.

Wolf, D.A., et al., A complex between EZF and the pr-related protein p130 is
specifically targeted by the simian virus 40 large T antigen during cell
transformation. Oncogene, 1995. 10(11): p. 2067-78.

Yoakim, M., et al., Interactions of polyomavirus middle T with the SH2 domains
of the pp85 subunit of phosphatidylinositol-3-kinase. J Virol, 1992. 66(9): p.
5485-91.

Yoo, W., M.E. Martin, and W.R. Folk, PEA] and PEA3 enhancer elements are
primary components of the polyomavirus late transcription initiator element. J
Virol, 1991. 65(10): p. 5391-400.

49

CHAPTER 2

ROLE OF POLYOMAVIRUS MIDDLE T C-TERMINAL
TYROSINES IN MT-MEDIATED SIGNALING

ABSTRACT

The polyomavirus middle T oncoprotein (MT) exerts its functions by binding to
and constitutively activating c-Src family tyrosine kinases and their downstream
signaling pathways. Previous studies have shown that MT itself is a Src substrate and that
C-temiinal tyrosines play a crucial role in linking Src activity to cellular signaling
intemiediates. Tyrosine Y315 was defined as the major Src substrate and shown to bind
phosphatidylinositol-3 kinase (PI3K) p85 subunit. Linkage to Ras has been attributed to
Y250, which was also shown to play a role in PI3K-dependent signaling. Y322 was
found to bind phospholipase C-yl. In the present studies I have characterized the role of
tyrosines Y258, Y288 and Y297 in Src-mediated functions, on their own and in
comparison with that of Y250, Y315 and Y322. I examined the effect of tyrosine to
phenylalanine (Y—* F) single and multiple MT mutations for their effects on Src binding,
Src kinase activity, MT substrate capacity and on MT capability to bind to p85. I found
that all mutants, including a sextuple mutant (Y6F) have the capacity to bind to and
activate c-Src. Furthennore, in the context of active Y250+Y315+Y322, now defined as
“Major” (M) tyrosines, mutation of the “minor” (m) tyrosines 258+288+297 has a very
minor effect, even with a triple mutation (Y3Fm). I showed that MT carrying a triple
major tyrosine mutation (Y3FM) has a residual activity, which is eliminated by mutating

the minor tyrosines. Thus, in the absence of active Major tyrosines, the minor tyrosines

50

have a substantial effect. The data show that mutation of both major and minor tyrosines
has an effect on Src activity, as determined by its capacity to phosphorylate enolase. This
decrease in Src activity affects the overall degree of both MT and p85 phosphorylation.
In addition, there are tyrosine specific effects in MT substrate capacity and in p85
binding. These direct comparisons allowed us to show that Y250, Y315, and Y322 have
relatively equal substrate capacities, and that MT containing a single active tyrosine
Y315, and to a lesser extent, Y250, (in the context of quintuple mutations of the other M

and m tyrosines) have the capacity to bind to p85 and induce its phosphorylation.

INTRODUCTION

The function of polyoma middle (MT) in the virus life cycle and cellular
transfomiation is mediated by the activation of various signaling pathways, in which the
interaction between MT and Src family tyrosine kinases plays a central role. The
association of MT with these kinases leads to the constitutive activation of their kinase
activity and results in the phosphorylation of MT C-terminal tyrosines (Figure 2-1). The
major phosphorylated tyrosines Y250, Y315 and Y322 [14, 16, 19, 23, 26] provide
binding sites for cellular signaling intemiediates, respectively, ShcA [5, 11], the p85
subunit of the phosphatidylinositol 3’kinase (PI3K) [29] and phospholipase C-yl (PLC-
yl) [28]. The phosphorylation of these intermediates initiates the activation of three
signaling pathways. In addition, there are three other “minor” tyrosines at the C-terminal
of MT, Y258, Y288 and Y297, whose functions have not been studied.

To study the functions of MT tyrosines in the polyomavirus life cycle, I mutated
the three major tyrosines and found no defect in tissue culture and a minor, if any,

replication defect in mice. This unexpected result prompted us to further mutate the

51

minor tyrosines. The virus expressing a sextuple mutated middle T showed a defect in
both tissue culture and in vivo replication. To begin to understand the function of the
minor tyrosines, I have examined the characteristics of middle T-Src complexes with a
panel of tyrosine mutants. The results show that the phosphorylation of Y250, Y315,
Y322, and at least one other tyrosine among Y258, Y288 and Y297 plays a role in the
activation of Src tyrosine kinase. 1 also demonstrate that tyrosine 250 or 315 alone can

interact with p85 but both tyrosines are required for efficient binding of p85 to MT.

MATERIALS AND METHODS

Cells, viruses and plasmids

Mouse NIH3T3 fibroblast cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM; GIBCO-BRL) supplemented with 10% heat inactivated bovine calf
serum (GIBCO-BRL) at 37°C and 5% C02.

Polyomavirus A2 (WTA2) is the wild-type strain [27]. A185 is a middle T/small
T defective mutant, which is derived from WTA2 by site-directed mutagenesis and
contains a 71 nucleotide out of frame deletion between nucleotide 646 and 717[18]. The
dl45 is a mutant harboring a 66 nucleotide deletion between nucleotide 1075 and 1140,
which deletes amino acids 281 to 302 in middle T antigens and also alters large T [1].
MT-ter is a middle T defective mutant with a stop codon inserted after amino acid 196 in
MT and therefore only expresses small T. 1387T is a mutant encoding a defective middle
T lacking the membrane-anchoring sequence [24]. Middle T C-terminal tyrosine mutants
were constructed by site-directed mutagenesis in the A2 background. Tyrosine residues

were mutated to phenylalanine by A to T single nucleotide mutations using WTA2 DNA

52

as template (pUC19-WTA2). Then, polyoma DNA was cut out by EcoRI digestion and
ligated into a circular genome. The ligated DNA was transfectcd into NIH3T3 cells using
LipofectAMINE 2000 (Invitrogen) to grow live virus. Upon the appearance of cytopathic
effect (CPE), the transfection cell Iysate was further used to infect NIH3T3 cells. Virus
was purified using a standard plaque assay technique and grown in NIH3T3 cells to
prepare virus stock. The titer of the virus was calculated using the plaque assay and by
comparison ofinput DNA ofa virus infection.

To generate retroviruses that express mutant MTs, the 540 bp DrdI-EcoRI
fragment of mutant polyomavirus DNA was cloned into the PINCO vector [13]
containing wild-type middle T cDNA (PINCO-MT/WT) to replace the wild type
sequence. The PINCO-MT plasmid was co-transfected into 293T human epithelial cells
with a packaging plasmid pSV-tlZ. After 6 hrs of transfection, 10% serum medium was
added. Virus stocks were collected every 12 hrs beginning at 60 hrs post transfection. The
titers of virus stocks were determined by MT and GF P expression.

All of the tyrosine mutant polyomaviruses and retroviruses were named by the
mutations they carried. Their properties with regard to the availability of tyrosines on MT
and potential binding sites for cellular proteins ShcA, PI3K and PLC-‘yl are listed in

Table 2-1.

Polyoma virus infection

Cells were grown to 50% confluence on 100 mm tissue culture plates followed by
24 hrs of starvation in 0.5% serum medium. After removal of medium, cells were

infected with virus at multiplicity of infection of 10 plaque forming units (PFU) per cell

53

in G0 state for 2 hrs. The infection lysate was removed prior to the addition of growth

SCI'UITI.

Retrovirus infection

Cells were grown in DMEM supplemented with 10% serum at 37°C and 5% CO;
until reaching confluence for 24 hrs. Cells were then trypsinized and plated at 8x105
cells/ 100 mm plate in 10% serum medium at 37°C for 4 hrs. Retrovirus infection was
carried out at 37°C for 3 hrs with 8ug/ml of polybrene. The infection lysate was removed

prior to the addition of growth medium.

Protein analysis

Infected cells were lysed in Hirt Buffer (10 mM Tris-HCl-IO mM EDTA-0.2%
SDS, [pH 76]). Proteins were precipitated with 1/10 volume of 5M NaCl at 4°C
overnight and collected by centrifugation at 14000 rpm for 30 min at 4°C. Proteins were
boiled in 1X protein sample buffer (5% SDS; 0.03% bromophenol blue; 20% glycerol;
5% fl-mercaptoethanol; 0.5 M Tris-HCI, [pH 6.8]) for 5 min. Alternatively, cells scraped
directly from plates were boiled in 1X protein sample buffer for 5 min. Proteins were
electrophoresed in 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride
membranes (Amersham). A polyclonal rat anti-tumor serum harvested as ascites fluid
was used as the primary antibody to detect three early proteins: large, middle, and small
T. It also reacts with a few cellular proteins which can serve as an internal loading control.
Goat anti-rat horseradish peroxidase (HRP; Pierce) was used as the secondary antibody.
A rabbit anti-MT antiserum (a gift from B. Schaffhausen) was also used as the primary

antibody and goat anti-rabbit HRP (Sigma) as the secondary antibody. A rabbit anti-

54

capsid antibody, (a gift from R. Garcea) was used to detect the capsid protein VPl, with
goat anti-rabbit HRP (Sigma) as the secondary antibody. Signals were visualized by
SuperSignal Enhanced Cherniluminescent Detection System (Pierce).

To detect c-Src, a mouse anti-v-Src monoclonal antibody (Oncogene) was used as
the primary antibody and goat anti-mouse horseradish peroxidase (American Qualex) as
the secondary antibody. To detect active c-Src, a rabbit anti-c-Src pY418 polyclonal
antibody (Biosource International) was used as the primary antibody and goat anti-rabbit

HRP (Sigma) as the secondary antibody.

Immunoprecipitation and Middle-T-associated kinase assay

The procedure of Schaffhausen and Benjamin was used [25]. Infected NIH 3T3
cells (2X 10° cells) were lysed with lysis buffer (50 mM Tris-HCI, pH 7.5; 150 mM NaCl;
1 mM EDTA, pH 8.0; 100 mM NaF; 10% glycerol; 1 mM MgC12; 1% NP-40; 1 mM
phenylmethylsulfonyl fluoride, 10 pg of leupectin per ml; 5 ug of aproteinin per ml; 1
mM sodium orthovanadate) and incubated on ice for 30 min. Lysates were centrifuged at
12,000 rpm for 10 min at 4°C. The pellets were removed. The antibodies were added to
the supernatant and then incubated at 4°C for 1 hr with rocking. Either the rat polyclonal
anti-tumor serum or a mouse monoclonal antibody PAb762 (a gift of S. Dilworth) was
used for immunoprecipitation of MT. 50 ul of Protein G-agarose beads (Santa Cruz
Biotech) was then added to the supernatant and incubated for 1 hr at 4°C with rocking.
The beads were washed twice in 0.5 M LiCl-0.l M Tris-HCI (pH 6.8) and finally once in
0.2 M Tris-HCI (pH 7.5)-5 mM MgC13. The immune complexes were suspended in 50 pl
of kinase buffer (20 mM Tris-HCI, pH 7.5; 5 mM MgC12; 20 uCi of[y—°2P] ATP) at 30°C

for 30 min. When specified, a Src family kinase selective inhibitor SU6656 (CalBiochem)

55

was used at 5 pM final concentration. 1 pg of acid-treated rabbit muscle enolase was
used per reaction as an indicator of Src kinase activity. To prepare acid-treated enolase, 2
mg of rabbit muscle enolase (Sigma) was mixed with 100 pl of 50 mM Hepes-NaOH
buffer, pH 7.0, 1 mM dithiothreitol, 1 mM MgC12 at 4°C for 10-30 min[8]. Equal volume
of 50 mM Acetic acid, pH 3.2 was added at 30°C for 5 min. Finally, 50 pl of glycerol
was added. Acid-treated enolase was aliquotted (~10 pg/ pl) and stored at -70°C.
Depending on a specific experiment, the kinase products were processed
differently following three protocols. (1) The beads were pelleted and washed twice in
0.5 M LiCl-0.l M Tris-HCI (pH 6.8) and finally once in 1X PBS, 20 mM NaF. The
proteins were dissociated from the beads by boiling for 10 min in 1X protein sample
buffer (5% SDS; 0.03% bromophenol blue; 20% glycerol; 5% fi-mercaptoethanol; 0.5 M
Tris-HCI, [pH 68]). The supernatant was loaded on a 10% SDS-polyacrylamide gel. (2)
When exogenous enolase and Src family inhibitor SU6656 were added to the kinase
reaction, 10 pl of 6X protein sample buffer was added to the reaction and boiled for 10
min. Beads were spun down and supernatant was loaded on a gel. (3) If a second
immunoprecipitation was required, beads were boiled for 2 min in 400 pl of elution
buffer (0.4% SDS, 50 mM Tris-HCl, [pH 7.5], 100 mM NaCl, 2 mM EDTA, [pH 8.0], 2
mM 2-6 mercaptoethanol). Iodoacetamide was added to a final concentration of 10 mM.
Samples were cooled down, beads were removed by spinning. 1/4 volume of 10% Triton
X-100 was added to the supernatant. The second immunoprecipitation was carried out
following the same procedure as the first immunoprecipitation. The beads were collected

and washed twice in 0.5 M LiCl-0.l M Tris-HCI (pH 6.8) and finally once in 1X PBS, 20

56

mM NaF. The proteins were dissociated from the beads by boiling in 1x protein sample
buffer. The supematant was loaded on a gel.

For KOH treatment, the 10% SDS-PAGE gel was rinsed briefly with 1X PBS and
then treated with 1N KOH at 55°C for 1 hr. After briefly rinsing again, the gel was
soaked in 25% Methanol + 5% Acetic acid for 30-45min. Finally, the gel was rinsed and
dried on a vacuum gel drier at 75°C for 1.5 hr and exposed to phosphorescent screens and
scanned with the Phosphorimager (Molecular Dynamics). The gel was then exposed to
X-ray films (Amersham Phamiacia Biotech) for up to 3 weeks at -70°C with an

intensifying screen to achieve darker exposure.

Pulse-Chase Assay

Serum-starved NIH3T3 cells (8x105 cells / 100 mm plate) were infected for 2 hrs
with virus at a multiplicity of infection (MOI) of 10 plaque forming units (PFU) per cell
and grown in 10% serum medium for 40 hrs at 37°C. Cells were washed with 1X PBS
twice, and starved with 1 m1 of wami methionine/cysteine free DMEM supplemented
with 10% dialyzed calf serum for 1 hr. Then 150 pCi of 35S-methionine (Perkin-Elmer)
was added for 2 hrs. After washing cells twice in warm 1X PBS, 10 ml of 10% serum
medium with 2 mM methionine was added. Cells were collected at O and 4 hrs after chase
and proteins immunoprecipitated with the polyclonal rat anti-tumor serum and Protein G-
agarose beads. Immunoprecipitates were boiled in 1X protein sample buffer and resolved

by 10% SDS-PAGE.

57

RESULTS

Ability of muta|_1t MT to interact with agd activate c-Src

MT C -terminal tyrosine mutants were generated in the PINCO retroviral
expression vector [13] as described in Materials and Methods. NIH3T3 cells were
infected with different PINCO-MT retroviruses. The expression of MT proteins was
examined by Western blotting (Figure 2-2). The levels of MT accumulation at 48 hrs
postinfection were high and apparently equal among all the viruses. Similar results were
obtained with polyomaviruses carrying the same mutations. The stability of the sextuple
mutant Y6F MT was further examined in a pulse-chase experiment. NIH3T3 cells were
infected with polyomavirus wild type WTA2, Y6F and a deletion mutant d145, used to
ascertain MT band identity. As shown in Figure 2-3A, similar levels of MT and other
viral early proteins were expressed by WTA2, Y6F at the time when the pulse phase
started; similar levels were also labeled during the 2 hr pulse period (Figure 2-3B).
Furthermore, the level of 35S-labeled wild type and Y6F MT proteins decreased to the
same extent over the 4 hr chase. This suggests that multiple Tyr-to-Phe mutations did not

change MT stability significantly.

The ability of mutant MT to bind to and activate c-Src was examined. MT was
immunoprecipitated from the lysatcs of NIH3T3 cells infected with retroviruses
expressing wild type (WT), the triple mutant in the three major tyrosines (Y3FM), the
triple mutant in the minor tyrosines (Y3Fm, and the sextuple mutant MT (Y6F). The
levels of co-immunoprecipitated c-Src were analyzed by Western blotting (Figure 2-4A).

c-Src protein was detected in all samples except that of the control vector, suggesting that

58

the mutant MTs are still able to bind to c-Src. The levels of c-Src associated with the
three mutant MTs appeared proportional to the levels of MT in the precipitates (Figure 2-
4C). The MT blot shows two closely spaced bands. The upper band is likely to represent
a 58k MT-species with higher Ser/Thr phosphorylation which has been previously shown
to be enriched in the MT-c-Src complexes [23]. Association with MT activates c-Src,
resulting in auto-phosphorylation of Tyr 418 (Tyr 416 in chicken), which can be
demonstrated by using a phosphopeptide-specific antibody [6, 7, 10]. The three mutant
MTs were found able to activate c-Src, as detected by the anti-Src pY418 antibody
(Figure 2-4B). Thus, the MT, c-Src and active c-Src levels appear proportional to each
other, suggesting that mutant MT has no overt defect either in c-Src-binding or in
inducing the autophosphorylation of c-Src. However, a small reduction in Src-binding

cannot be eliminated by the present experiments.

The activation of c-Src was further examined in an in vitro kinase assay. The
immunocomplex associated with MT contains several cellular proteins with similar
molecular weight, including MT (56 kDa), c-Src (60 kDa), PP2A regulatory A subunit
(60 kDa), which are at stoichiometric levels, ShcA (46, 52 and 66 kDa three isoforms) at
low abundance, as well as IgG heavy chain (55 kDa) and enolase (47 kDa) which are
added to the kinase reaction. All of these proteins except for PP2A A subunit are
phosphorylated by c-Src, thus resulting in a relatively high background. An unknown 50
kDa cellular protein can also be immunoprecipitated by rat anti-tumor serum and
phosphorylated. Therefore kinase assay protocols were modified to suit the specific
objective of each experiment as described in Materials and Methods. In some early

experiments, two rat anti-tumor sera (rat 11 and rat 18) were used for

59

immunoprecipitation (Figure 2—7A and Figure 2-7B). High background was observed due
to the phosphorylation of IgG heavy chain. The high level of IgG also caused distortion
of protein bands around the 55 kDa region on the gel. The presence of the phosphorylated
50 kDa cellular protein pulled down by rat antiserum also interfered with the quantitation
of enolase phosphorylation. A second immunoprecipitation after the kinase reaction using
the same antiserum was performed to clean up the phosphorylated IgG heavy chain and
the cellular protein, therefore reducing background around MT. However, double IP also
decreases the intensity of the phosphorylation signal due to the loss of phosphoproteins
during the second IP. Furthermore, the double IP protocol is not applicable when
quantitation of enolase phosphorylation is required, because the second IP requires
removing all supernatant after the kinase reaction, which contains phosphorylated enolase.
A mouse monoclonal antibody against MT, PAb762 was found to yield better results than
the rat antiserum, since the amount of total IgG in the immunoprecipitates can be greatly
reduced. This antibody also does not recognize the 50 kDa cellular protein, therefore does
not interfere with enolase quantitation. This antibody was used in later experiments

(Figure 2-5A and Figure 2-6A, B, C).

The results of a typical experiment are shown in Figure 2-5A. Cells were infected
with retroviruses expressing various mutant MTs, resulting in equal levels of MT
expression, as determined by Western blotting (Figure 2-5B). MT-c-Src complexes were
immunoprecipitated with an anti-T antigen antibody and subjected to an in vitro kinase
assay as described in Materials and Methods (Protocol 2) The phosphorylation of enolase,
an exogenous substrate for c-Src [8, 17, 20], was measured as an indicator of c-Src kinase

activity (Figure 2-5A). As shown, uninfected cells and control vector infected cells

60

showed no activity. In contrast, wild type and mutant extracts all demonstrated kinase
activity, but of differing levels. The level of enolase phosphorylation in the
immunoprecipitates containing Y3Fm, Y3FM and Y6F MT decreased to 91%, 50% and
37% of wild type level, respectively. Similar results were found in other experiments; for
example, in the experiment shown in Figure 2-6A these levels were 99%, 40% and 23%.
This suggests that the ability of MT to activate c-Src is reduced by the tyrosine-to-
phenylalanine mutations. The variability from experiment to experiment is presumably
due, at least in part, to the relatively high background in this gel region, as discussed
above. In experiments in which a second immunoprecipitation was used to further purify
the kinase reaction products, higher ratios were obtained between the wild type and Y3FM
and similarly with Y6F mutants (Figure 2-7A, B). When each single major tyrosine alone
(i.e. in a quintuple mutant background) was evaluated for its ability to activate Src kinase
activity, the pattern showed small differences with Y250> Y315>Y322, as judged by the

levels of enolase phosphorylation (Figure 2-6B, C).

To confinn that the MT-associated activity was indeed mediated by a tyrosine
kinase, the gel was treated with 1N KOH at 55°C for 1 hr [9]. Alkaline treatment
selectively removes serine/threonine-bound phosphate, due to the relative stability of the
phosphodiester bond of phosphotyrosine under alkaline conditions [22]. No obvious
changes in the phosphorylation pattern was found between gels with and without KOH
treatment (Data not shown), indicating that the most of the proteins were phosphorylated
at tyrosine residues. However this is not a very stringent method [9]. To further examine
the specificity ofthe kinase reaction, I made use ofa selective inhibitor for four ofthe Src

family kinases. Src, Fyn, Yes and Lyn [2, 3]. In the presence of SU6656, the

61

phosphorylation of most protein species, including enolase, was greatly reduced (Figure
2-5A). This result implies that the enolase and MT phosphorylation, as well as that of
other known and unknown visible proteins, is mediated by Src family kinases. No

activity was detected in the control vector.

MT as substrate for Src phosphorylatiog

The capacity of each mutant MT to serve as substrate for Src in vitro
phosphorylation was evaluated (Figures 2-5A, 2-6A). To make a quantitative assessment,
the mutant specific-Src activity was taken into account by nomializing MT against
enolase phosphorylation. Compared to wild type MT, the ratio of MT/enolase
phosphorylation for Y3Fm, Y3FM and Y6F MT decreased to 59-89%, 25-35% and 19-
18%, respectively, in experiments shown in Figures 2-5A and Figure 2-6A. Similar

results were also obtained in transfections with a pMEX-plasmid vector.

The decrease in Y6F middle T phosphorylation suggests that the majority of MT
in vitro phosphorylation sites are at its C-terminal half. These ratios also show that Y250,
Y315 and Y322 are major phosphorylation sites. Y258, Y288 and Y297 are minor
phosphorylation sites on their own, but have a substantially higher in vitro substrate
capacity in the absence of phosphorylation at the major sites. The contribution of each
major tyrosine 250, 315 and 322 in MT phosphorylation was tested using quintuple
mutant MTs (Y5F-250+, Y5F-315+ and Y5F-322+) (Figure 2-6B). The phosphorylation of
these three tyrosines was relatively equal, suggesting that these three tyrosines are equal
acceptors for in vitro phosphorylation by Src. The quadruple mutants MT (Y4F-
250+/315+, Y4F-250+/322+ and Y4F-315+/322+) (Figure 2-6C) showed increased MT

phosphorylation close to that of Y3Fm MT.

62

The study of MT-dependent kinase activity presented so far used a retroviral
expression vector to drive MT expression. This activity was compared to that derived
during infections with polyomavirus, carrying the same mutations (Figure 2-7A, B). This
strategy has the advantage of leading to higher levels of MT expression and hence a
higher signal-to-noise ratio in the kinase reaction. In this case, the phosphorylation
products were also further purified by a second immunoprecipitation. Polyomaviruses
with single or multiple major tyrosine mutations were examined (Figure 2-7A). This
experiment was carried out using rat polyclonal antiserum (rat 1 1) following the protocol
(1) described in the legend, in which large T antigen is co-precipitated and
phosphorylated [12, 25]. The MT fragment produced by 1387T was also phosphorylated.
The intensity of the LT band serves as an indicator of the level of MT in the
immunoprecipitate. LT levels are proportional to the levels of active Src. The levels of
co-immunoprecipitated active Src were in the same range, except in the case of Y6F and
to a lesser extent that of Y3FM, due to slightly lower MT expression. As expected, no Src
was pulled down in the case of MT-ter and 1387T, mutants with a terminatiOn codon
after amino acid 196 and 384, respectively, nor in 3 WT infection extract
immunoprecipitated with a normal control antibody. Loss of each of the major tyrosines,
Y250, Y315 and Y322 resulted in a ~2-fold loss in MT phosphorylation in each case,
while loss of two of these tyrosines caused a 4-6-fold drop; the bigger drop for the
Y250/322E double mutant is likely to reflect at least in part the lower level of active Src
in this extract. Loss of two minor tyrosines (Y288 + Y297) in the d145 deletion mutant
resulted in a 2.5-fold loss in MT phosphorylation. As shown in Figure 2-7A, despite the

co-expression of the other early polyoma proteins, large T and small T antigens, the

63

pattern obtained is qualitatively very similar, except for the presence of phosphorylated
LT. The low expression of Y6F MT was corrected in another experiment (Figure 2-7B).
In this case, a 25-fold difference in MT phosphorylation was observed between wild type
and Y6F MT, even though higher MT expression was obtained in the Y6F infection (due

to slightly off dosage).

Based upon Western blotting results, the band above MT in these
electrophoregrams of kinase products (e. g. Figure 5A) corresponds to the c-Src position. I
assume that its low level of in vitro phosphorylation is reflective of its in vivo
phosphorylated status, given that only MT-complexed c-Src and hence only active,

phosphorylated c-Src is pulled down by anti-MT antibodies.

R_equiremerlt of MT tyrosines for PI3K binding

The role of the major tyrosines in the recruitment p85/PI3K to the MT-c-Src
complex was examined indirectly, by way of p85 phosphorylation in the kinase assay.
For this purpose, tyrosines 250, 315 and 322 were tested individually in quintuple
mutants and in pairs in quadruple mutants to determine if single tyrosines are which is
sufficient for p85 binding. The results of in vitro kinase assays are shown in Figures 2-5A
and Figure 2-6 (panel A, B and C), and Figure 2-8A, in which the p85 position is
indicated by an arrow. The size of the phosphorylated ~85 kDa band was determined
with molecular weight markers, and matches the migration of the p85 subunit of PI3K
determined using anti-p85 antibody in a Western blot run in parallel. As previously
demonstrated, Y315F mutation resulted in the loss of most of the p85 signal, a smaller
reduction was observed with Y250F mutation and Y322F mutation had no effect. The

triple mutation of the minor tyrosines also had no effect. The concomitant mutation of

64

Y315 and Y250 totally abolished the p85 signal, as shown in the immunoprecipitates
from cells expressing Y5F-322+, Y3FM and Y6F middle T (Figure 2-6A, B). In a
corollary analysis, quintuply mutated MT that contained only Y315 showed the capacity
to bind p85, as seen with Y5F-315+; this Y315 capacity is also seen with Y4F-315+/322+
(Figure 2-6C). Binding was also detected, albeit to a lower level, in the case of Y5F
containing only Y250, as well as with Y4F-250+/322+ (Figure 6B and 6C long term
exposure). With both Y250 and Y315 intact, mutant MTs were able to bind p85 as
efficiently as wild type MT, as seen with Y4F-250+/315+, Y3Fm, d145, and Y322F. The
presence of Tyr 322 or that of the minor tyrosines did not facilitate p85 binding. The
difference in the levels of MT-bound phosphorylated p85 may be the result of changes in
both p85 binding to MT or in Src kinase activity. However, with similar or even lower
kinase activity judged by enolase phosphorylation, the levels of phosphorylated p85 in
samples expressing Y5F-315+ and Y4F-315+/322+ MT were always higher than those
expressing Y5F-250+ and Y4F-250+/322+ MT, suggesting that these differences are not
likely due to lower Src kinase activity. The comparison of the levels of bound

phosphorylated p85 is summarized in Table 2-2.

DISCUSSION

In this study, the effect of tyrosine mutations on the capacity of MT to bind to and
activate Src tyrosine kinase and the capacity of these tyrosines to serve as phosphate
acceptor for Src in vitro phosphorylation were examined. Three tyrosines, Y258, Y288
and Y298, termed minor, that had not been tested previously in details were tested, and

the well studied tyrosines Y250, Y315 and Y322, termed major, were reexamined in this

65

context. Two new conclusions can be drawn from this work. Tyrosine mutations were
shown to impact Src enzymatic activity, while having no detectable effect on Src binding.
Furthermore, the minor tyrosines were found to play a substantial role in modulating Src

activity, which could be revealed only in the absence of the major tyrosines.

MT six carboxyl-tenninal tyrosines were studied in parallel by tyrosine to
phenylalanine mutations, all generated in the A2 genetic background. The minor
tyrosines were studied as a block, either in the presence of functional major tyrosines by
comparing WT to Y3Fm, or in their absence, in comparisons of Y3FM and Y6F. In
contrast, the major tyrosines were studied one at a time, either as single mutations in the
context of functional other tyrosines (YlFs), or as single functional tyrosines in the
context of quintuple mutations (Y5Fs). This study represents the most comprehensive
direct comparison to date. All mutants were found to be stable, and normal in their
capacity to bind to c-Src and to induce its autophosphorylation [6]. This point was
established in comparisons of MT immunoprecipitates of wild type, Y3Fm-, Y3FM-, and
Y6F-MT expressing extracts, analyzed by western blotting with antibodies against MT
and total c-Src. In each case, the Src levels were proportional to MT levels. Furthermore,
the levels of activated Src probed with an antibody directed against Src- phosphopeptide
spanning Tyr 418 were proportional to the level of total Src. These results are not
surprising, since previous studies of double mutants Y250/315F, Y315/322F have not
uncovered defects in Src-binding and since binding to MT activates Src, resulting in
autophosphorylation. However, when Src enzymatic activity was tested on a heterologous
substrate, enolase, in an in vitro kinase assay, the mutations of the three major tyrosines

led to a reproducible 2-2.5-fold decrease in enolase phosphorylation. In contrast, triple

66

mutation of the minor tyrosines did not lead to a decrease in Src activity, in the context of
three intact major tyrosines; however, a 1.5-2-fold decrease was observed when the minor
tyrosines were mutated in the context of absence of the major tyrosines. Thus Src
complexed to the sextuple Y6F MT shows an overall 3-4-fold decrease in activity
compared to wild type. The restoration of single major tyrosines into the Y6F background
led to an increase in activity, further increased by restoring two major tyrosines. The
contrast between the normal capacity for autophosphorylation of Src complexed with
multiply mutated MT and the decreased capacity for heterologous phosphorylation is
intriguing. It may reflect differences in the assay: the autophosphorylation test measures a
steady state following an undetermined, but likely long in vivo interaction time, while
enolase phosphorylation was detected in a 30-minute reaction. Since the c-Src binding
site has been mapped to MT to amino acid 185 to 210 [4], which is not altered by the
tyrosine mutations, the exact mechanism for this regulatory effect of MT on c-Src activity
is unknown at present. I note that an effect of tyrosine mutation on Src activity on enolase
was suggested in early studies [19]. These experiments reported large differences based
on studies of Y250 and Y315 mutations. It is possible that these different results were due
to using an MT transformed cell line in their study instead of using polyomavirus and
retrovirus infection.

The enolase phosphorylation test revealed a function of the minor tyrosines in this
process that was not detectable in the presence of nomial major tyrosines. A similar
situation has been documented in the case of phosphorylated Y315, whose major,
detectable role is to bind p85 subunit of PI3K, which provides a consensus binding motif

for p85 via one ofits SH2 domain [15]. However, when the p85 binding motif is altered,

67

phosphorylated Y315 appears to bind an unknown partner, which participates in a
signaling that contributes toward transfomiation. Similarly, Y322 may help in p85
binding, while it participates in PLCy—l binding in the absence of p85 binding [30]. As
will be discussed below, the minor tyrosines are a weak Src substrate. Thus it is
conceivable that their contribution toward Src-activity takes place in the absence of
phosphorylation. However, it is equally possible that these minor tyrosines are only
phosphorylated in the absence of major tyrosine phosphorylation, and that MT
phosphorylation is at the root of enhancing Src activity, in addition to creating
phosphotyrosine- and SH2-binding sites. The residual function of Y3FM MT may be
mediated by the binding of one or more of the minor tyrosines to signaling intermediates,
or by Src trans-kinase activity.

The capacity of MT tyrosine mutants to serve as Src substrates was also
detemiined. To normalize for reduced Src activity, the ratio of MT/enolase
phosphorylation was calculated for each mutant tested. This strategy also corrects for
unavoidable occasional differences in MT levels. The loss of phosphorylation caused by
each major Y—> F mutation in the wild type context was examined, as well as the
contribution of single major tyrosine in a quintuple mutant context. The results confirm
previous findings, that the major tyrosines are primary substrate sites while the minor
tyrosines are weak substrate sites. Nevertheless, the phosphorylation of one or more
among the minor tyrosines is clearly detectable. Evidence for Y297 phosphorylation has
previously been presented [16]. Our results with dl45 deletion mutant, which
encompasses Y288 and Y297, are in agreement. In studies of single major tyrosine

mutations I found that the three major tyrosines are equally good substrates for the in

68

vitro kinase. This was a consistent finding. It was demonstrated both with a MT
retroviral vector and in polyoma viral infections when analyzing single mutants, as well
as in the context of quintuple mutants with a single functional tyrosine (retroviral vector
only). Additionally the behavior of double mutants also supports this observation. The
results suggest a simple augmenting pattern. A number of previous reports have
suggested Y315 as the primary phosphorylation site in vitro as well as in vivo [23] and
Y250 as another primary site in vitro [16], whereas Y322 as a secondary site in vitro [26].
Several factors might be accounted for the observed differences, such as precise
conditions used for cell growth and infection, antibodies used for immunoprecipitation,
61C.

The in vitro kinase electrophoregrams easily demonstrate the presence of the p85
subunit of the PI3K; the band intensity reflects the combination of levels of bound protein
and phosphorylation efficiency. The results confirm previous findings that pY315
provides a major binding site [29] and pY250 a minor site. Recent studies showed that
tyrosine 250 can recruit p85 directly [15] or indirectly through Grb2-Gab1 interaction
[21]. Experiments with the quintuple mutants demonstrate that Y315 alone and to a lesser
extent Y250 are able to bind p85, and that a quadruple mutant containing both Y250 and
Y315 binds as efficiently as the wild type.

The other MT-Src partners were not reliably detected in these electrophoregrams.
A faint band corresponding to Src size was seen in wild type and Y3Fm extracts. As
mentioned in the results section it is expected that most of the Src protein found in
association with MT is activated and thus phosphorylated in vivo, as suggested by the

results of blots with the antibody directed against the Sre activation site. A band

69

corresponding to the position of PLC—yl was observed in very active extracts of cells
infected with wild type and dl45 polyomavirus; it was absent in Y6F. Its intensity was
considerably lower than that displayed by p85. This may reflect competition between

PI3K and PLC-‘yl for binding in the Y315/Y322 region.

SUMMARY

In summary, the present experiments have revealed that the mutations of MT
tyrosines affect the activity of the MT-associated Src kinase, and that the minor tyrosine
Y258, Y288 and Y297 play a role in this MT function. The effect oftyrosine mutations
on Src-enzymatic activity affects MT phosphorylation and thus is likely to impact all
aspects of MT signaling, via decreases in binding of the MT-associated signaling
intermediates. A strong biological support for the role of the minor tyrosines was
obtained in the infections of mice, in which the replication and survival of polyomavirus
strains carrying the Y6F mutation were shown to be poorer than that of a strain the Y3FM

mutation (See Chapter 4).

70

APPENDIX 2: FIGURES AND TABLES FOR CHAPTER 2

71

Figure 2-1 A schematic representation of middle T

Tyrosines on middle T are presented above the line. The position of DnaJ domain,
intron and membrane insertion domain and the binding sites for MT-associated proteins
including PP2A, c-Src, ShcA, 14-3-3, PI3K and PLC-yl are marked below the line. The
signaling pathways activated by these proteins are shown. The common region shared

between MT and ST is shown at the bottom.

9:55:52

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73

Table 2-1 Tyrosine mutant middle T

This table lists the tyrosine mutant MTs used in this study. Six C-terminal
tyrosines were mutated to phenylalanine in various combinations to generate single,
double, triple, quadruple, quintuple and sextuple mutant MT. Tyrosines 250, 315 and 322
are referred to as the major tyrosines and tyrosines 258, 288 and 297 as the minor
tyrosines. The capability of each mutant MT to bind ShcA, PI3K and PLC-yl are

indicated.

74

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Available C- Binding sites for cellular
Mutations Viruses . . proteins
terminal tyrosrnes ShcA PI3K PLC-y1
. 250, 258, 288,
Wild Type WT 297’ 315’ 322 + + +
258, 288, 297,
YZSOF 315,322 ' + +
Single 250, 258, 288,
Mutants Y315F 297,322 + ' +
250, 258, 288,
Y322F 297’ 315 + + -
Y250/315F 258, 288, 297, 322 - - +
Double
Mutants Y250/322F 258, 288, 297, 315 - + -
Y315/322F 250, 258, 288, 297 + - -
Triple Y3FM 258, 288, 297 - - -
Mutants Y3Fm 250, 315, 322 + + +
Y4F-250‘7315+ 250, 315 + + -
Quadruple + +
Mutants Y4F-250 /322 250, 322 + - +
Y4F-315‘l325” 315, 322 - + +
Y5F-250+ 250 + + -
Quintuple +
Mutants Y5F'315 315 - + -
Y5F-322+ 322 — - +
Sextuple
I Mutant Y6F ' ' ' '

 

75

 

 

 

.-
‘r‘l'
.‘l‘ l!

.i~‘
V

1.. t ‘
MET.

on l‘
H)
it“

Figure 2-2 Analysis of tyrosine mutant middle T expression
Cells were infected with retroviruses expression different tyrosine mutant MTs as
described in Materials and Methods. Proteins were extracted at 72 hours postinfection.

The Western blot was probed with a polyclonal rat anti-tumor serum.

76

JESS/+9 L8‘:|17A

+ZZ€/+OQZ':|VA
+9 L€/+OSZ‘:I 17A
+ZZ€':I§A
+9 LS'dSA
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77

MT———+

Figure 2-3 Pulse-chase analysis ofthe stability of Y6F middle T

Cells were infected with polyomavirus WTA2, Y6F and dl45. The dl45 virus has
a deletion in MT from amino acid 281 to 302, which was included as a reference to
identify MT protein. Cells were infected as described in Materials and Methods. (A) A
Western blot of early proteins at 40 hrs postinfection. (B) In the meantime, cells on
duplicated plates were pulse-labeled for 2 hrs and chased for 4 hrs. Proteins were
collected immediately before and after the chase phase. The three viral early proteins
were immunoprecipitated with the polyclonal rat anti—tumor serum, and subjected to

reducing SDS-PAGE.

78

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:3

:5

9:: 3ch

.m

l l is:

'I‘l:

u_©> N<._.>>

79

Figure 2-4 Analysis of c-Src activation in cells expressing mutant MTs

Cells were infected with retroviruses expressing different tyrosine mutant MT.
Proteins were extracted at 40 hrs postinfection and immunoprecipitated with a mouse
monoclonal anti-T antibody (PAb762). The Western blot was probed sequentially with
(A) a mouse anti-v-Src antibody; (B) a rabbit anti-c-Src pY418 antibody; and (C) a

polyclonal rabbit anti-T serum.

80

:2 ll.

 

, l. a. a a a.
33305.01 All... rt-
. I .. .

81

Figure 2-5 MT-associated in vitro kinase assay and the effect of Src inhibitor SU6656

Cells were infected with retroviruses expressing different mutant MTs as
described in Materials and Methods. Proteins were extracted at 40 hrs postinfection. (A)
In vitro kinase assay. A total of 2 x 106 cells were immunoprecipitated with the
monoclonal antibody (PAb762), and a kinase assay was perfomied as described in
Materials and Methods (Protocol 2). An uninfected cell control and a vector control were
also immunoprecipitated at the same time. A Src family selective inhibitor SU6656 was
added to the duplicated samples. An exogenous Src substrate, enolase was added as an
indicator of Src kinase activity. The bands representing phosphorylated middle T, enolase
and the p85 subunit of PI3K are indicated. The intensity of phosphorylated enolase and
MT were counted. The counts of enolase as well as the ratio of counts between MT and
enolase were calculated and compared to that of wild type MT. (B) A Western blot of
proteins from cell lysates (l/20th of total cells). This blot was probed with the rat

polyclonal serum. MT is identified with an arrow.

82

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I. ._.I. mwa
- + - + - + - + - + omomDm
am; f? E? is ES

83

Figure 2-6 Analysis of Src activity and MT phosphorylation by MT-associated in
vitro kinase assay

Cells were infected and proteins extracted as described in Materials and Methods.
In vitro kinase assay was perfomied as described in the legend to Figure 2-5. The bands
representing phosphorylated middle T, enolase and the p85 subunit of PI3K are indicated.
The relative ratio of enolase and MT phosphorylation of mutant/WT middle T are
calculated. (A) Vector, WT+ Src inhibitor SU6656, WT, Y3FM, Y3Fm and Y6F; (B)
Tyrosine quintuple mutants Y5F-250+, Y5F-315+ and Y5F-322+; (C) Tyrosine quadruple
mutants Y4F-250+/315+, Y4F-250+/322+; Y4F—315+/322+. A two-week exposure of p85

in panel (B) and (C) is shown underneath

84

 

 

 

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mm em 8 E 3 mm mm mm 3 OS 3.3.0.205
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a a a Q m... A». s m
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.o .<

85

Figure 2-7 MT phosphorylation in cells infected with polyomaviruses by in vitro
kinase assay

Cells were infected as described in Materials and Methods. Proteins were
extracted at 40 hrs postinfection. In vitro kinase assay. A total of 2 x 10" cells were
immunoprecipitated with the polyclonal antiserum, and an in vitro kinase assay was
perfomted, followed by a second immunopreeipitation as described in Materials and
Methods (Protocol 3). The bands representing phosphorylated large T, middle T, p85 and
cellular protein are indicated. The reduction of MT phosphorylation of mutants compared
to wild type A2 is presented. (A) Cells were infected with different mutant
polyomaviruses. Rat polyclonal antiserum (ratl l) was used for immunoprecipitation. An
uninfected cell control precipitated with immune serum (Mock) and A2 infected cell
precipitated with pre-immune serum were included. A Western blot of extracts of the
same infected cells was probed with anti-c-Src p418 antibody is shown at the bottom.
Given its size. similar to dl45 MT, the band in 1387T infected sample is assumed to be
MT 1387T fragment. This experiment was performed by Dr. Li Chen. (B) Cells were
infected with WTA2, Y6F and dl45. Rat polyclonal antiserum (rat18) was used for
immunoprecipitation. An uninfected cell control was precipitated with immune serum.
The bands representing phosphorylated large T, middle T and p85 are indicated. A
Western blot of proteins from cell lysates (l/20th of total cells) was probed with the rat

polyclonal serum. MT is identified with an arrow.

86

A. “c’

D

E

c E

E a“:

V L

.25 CL

0 l—

2 E
-

Fold Reduction

LT

p85

MT

Cellular

Fold Reduction

MT

 

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an

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1 20

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ill
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167

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(a)
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Infection
Y6F dl45 WTA2
Halal...
mm ‘ ' mm

    

..,olniiu........

 

87

Figure 2-8 Requirement of MT tyrosines for p85 binding

Cells were infected with different polyomavirus mutants as described in Materials
and Methods. Proteins were extracted at 40 hrs postinfection. (A) In vitro kinase assay
was perfomied as described in the legend to Figure 2-7. Only the bands representing
phosphorylated p85 are presented. (B) A Western blot of proteins from cell lysates
(l/20th of total cells). This blot was probed with the rat polyclonal serum. MT is

identified with an arrow.

88

peloeluwn

SVIP

:IQA

stx

LU

:IEA

:lZZE/S LEA
:lZZE/OQZA

:lSLS/OQZA

:lZZ8A

:lQLSA
3093A

iN\

_ p85

89

MT

Cm‘

Table 2-2 Summary of the levels of phosphorylated p85 in the in vitro kinase assays

The levels of phosphorylated p85 were compared among different mutant MTs.
Given the variation in the levels of MT expression, the relative levels of p85 were
adjusted accordingly. Results from retrovirus infection (Figure 2—6A, B and C) and

polyomavirus infection (Figure 2-8A) are summarized.

90

 

 

 

 

 

 

 

 

 

 

 

 

Retrovirus p85 Level Polyomavirus
WT ++++ WT
Y3Fm ++++ Y3Fm
Y4F-250+/31 5+ ++++ Y322F
Y4F-315"/322+ ++ Y250F
Y5F-31 5+ ++ Y250/322F
Y4F-250"/322+ -/+ Y315F
Y5F-250+ -/+ Y315/322F
Y5F-322" - Y250/315F
Y3FM - Y3FM
Y6F - Y6F

 

 

 

91

 

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10.

Bendig, M.M., T. Thomas, and W.R. Folk, Viable deletion mutant in the medium
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Blake, RA, et al., 5 U665 6. a selective srcfamily kinase inhibitor, used to probe
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Bowman, T., et al., Stat3-mediated Myc expression is required for Src
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Campbell, K.S., et al., Polyoma middle tumor antigen interacts with SHC protein
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Cooper, .1 .A. and T. Hunter, Changes in protein phosphorylation in Rous sarcoma
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Hong, Y.K., et al., A new class of mutations reveals a novel function for the
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94

CHAPTER 3

THE ROLE OF MT C-TERMINAL TYROSINES IN VIRAL DNA
REPLICATION AND GENE EXPRESSION IN CELL CULTURE

ABSTRACT

I have been studying the effect of middle T on viral DNA replication in NIH3T3
cells. Here I describe the role of MT C-temiinal tyrosines on viral genome accumulation
and gene expression in cell culture. Infection of NIH3T3 cells with polyomaviruses
expressing different tyrosine mutant MTs demonstrates that a tyrosine sextuple mutant
(Y6F) has a severe defect in both viral genome accumulation and gene expression. The
major tyrosine triple mutant (Y3FM) has a moderate defect, whereas the minor tyrosine
mutant (Y3Fm) shows only a minor defect. The capability of different mutant MT to
complement the MT'/ST' defective virus A185 in genome accumulation and gene
expression is also consistent with viral infection data. TheMT complementation study
also shows that the ef feet of the major tyrosines is augmenting. Furthermore, the ability
of MTs to promote viral DNA replication is reduced but not abolished in the presence of
abundant LT. Finally, I show that all mutant MTs can activate polyoma enhancer-origin

dependent gene transcription to different degrees.
INTRODUCTION

Polyomavirus MT plays a major role in viral lytic cycle: it strongly upregulates
the expression of both early and late genes [7, 10, 18, 31], as well as DNA replication [5,

6]. Most of these functions are thought to be mediated by transcription factors, i.e.,

95

PEAl/AP-l and PEA3/e-Ets [16, 25, 31] through the activation ofthe viral enhancer. By
increasing LT expression, which is ultimately responsible for viral DNA replication, MT
indirectly promotes viral DNA replication. PEAI /AP-1 and PEA3/c-Ets have been shown
to bind to the polyoma enhancer and directly stimulate DNA replication [1, 19, 30],
therefore suggesting another mechanism for MT regulation of viral DNA replication. MT
C-temiinal tyrosines 250, 315 and 322 are involved in MT mediated signaling and are
able to activate different signaling pathways leading to the activation of API and

PEA3/c-ets. This function is likely to represent the requirement for MT in the lytic cycle.

Here, I investigate the involvement of these tyrosines in the viral lytic cycle and
demonstrate the role of these major tyrosines in viral DNA replication and gene
expression in cell culture. I also expand the study to three minor C-terminal tyrosines 258,
288 and 297 for their potential roles in MT function. The results from this study suggest
that these six tyrosines are involved in MT regulation of viral DNA replication and gene
expression. Tyrosines 250. 315 and 322 play a major role whereas tyrosines 258, 288 and

297 play only a minor role.

MATERIALS AND METHODS

Cells, viruses and infections

Polyomavirus WTA2, Y3FM, Y3Fm, Y6F, MT-ter and A185 have been described
previously. NIH3T3 cells were grown to 50% confluence on 35 mm tissue culture plates
followed by 24 hrs of starvation in 0.5% serum medium. After removal of medium, cells
were infected in the Go state for 2 hrs. The infection lysate was removed prior to the

addition of 0.5% serum medium.

96

Retroviruses expressing various mutant MTs have been described previously.
NIH3T3 or NIH3T3-LT (stably expresses LT) cells were grown in DMEM with 10%
serum at 37°C and 5% C02 until reaching confluence for 24 hrs. Cells were then
trypsinized and plated at 2x105 cells/35 mm plate in 10% serum medium at 37°C for 4 hrs.
Retrovirus infection was carried 0th at 37"C for 3 hrs with 8ug/m1 of polybrene. The

infection lysate was removed prior to the addition of 0.5% serum medium.

Preparation and analysis of DNA

Infected cells were lysed at 6 hrs and 48 hrs postinfection in Hirt Buffer (10 mM
Tris-HCl-IO mM EDTA, 0.2% SDS, [pH 76]). Proteins were precipitated with 1/10
volume of 5M NaCl at 40C overnight. Proteins were isolated for Western blot analysis by
centrifugation at 14000 rpm for 30 min at 40C. DNA was extracted with phenol-
chlorofomi. DNA was digested with the restriction endonuclease EcoRI (Gibco-BRL),
which linearizes the polyomavirus genome. Digested DNA was electrophoresed on 1%
agarose gels, stained with ethidium bromide, and blotted onto nylon membranes
(Amersham Phamiacia Biotech). Hybridization was carried out at 650C in 1X Denhardt’s
solution, 2X SSPE (1X SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH
7.7]) by standard procedures with a 32P-radiolabeled probe containing the whole
polyomavirus genome. Hybridization probes were labeled with [oz-32P] dCTP using
random primer DNA labeling kit (Invitrogen). The hybridized blots were washed under
stringent conditions. The blots were exposed to phosphorescent screens and scanned with
a Phosphorimager (Molecular Dynamics).The blots were then exposed to X-ray films
(Amersham Phamtacia Biotech) for up to 3 weeks at -700C with an intensifying screen to

achieve darker exposures.

97

Protein analysis

Proteins from Hirt extraction (see DNA analysis) or cells scraped directly from
plates were boiled in protein sample buffer (5% SDS; 0.03% bromophenol blue; 20%
glycerol; 5% B-mercaptoethanol; 0.5 M Tris-HCl, [pH 6.8]) for 5 min. Proteins were
electrophoresed in 10% SDS-PAGE and electroblotted onto polyvinylidene difluoride
membranes (Amersham). A polyclonal rat anti-tumor serum, harvested as ascites fluid,
was used as the primary antibody to detect three early proteins: large, middle, and small
T. It also reacts with a few cellular proteins which can serve as an internal loading control.
Goat anti-rat horseradish peroxidase (HRP; Pierce) was used as the secondary antibody.
A rabbit anti-MT antiserum (a gift from B. Schaffliausen) was also used as the primary
antibody and goat anti-rabbit HRP (Sigma) as the secondary antibody. A rabbit anti-
capsid antibody, (a gift from R. Garcea) was used to detect the capsid protein VPl, with
goat anti-rabbit HRP (Sigma) as the secondary antibody. Signals were visualized by

SuperSignal Enhanced C hemiluminescent Detection System (Pierce).

Luciferase reporter gene assay

EOL cells are a stable NIH3T3 cell line that contains an integrated luciferase gene
under the control of the polyomavirus enhancer-origin. EOL cells were grown in DMEM
with 10% serum at 37”C and 5% C02 until reaching confluence for 24 hrs. Cells were
then trypsinized and plated at 2x105 cells/35 mm in 10% serum medium at 370C for 4 hrs.
Retrovirus infection was carried out at 370C for 3 hrs with Sag/ml of polybrene. The
infection lysate was removed prior to the addition of 2% serum medium for 18 hrs.
Growth medium was changed to 0.5% for 48 hrs. Cells were washed with cold 1x PBS

and lysed in 150 pl of 1x reporter lysis buffer (Promega). Samples were mixed by

98

vortexing for 15 sec and centrifuged at 14000 rpm for 2 min at 4°C. 20 ul of the extract
was assayed for luciferase activity with 100 pl of Luciferase reagent (Promega) using a

TD-20e luminometer (Turner).

RESULTS

Effect of MT tyrosine mutations on viral genome accumulation during

polyomavirus infection

I first studied the effect of different tyrosine mutant polyomaviruses in viral lytic
infection. NIH3T3 cells were infected with wild type A2 (WTA2) or Y3FM, Y3Fm, Y6F,
MT-ter and A185. The mutant properties with respect to the expression of early viral
proteins are shown in Table 3-1. Input and output viral DNA was collected at 6 and 48

hrs post infection, respectively. Viral DNA was analyzed by Southern blotting.

During 48hrs of infection, the WTA2 genome amplified 900 fold, Y3FM 100 fold,
Y3Fm 400 fold, Y6F 130 fold; MT-ter 33 fold and A185 8 fold (Figure 3-1). Thus,
relative to WTA2, Y6F showed a 7-fold, Y3Fm a 2.5-fold and Y3FM a 9-fold defect in
genome accumulation. The slightly stronger defect of Y3FM compared to Y6F is likely
due to variations in DNA dilution and loading, since the accumulation of Y3FM genomes
in other experiments was usually similar or slightly higher that that of Y6F. A185 showed
a more than 100 fold defect in genome accumulation, consistent with published data [5].
MT-ter had a 30 fold defect compared to WTA2, but had a 4-fold increase compared to
A185. Thus small T makes a small and MT large contribution to the amplification of the

viral genome This result conflmied the role of MT [5] [12]and ST [2, 20, 29] in viral

99

DNA replication. Interestingly, the defect of Y6F was less severe than that of MT-ter,
which suggested that residuals other than the six tyrosines on MT were involved in
regulating viral genome accumulation. The 2.5-fold defect of Y3Fm indicated a new role
of the three minor tyrosines (tyrosines 258, 288 and 297) in MT function. However, these
tyrosines play only a minor role in viral genome accumulation since Y3Fm had a weaker

defect than Y3FM.

Effect of MT tyrosine mutations 09 viral gene expression during polyomavirus

infection

The effect of MT tyrosine mutations on viral early and late gene expression was
examined. NIH3T3 cells were infected with WTA2 or Y3FM, Y3Fm, Y6F, MT-ter and
A185. Viral early and late protein expression was analyzed by Western blotting (Figure
3-2). Compared to WTA2, the expression of LT in A185 was much lower, indicating the
requirement of MT and ST for efficient early gene expression. In all other mutants, the
expression of LT was only slightly reduced. However, the pattern showed WT = Y3Fm >
Y4F > Y5F 2Y3FM > Y6F. Levels of MT and ST in WTA2, Y3FM, Y3Fm and Y6F were
also only slightly different. As expected, MT-ter only expressed ST. Larger differences
were observed in the level of late protein VPI. Y3FM showed decreased VPl expression
to a level similar to Y6F, while the level of VP] in Y3Fm was similar to that in A2. These
results suggested that Y6F and Y3FM MT are defective in upregulating viral late gene
expression, while Y3Fm MT was not. Therefore, as they did in genome accumulation, the
major tyrosines also played a more important role in regulating viral gene expression than
the minor tyrosines. The relatively high level of VPl in A185 was due to the high level of

empty capsids in the A185 virus stock that entered the cells and persisted during infection.

100

Complementation of A185 genome accumulation and gene expression by wild tygg

and mutant MT

In polyomavirus infected cells, both WTA2 and all the tyrosine mutant viruses
also express ST. In order to study only the effect of MT, 1 expressed different tyrosine
mutant MTs in NIH3T3 cells by retrovirus infection, followed by A185 infection as
described in the Materials and Methods. DNA and protein samples were collected and
analyzed by Southern blotting and Western blotting. The ability of different tyrosine
mutant MT to complement the defect of A185 in genome accumulation and gene

expression was assessed.

Figure 3-3A showed the levels of A1 85 genomes after 48 hrs of complementation
by various MT mutants or control vector. The quantitation of the A185 genome increase
is presented in Figure 3-3B. Compared to the control vector, WT MT increased the A185
genome levels by 6 fold; Y3Fm by 4 fold and Y3FM MT by 1.5 fold, while the expression
of Y6F MT failed to increase the A185 genome level. Y5F-250+, Y5F-315+ and Y5F-
322.. MT increased the A185 genome level by 2-2.5 fold, whereas Y4F-25077315: Y4F-

250+/322+ and Y4F-315+/322+ MT increased the A185 genome level by 3.6-4.4 fold.

The expression of the various mutant MTs was high and relatively equal at 8 hrs
(Figure 3-4A) and 48 hrs (Figure 3-4B) post A185 infection. Compared to control vector,
the expression of WT and Y3Fm MT increased the expression ofA185 LT. But Y6F MT
failed to do so. Y3FM, quadruple mutants (Y4F) and quintuple mutants (YSF) MT also
increased LT expression to various degrees. A similar pattern was seen for VP]

expression, though the differences were small due to the high level of VP] in A185 stock.

101

Both genome accumulation and protein expression results suggest that the six C-
temiinal tyrosines are involved in the MT regulation of both viral genome accumulation
and gene expression. Among them, tyrosine Y250, Y315 and Y322 play a major role and

tyrosine 258, 288 and 297 play only minor role.

The effect of MT on DNA replication could be either through LT or through
transcription factors that bind to the polyoma enhancer or a combination of both. In order
to separate these two effects, the effect of MT on A185 replication was compared in
NIH3T3 and an NIH3T3—derived LT-expressing cell line. Cells were infected by
retroviral vectors expressing WT or Y6F MT followed by A185. DNA and protein
samples were analyzed by Southem and Western blotting (Figure 3-5A). Western blots
continued the relatively equal levels of MT expression in these cells (Figure 3-SB). In
NIH3T3 cells, the expression of WT MT increased the A185 genome levels by 50 fold
(Figure 3-5A, left panel), while expression of Y6F MT led to a 5-fold increase. Thus,
Y6F MT showed a lO-fold defect in the ability to complement the A185 replication
defect. These results are similar to those shown in Figure 3-3A except that in that
experiment the replication of Al 85 in the presence of Y6F MT was the same as that when
there was no MT expression at all. This difference is likely due to the smaller scale ofthe
overall genome accumulation in that experiment, since there was only 6 fold difference in
A185 replication between with and without WT MT. I have observed a wide range of
A185 replication defect, depending on a variety of factors, such as status of cells upon
infection, batch of serum, etc. The small increase of A185 genome in the presence of Y6F
MT suggests that MT functions other than those mediated by the six tyrosines may play a

role in regulating viral genome accumulation.

102

Under conditions of high level LT expression in the NIH-LT cell line, the
expression of WT and Y6F MT was still able to increase the level of A185 genomes by
5.6 and 2 fold, respectively (Figure 3-5B, right panel). Therefore, the effect of MT in
A185 genome accumulation is not solely mediated through an increase in LT expression.
Transcription factors activated by MT may also participate directly in DNA replication

through pol yoma enhancer.
MT activation of gene expression through polyoma enhancer

The ability of each tyrosine to activate the polyoma enhancer-dependent
transcription was examined using a reporter gene assay. Retrovirus infection was carried
out in EOL cells. an NIH3T3 cell line with an integrated -luciferase gene under the
control of polyoma enhancer-origin sequence. Cell lysates were collected and were
subjected to luciferase assay. As shows in Figure 3-6, the expression of wild type
increased the luciferase activity by 3.7 fold. Y3Fm MT was able to increase the luciferase
activity to a level similar to that of WT MT. Removal of all three major tyrosines in
Y3FM and Y6F MT reduced the luciferase activity to close to that of control vector. The
presence of single major tyrosines caused only minor, if any, effects as shown in YSF
mutants. Y4F-2507315+ and Y4F-250732? MT increased luciferase activity by 2.4 and
1.8 fold, respectively. Interestingly, Y4F-315+/322+ MT activated the luciferase activity
to close to that of wild type level. The significance of this finding remains to be

detemiined.

103

DISCUSSION

The role of MT in polyomavirus replication has been demonstrated both in mice
[12] and in cell culture [5, 6]. Possible mechanisms have been proposed: (1) MT can
indirectly regulate viral DNA replication by increasing LT expression through the
activation of polyoma early promoter; (2) MT can activate transcription factors (i.e.
PEAl/AP-l and PEA3/c-ets‘), which bind to the polyoma enhancer and stimulate DNA
replication directly. Both processes are mediated by the activation of MT induced
signaling pathways. which requires the phosphorylation of several MT C-terminal
tyrosines. In this study, I investigate the role of six C-temiinal tyrosines in viral genome
accumulation and gene expression in cell culture. The following conclusions can be
drawn from the results presented above: ( 1) Tyrosine 250, 315 and 322 play a major role
among the six tyrosines being studied, while tyrosine 258, 288 and 297 all together play
only a minor role. Each of the three major tyrosines contributes relatively equal to viral
genome accumulation. (2) The six C-terminal tyrosines could not solely account for the
defect caused by the loss of MT. (3) The effect of MT on DNA replication is attributed to
an increase in LT expression as well as the activation of transcription factors that bind to

polyoma enhancer and regulate DNA replication.

In this study, I used two approaches to examine the effect of MT tyrosine
mutation on viral genome accumulation and gene expression. One approach used mutant
viruses in a lytic infection, which more closely represents polyomavirus infection in
nature. The other approach was to complement the MT—/ST- defective virus A185 with
different mutant MTs by retrovirus infection. The MT complementation experiments

eliminated the effect of ST on DNA replication, so that the role of MT in viral replication

104

and gene expression could be better isolated. To further separate the effect of MT on LT
expression, MT complementation of A185 replication was carried out in a LT-expressing
cell line. In the presence of abundant LT, MT was still able to increase the A185 genomes,
though the magnitude was reduced (Figure 3-5B). This suggests the more direct role of

MT on viral replication through transcription factors bound to polyoma enhancer.

Our results demonstrated that the function of C-terminal tyrosines in genome
accumulation was parallel to their function in gene expression, both in terms of
increasing early and late gene expression (Figure 3-4B) and activating polyoma
enhancer-dependent transcription (Figure 3-6). However, in mutant polyomavirus
infected cells, the differences of LT expression between WTA2, Y3FM, Y3Fm, Y6F, MT-
ter were small (Figure 3-2), while in the MT complementation experiment, the
differences in LT levels were obvious (Figure 3-4B). One possible explanation is that
WTA2, Y3FM, Y3Fm, Y6F, MT-ter all expressed high levels of LT. Since LT can
autoregulate its own synthesis, viruses expressing higher levels of LT will exert a
stronger repression of its own LT synthesis, and therefore offsetting the MT effect on LT
expression. A185 expresses very low levels of LT, so the promoting effect of MT on LT

expression becomes distinguishable.

Tyrosine 250 is required [11, 15] and tyrosine 322 is dispensable [28] for cellular
transfomiation. Tyrosine 315 is not essential for transformation, but mutation of tyrosine
315 to phenylalanine greatly reduces transfomiation efficiency [4, 17, 21]. In our study,
the effect of each major tyrosine on viral genome accumulation and gene expression is
similar, demonstrating a difference in the function of these tyrosines in viral replication

and cellular transformation.

105

 

One interesting observation is that Y6F MT still displays partial capacity to
promote genome accumulation and gene expression. Several possibilities can be
speculated. (1) There may be binding sites for other proteins at the C-terminus of MT.
For example, Serine 257 when phosphorylated, binds 14-3-3 [9, 22, 23]. 14-3-3 has been
shown to bind PI3K [3], cdc25 [8] and Raf-l [l4]. l4-3-3 is involved in the Ras-
dependent activation of Raf-l [13, 26]. Mutation ofthe 14-3-3 binding site on MT does
not affect MT transforming ability, but alters tumor profile [9]. (2) Other functions at the
N-temiinus of MT may also be involved. The DnaJ domain between residues 42 and 47
interacts with Hsc70 [24]. Hsc70 functions as a chaperone protein to promote the proper
folding of proteins. This sequence is shared by all T antigens. It is possible that
membrane-bound MT promotes the interaction of Hsc70 and its binding partner, which is
involved in MT signaling. This interaction is abolished when MT does not bind to the cell
membrane. The interaction between MT and PP2A is also essential for MT function. (3)
The status of the viral minichromosome might be another contributing factor. hr-t
mutants showed decreased level of histone H3 and H4 acetylation [27] compared to wild
type virus, which implied a difference existed in chromatin structure. This difference may
have strong impact on viral early gene expression. The 4- to l6-hrs delay of A185 in
large T expression compared to wild type virus [5] provides evidence to support this
hypothesis. The exact mechanism of how MT regulates viral replication is still under
investigation. The functions proposed above may reflect the involvement of MT in
various aspects of the polyomavirus lytic cycle, such as virus entry, early and late gene

expression, viral DNA replication and virus encapsidation.

106

 

 

SUMMARY

In summary, this study demonstrates the role of C-temiinal tyrosines in viral
genome accumulation and gene expression and presents a possible mechanism for the
role of MT in regulating DNA replication and gene expression. By activating different
signaling pathways through these tyrosines, like it does for transformation, MT activates
transcription factors (i.e. PEAl/AP-l and PEA3/c-ets), which bind to the polyoma
enhancer to either directly stimulate DNA replication, or indirectly through the activation
of LT transcription. Although MT likely utilizes the same pathways for its functions in
viral replication and transformation, the requirements for these pathways in the two
processes are different. In transformation, the ShcA-Ras-MAPK pathway plays a more
important role than the PI3K pathway, and the PLC-yl pathway is dispensable. In viral

DNA replication, those three pathways contribute equally.

107

APPENDIX 3: FIGURES AND TABLES FOR CHAPTER 3

108

 

 

Table 3-1 Properties of polyomaviruses used in cell culture studies
This table lists the polyomaviruses used in this study and the properties of viral

early proteins. WT = wild type; Mut = mutant; - = not expressed.

Notes:

a. X stands for the number of tyrosines that are mutated.

b. Single nucleotide (A to T) change in MT coding sequence does not affect LT amino
acid sequence.

c. MT-ter expresses wild type ST as well as a truncated MT fragment which has the same

amino acid sequence as ST, except that ST has 4 extra amino acids at the C-terminus.

109

 

 

 

 

 

 

 

 

 

Mutant LT MT ST
A2 WT WT WT
\era WTh Mut WT
MT-ter WT " WTC

A1 85 WT — _

 

 

 

 

 

110

E-

Figure 3-1 Comparison of viral genome levels in wild type and mutant polyomavirus
infected cells

Cells were infected as described in Materials and Methods. Viral DNA was
extracted at 6 and 48 hrs postinfection, digested with EcoRI, which linearizes the viral
genome, processed for Southem blotting, and hybridized to a 32P-labeled polyomavirus
genomic probe as described in Materials and Methods. 1/2 of 6—hr DNA was loaded. 1/10
of 48-hr DNA from WTA2, Y3FM and Y3Fm and 1/3 of those from Y6F, MT-ter and
A185 were loaded as shown. Hybridized counts were detemiined with a PhosphorImager
and corrected for dilution, and the increase relative to the input is shown. The properties
ofeach virus with respect to the expression of MT and ST are indicated. “+” = wild type;

“M" = mutated; “-" = no expression.

111

 

 

 

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Figure 3-2 Analysis of the early viral proteins

Cells were infected as described in Materials and Methods. Proteins were
extracted at 48 hrs postinfection. (A) The Western blot was probed with the rat
polyclonal anti-tumor serum. The three early polyomavirus proteins are identified on the
left as large T (LT), middle T (MT), and small T (ST). This antiserum also detects two
cellular proteins (identified with arrows), which serve as loading controls. (B) The blot

shown in panel A was stripped and probed with a polyclonal rabbit anti-VP] antibody.

Notes:

a. The level of VP] in A185 infected cells is due to the entry and persistence of empty

capsids in the A185 stock during infection.

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LT
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B.

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114

 

Figure 3-3 Complementation of MTs in A185 genome accumulation

Cells were infected with retroviruses expressing different mutant MT for 20 hrs,
then infected with A185 as described in Materials and Methods. DNA and proteins were
extracted at 6 and 48 hrs post A185 infection. Duplicate samples were analyzed. (A)
Southern blot was performed as described in the legend to Figure 3-2. The increase of
A185 genomes is indicated. (B) A graphic presentation of the increase of Al 85 genomes
in the presence of different mutant MTs, detemiined in the panel A. Duplicate samples

are presented.

Note: Only one DNA sample of WT MT-expressing cells was analyzed.

115

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Figure 3-4 C omplementation of MTs on A185 viral early and late protein expression
Protein samples from the same experiment (as shown in Figure 3-3) were
analyzed by Western blotting with the rat polyclonal anti-tumor serum for early and late
protein expressions as described in the legend to Figure 3-2 (A) Protein samples at 6 hrs
post A185 infection. MT is indicated on the left. Two cellular proteins are also detected.
No LT is detected at this time. (B) Protein samples at 48hrs post A185 infection. LT and
MT are indicated on the left. The blot was stripped and re-probed with a polyclonal rabbit ._—

anti-VPI antibody as shown at the bottom.

Notes:

 

a. The level of VP] in A185 infected cells is due to the entry and persistence of empty

capsids in the A185 stock during infection.

117

 

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118

Figure 3-5 Comparisons of MT complementation in NHI3T3 and NIH-LT cell lines
MT complementation of A185 genome accumulation was compared in NIH3T3
cells (A) and NIH-LT cell line (B). Cells were infected with retroviruses expressing WT
and Y6F middle, then infected with A185 as described in the legend to Figure 3-3. DNA
and proteins were extracted at 6 and 48 hrs post A185 infection and analyzed by Southern

blot and Western blot as described in the legend to Figure 3-3 and Figure 3-4.

119

 

 

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Figure 3-6 MT activation of gene expression through polyoma enhancer

NIH3T3 cells containing an integrated luciferase gene under the control of the
polyoma enhance-ori gin (EOL) were infected with retroviruses as described in Materials
and Methods. Luciferase activities were measured at 60 hrs postinfection and expressed
as luciferase relative light unit with basal level (uninfected cells) set at 1. The increase in

luciferase activity (average of duplicated measurements) is indicated above the column.

121

 

 

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Murakami, Y., et al., The nuclear protooncogenes c-jun and c-fos as regulators of
DNA replication. Proc Natl Acad Sci U S A, 1991. 88(9): p. 3947-51.

Nilsson, S.V. and G. Magnusson, T-antigen expression by polyoma mutants with
modified RNA splicing. Embo J, 1983. 2(12): p. 2095-101.

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21.

23.

24.

25.

26.

27.

28.

29.

30.

Oostra, B.A., et al., Transforming activity of polyoma virus middle-Tantigen
probed by site-directed mutagenesis. Nature, 1983. 304(5925): p. 456-9.

Pallas, D.C., et al., Cellular proteins that associate with the middle and small T
antigens of polyomavirus. J Virol, 1988. 62(1 1): p. 3934-40.

Pallas, D.C., et al., Association of polyomavirus middle tumor antigen with 14-3-3
proteins. Science, 1994. 265(5171): p. 535-7.

Pallas, D.C., W. Morgan, and TM. Roberts, The cellular proteins which can
associate specifically with polyomavirus middle Tantigen in human 293 cells
include the major human 70-kilodalton heat shock proteins. J Virol, 1989. 63(11):
p. 4533-9.

Piette, J. and M. Yaniv, Two different factors bind to the alpha-domain of the

polyoma virus enhancer, one of which also interacts with the S V40 and c-fos
enhancers. Embo J, 1987. 6(5): p. 1331-7.

Roy, S., et al., 1 4-3-3 facilitates Ras-dependent Raf-l activation in vitro and in
vivo. Mol Cell Biol, 1998. 18(7): p. 3947-55.

Sehafthausen, BS. and T.L. Benjamin, Deficiency in histone acetylation in

nontransforming host range mutants of polyoma virus. Proc Natl Acad Sci U S A,
1976. 73(4): p. 1092-6.

Schafthausen, B.S., et al., Residual transforming activity of FY l l 78T, a mutant
lacking the principal in vitro tyrosine phosphorylation site, is not affected by

removal of the secondary tyrosine phosphorylation site at residue 322. Virology,
1985. 143(2): p. 671-5.

Turler, H. and C. Salomon, Small and middle Tantigens contribute to lytic and
abortive polyomavirus infection. J Virol, 1985. 53(2): p. 579-86.

Veldman, G.M., S. Lupton, and R. Kamen, Polyomavirus enhancer contains
multiple redundant sequence elements that activate both DNA replication and
gene expression. Mol Cell Biol, 1985. 5(4): p. 649-58.

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31. Yoo, W., M.E. Martin, and W.R. Folk, PEA] and PEA3 enhancer elements are
primary components of tlte polvomavirus late transcription initiator element. J
Virol, 1991. 65(10): p. 5391-400.

126

 

 

CHAPTER 4

THE ROLE OF MT C-TERMINAL TYROSINES IN GENOME
ACCUMULATION AND PERSISTENCE IN MICE

ABSTRACT

In this study, I examine the function of middle T C-terminal tyrosines in viral
replication in mice. A tyrosine sextuple mutant (Y6F) shows a 7-10 fold defect in viral
genome accumulation in the acute infection of both neonatal and 4-week old mice. This
defect becomes more severe in the 4-week persistent infection. The major tyrosine triple
mutant (Y3FM) shows a 2.4 fold defect, whereas the minor tyrosine triple mutant (Y3Fm)
shows no defect in neonatal mice infection. Furthemiore, a mild maturation defect is

found in Y6F.

INTRODUCTION

MT has been shown to play important roles in viral DNA replication and gene
expression in tissue culture [6, 7], as well as virus growth, persistence and tumor
induction in mice [9, 16]. In the previous chapter, 1 demonstrated the role of six MT C-
temiinal tyrosines in viral genome accumulation and gene expression in cell culture. Here,
I extend the study into the natural host of the virus -- the mouse. The interest was in
comparing the function of middle T tyrosines in oncogenesis and in virus replication in
mice. Mutant viruses were examined in both neonatal and 4-week young adult mice for

possible defects in genome accumulation. Y6F was also examined for possible defects in

127

 

 

viral persistence and maturation. The results show that Y6F had a 7-10 fold defect in viral
genome accumulation in both neonatal and 4-week old mice. This defect was more severe
in persistent infection. In neonatal mouse acute infection, Y3FM showed only a 2.4 fold
defect while no defect was found in Y3Fm. Moreover, a mild maturation defect was found
in Y6F. These results demonstrate that the MT C-tenninal tyrosines play important roles

in viral replication in mice.

MATERIALS AND METHODS

Animal and polyomavirus infection

Balb/C mice at 4 weeks of age or in mid-pregnancy were purchased from the
National Cancer Institute, Frederick. For neonatal mouse acute infection, mice were
infected within 24 hrs of birth with me" PFU of each virus in 50 pl of 1x PBS
subcutaneously in the dorsal region for 6 days. For young adult mouse acute infection,

the same amount of virus was injected into 4-week old mice intraperitoneally.

Preparation and analysis of DNA

Mice were sacrificed and organs (skins, ribs, kidneys and lungs) were placed in
digestion buffer (100 mM NaCl, 10 mM Tris [pH 8.0], 25 mM EDTA [pH 8.0], 0.5%
SDS) containing 100 pg of proteinase K per ml, homogenized for 10 sec at 30,000 rpm
with a Tekmar-Tissumizer, and digested overnight in a 500C shaking water bath.
Digested tissues were extracted with phenol-chloroform. Total DNA was precipitated
with the addition of 1/10 volume of 3M sodium acetate and equal volume of isopropanol

at -200C for 1 hr. DNA was collected by centrifugation at 40C and 10,000 rpm (Sorvall

128

 

 

SA-600) for 30 min. DNA samples were dried under vacuum and resuspended in TE
(lOmM Tris [pH 8.0] and 1mM EDTA [ph 8.0]). DNA concentrations were measured by
absorbance at 260 nm with a spectrophotometer. For each sample, 1.0 u g of total DNA
was digested with EcoRI (for single virus infection) or EcoRI +NheI (for mixed infection)
and electrophresed on 1% agarose gels. DNA was transferred to Hybond nylon
membrane (Amersham Pharmacia Biotech), UV crosslinked (Stratagene Stratalinker;
1200 micro-joules) and the blots were hybridized to a 32P-radiolabeled polyomavirus
genomic probe. For single virus infection, the blots were stripped and re-probed with a
probe hybridizing to the immunoglobulin gene uj region.

The hybridization was carried out at 650C for 24-48 hrs in 1X Denhardt’s
solution, 2X SSPE (1X SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH
7.7]). The hybridized blots were washed under stringent conditions. The blots were
exposed to phosphorescent screens and scanned with the Phosphorimager (Molecular
Dynamics). The blots were then exposed to X-ray films (Amersham Pharmacia Biotech)
for up to 3 weeks at -70oC with an intensifying screen to achieve darker exposures.

Statistic analysis was carried out by ANOVA method using SAS software package.

Live virus production

The amount of virus in the organs was determined by plaque assay using NIH 3T3
cells. Organs were placed in digestion buffer (100 mM NaCl, 10 mM Tris, [pH 8.0], 25
mM EDTA, [pH 8.0]) and homogenized as described above. The homogenates were
frozen and thawed 4-5 times and clarified by centrifugation. The supernatant was used to
infect NIH3T3 cells at 80% confluence. The infection was carried out at 370C for 1 hr.

Infected cells were fed with medium containing 5% serum and 0.9% agar and incubated

129

 

 

for 7 days at 370C. The plates were stained with neutral red. The plaques were picked
and resuspended in 20 ul of ddHZO. Following incubation at 450C for 30 minutes, agar
was removed by centrifugation. 10 ul of supernatant was used as the template to amplify
a fragment of viral genome (nucleotide 41-1215) by PCR. PCR products were digested
with Xhol, which cut the AZ—RE DNA into two fragments (555 and 619 bp), but does not

cut Y6F DNA (1 174 bp).

RESULTS

fle role of MT C-terminal tyrosines in genome accumulation in neonatal mice acute

infection

To examine the role of three major tyrosines Y250, Y315 and Y322 in
polyomavirus replication in neonatal mice, tyrosine to phenylalanine mutants were used.
These mutants also express small T protein. For comparison, a mutant named MT-ter
(MT'/ST+) which expresses ST and a MT fragment that is almost identical to ST and a

mutant that produces neither MT nor ST, A185 (MT/ST) were also included.

The accumulation of viral genomes was used to track viral replication. Balb/C
neonatal mice were infected, sacrificed 6 days after infection and total DNA was
analyzed by Southem blotting. The level of viral genomes was compared. Figure 4-1
shows viral genome accumulation in the skin close to the site of injection. In the case of
all tyrosine mutants tested, single (YlF), double (Y2F) and triple (Y3FM) mutants,
genome levels were comparable to that obtained with WTA2, indicating no detectable

defect. In contrast, MT—ter showed a 14-fold and A185 a 30-fold reduction in viral

130

 

1 -

genome levels. Skin distal to the site of injection was also analyzed as well as other
organs such as the bone (ribs), the kidney and the lung and the results are summarized in
Table 4-2. Since results from single (Y1 F) and double (Y2F) mutants were similar to that
of Y3FM. only the data for Y3FM is shown. Overall, the levels of viral genome were high
in skin, bone (ribs) and moderately high in kidney. But the genome levels in lung were
relatively low even after 3 weeks of exposure, therefore the results were not included in
Table 2. The genome tissue distribution agrees with the results from a previous study that
demonstrated organ- specific replication pattern of polyomavirus infection [26]. Tyrosine

mutants did not show an alteration of this pattern.

As shown in Table 4-2, in all organs studied, the Py/uj ratio for Y3 FM was within
a 2-fold range of that obtained with wild type virus, indicating no severe defect. MT-ter
showed a 14-50 fold reduction, demonstrating the requirement of MT for efficient viral
replication in mice. A185 showed the strongest defect with a 30-100 fold reduction. A 2-
fold advantage of MT-ter over A185 in viral genome accumulation suggests a small role

for ST compared to that of MT.

Given the lack of a severe defect for Y3FM in genome accumulation, a sextuple
mutant Y6F was tested, in which all 6 C -temiinal tyrosines were mutated to
phenylalanine. Furthennore, a new strategy was developed to avoid variations between or
within litters, or other experimental variations such as leakage of the virus input drop
during infection, DNA recovery or loading. The strategy involved the use ofa mixture of
mutant with a reference wild type and allows direct comparisons in a single animal. One
possible drawback of this strategy is that the expression of wild type MT may

complement the defect of the mutant MT protein if the two viruses infect the same cell.

131

- i' «I. «1?

 

 

However, the probability of complementation should remain low, as demonstrated in
Figure 4-5. The reference wild type, called A2-RE, is derived from WTA2 and contains

added restriction endonuclease sites (e. g. NheI, XhoI).

To determine the ratio of the two viruses in the input, NIH3T3 cells were infected
with the virus mixture and DNA was harvested prior to the onset of DNA replication,
digested with Nhel or Xhol and the genome ratio was calculated from the Southern blots.
(Figure 4-2, input). The virus mixture was used to infect five litters of Balb/C neonatal
mice subcutaneously for a 6-day acute infection. Total DNA extracted from various
organs was analyzed by Southern blot (Figure 4-2). Both viruses achieved disseminated
infection in the organs examined. Following the 6-day acute infection, A2-RE genome
levels were higher than those found for Y6F by 4 to 18 fold, indicating a defect of Y6F in
viral genome accumulation. Substantial variations between animals were observed,
despite the mixed infection strategy. However, the defect in Y6F was relatively consistent
in all organs studied, indicating that the Y6F defect is not organ-specific. The smallest
defect was found in the skin close to the site of injection (average ratio of 6.2) compared
to distant skin (ratio = 9.8) and other distant organs (bone = 9.4) and kidney (ratio = 11.8).

This is indicative ofa potential defect in dissemination (see below).

To further examine the role of the C-tenninal tyrosines, a side-by-side comparison
of different mutants was carried out using the mixed infection strategy. In this experiment,
Y3FM, Y3Fm, Y6F, MT-ter as well as WTA2 were included. Balb/C neonatal mice were
infected and total DNA from skin close to the site of injection was analyzed by Southern
blotting. Figure 4-3A showed a skin (close) sample results from 4 individual mice of

each mixed infection. The overall results are summarized in Figure 4-3B. Both Y6F and

132

MT-ter had 7.6-fold defect in viral genome accumulation compared to WTA2, Y3Fm and
Y3FM (p<0.001). Y3FM showed only a 2.4-fold defect (p=0.08), which was not found in

single virus infection (Figure 4-1). No defect was found with Y3Fm.

The role of MT C-termifinal tyrosines in viral genome accumulation ill—neonatal mice

persistent infection

The persistence of viral infection following infection of neonatal mice was also
examined. Mice from the same litters as those examined in the 6-day acute infection (and
shown in Figure 4-2) were kept alive for 4 weeks. Total DNA from the same set of
organs of individual mice was analyzed by Southern blotting (Figure 4-4). An up to 100-
fold decrease in the Y6F genome was observed. In most of the samples, the Y6F genome
was barely detectable. The A2-RE/Y6F ratio was larger in samples where overall higher
levels of viral genomes were maintained. These results showed that the defect of Y6F

becomes more dramatic when followed through even a short persistence phase.

The role of MT C-terminal tyrosines infi viral gegome accumulation in 4-week young

adult mice

Since the replication potential of polyomavirus in mouse progressively decreases
with increased age of the mouse, I tested if tyrosine mutations also caused defects in adult
mice. Previous studies in our lab demonstrated that replication potential declines with the
age ofthe mouse in certain organs (kidney, liver and lung) with a marked drop around 3

weeks ofage. Therefore mice of4 weeks of age were used for adult mouse infection.

133

 

 

Balb/C mice at 4 weeks of age were infected with WTA2, A185 or doubly
infected with WTA2 and A185 and sacrificed at 3, 5 and 7 days postinfection. These
times were chosen to determine an optimal period of infection prior to the onset of the
antiviral immune response that would clear the virus; both single and mixed infections

were used to examine the possibility of complementation.

Total DNA from ribs was digested with Mspl and analyzed by Southern blotting
(Figure 4-5). Mspl digests the polyoma genome into 8 fragments. Because A185 has a 71
nucleotide deletion in Mspl fragment 4, the fragment 4 could be used to track the
replication of each virus. In both WTA2 single and WTA2+A185 mixed infection,
maximal genome levels were obtained at 5 days postinfection and decreased at 7 days
postinfection despite the variations between two individual mice. Therefore, a 5-day
infection period was used in adult mice infection in the following experiments. Compared
to WTA2, A185 showed a severe defect in viral genome accumulation (Figure 4-5). The
level of A185 fragment 4 in WTA2/A185 mixed infection was only slightly higher (less
than 2 fold) than that in A185 single infection, suggesting that mixed infection does not

cause serious complications.

Following the established protocol for adult mice mixed infection, Y6F was tested
for a defect in genome accumulation. An up to 12-fold A2-RE/Y6F ratio was observed in
organs studied (Figure 4-6). This defect was similar to that observed in neonatal mice

acute infection, therefore was not age-specific.

134

 

 

Genome analysis of live virus production of polyomavirus AZ-RE and Y6F

Polyoma middle T-defective hr-t mutants are defective in VPl phosphorylation,
resulting in defective virion maturation [10, 1 1]. Differences in the Y6F /WT ratio in skin
close versus distant to the injection suggested the possibility of a maturation defect. To
further test this possibility, levels of live virus in skin samples from both neonatal and
young adult mice in the A2-RE/Y6F acute infection were measured. To this end, a plaque
assay was carried out in NIH3T3 cells using tissue lysates. DNA from the plaques was
used as a template to amplify a fragment of the viral genome spanning the XhoI site in
the A2-RE genome. Thus, the parental origin of the viral genome in each plaque was

determined (Figure 4-7).

In neonatal mice, 110 plaques were analyzed, among which 102 contained the
A2-RE genome and 8 contained the Y6F genome. ln 4-week young adult mice, 65
plaques were analyzed, among which 59 the contained A2-RE genome and 4 contained
the Y6F genome. The ratio of A2-RE to Y6F genome was 13:1 and 15:1 in neonatal and
4-week young adult mice, respectively. AZ-RE infected cells usually form larger plaques
at earlier times than Y6F infected cells. Larger A2-RE plaques sometimes merge with
small Y6F plaques, which lead to the presence of both genomes in a single plaque (Figure
4-7, arrows). In such cases, the plaque was treated as two, one attributed each virus.
These 13-15:1 plaque ratios represent the accurate ratio of parental genomes in live,
infectious viral particles of A2-RE and Y6F in the infected mouse skin. These live virus
ratios are about 2-fold higher than the viral genome ratios in skin close to the site of
infection (Figure 4-2 and Figure 4-6), and suggest that the Y6F mutant displays a modest

defect in virion assembly.

135

 

 

 

DISCUSSION

'The purpose of encoding a potent oncoprotein MT during polyomavirus evolution
would be to provide advantages for virus growth and propagation, instead of causing
tumor in its host. However the role of MT on virus replication in its natural host hasn’t
been studied extensively. In Chapter 3 of this thesis. 1 demonstrated the role of six C-
temtinal tyrosines in viral genome accumulation and gene expression in cell culture. Here,
I further examined the replication of different tyrosine mutants in their natural host, both
neonatal and young adult mice. The following conclusions can be drawn from the results
presented above: (1) The six C-terminal tyrosines are required for efficient viral genome
accumulation in mice, at both the neonatal and adult stage. The role of the minor
tyrosines is very evident. (2) As expected, the loss of six tyrosines causes more profound
defect in virus persistence than in acute infection. (3) The signaling events induced by
these six tyrosines may have a small effect on maturation. (4) The age- and organ-

specific replication pattern is not affected by tyrosine mutations.

In mice infected as neonates, the peak in virus titer is reached at 7-10 days
postinfection [20, 21], which coincides with the onset of the immune response. This
period represents the acute phase of polyomavirus infection. At 2 weeks postinfection,
the immune response clears most of the viruses and infected cells, resulting in a dramatic
decline in virus titer. Therefore in our study, mice were sacrificed at 6 days and 4 weeks
postinfection, the former representing the acute phase and the latter a rather short period
(approx 2.5 weeks) of the persistent phase of infection. The age of the mice at the time of

infection also has an impact on viral replication [26], characterized by a marked decline

136

 

 

 

in viral replication potential in mice infected around 3 weeks of age [27] Therefore 4-

week old mice were chosen for adult mouse infection.

Early studies by Freund et. al. demonstrated the role of MT in viral replication
and persistence in mice [7, 9]. This is further confirmed by our results showing the defect
in A185 in genome accumulation. Removing all six tyrosines caused a similar defect in
viral genome accumulation as MT-ter. These results not only demonstrate the important
role of these tyrosines in viral genome accumulation, but also suggest that these tyrosines
accounts for the overall function of MT in viral replication in mice. These results are
inconsistent with the results from cell culture experiments presented in Chapter 3, which
showed MT-ter had a stronger defect than Y6F. Although I cannot rule out the possibility
that the variations in the mouse experiment masked the actual difference between Y6F
and MT-ter, it is possible that the presence of different cell types in organs may lead to
more complex regulation of viral replication in mice than in cell culture. The smaller

defect on3FM in mice (2.4 fold) than in cell culture (9 fold) provides another example.

The exact degrees of defect among Y3 FM, Y3Fm and Y6F are difficult to estimate,
due to the inconsistent results from cell culture and mice experiments. The functions of
the major and minor tyrosines are likely redundant instead of being distinct, with the
major tyrosines being more important. In such case, removing the minor tyrosines does
not cause a severe defect in viral replication. When the major tyrosines are removed, the
minor tyrosines can still partially compensate for the function. It is only when all six
tyrosines are removed that the defect becomes apparent, especially when the growth
conditions for the viruses are more stringent, e.g., a low level of serum in cell culture.

The defect in Y6F genome accumulation is more severe in the 4-week persistent infection

137

 

 

than in acute infection. This is probably due to the accumulation of a small defect over
multiple cycles of infection, or the decreased replication potential in adult mice, which

pose more stringent conditions for viral growth, or a combination of both.

One interesting finding is that mutation of the tyrosine Y250 and Y315 did not
cause a severe defect in viral genome accumulation in mice. Since tyrosine Y250 [8, 14]
and tyrosine Y315 are important for transformation [4, 17, 18], our results suggest that
the mechanisms of MT for regulating viral replication and cellular transformation are

different.

By studying mice infected at the neonatal or adult stage, an organ- and age-
specific pattern of viral replication was discovered [26]. In kidney, liver and lung, viral
replication was very high after infection of neonatal mice but very low after infection of
adults, while in mammary gland, skin and bone, viral replication was very high in
neonates and moderately high in adults. The results presented here confirmed the viral
replication pattern in neonatal and adult infection. But the six tyrosine mutation does not
alter the age- and organ-specific viral replication pattern. The polyoma enhancer plays an
important role in detemtining the age- and organ-specific pattern of polyomavirus
replication in the mouse [2, 26]. The B enhancer domain mediates polyomavirus
replication in most organs. in particular in kidney, at the neonatal stage but is not required
in those organs which can sustain replication in the adult, i.e., mammary gland, skin and
bone. The A enhancer domain mediates polyomavirus replication in the adult stage. In
the alpha core ofthe A domain, there are binding sites for transcription factors PEAl/AP-
l, PEA2 and PEA3/c-ets [15, 19, 23-25]. PEAl/AP-l and PEA3/c-ets are proposed to

mediate the role of MT and ST on viral DNA replication through the polyoma enhancer

138

 

 

 

[7]. The organs that support high levels of viral replication and persistent infection in
adults are also the organs that develop tumors [27], suggesting that sustained viral
genome replication and/or gene expression are required for oncogenesis. The present
results imply that these six tyrosines are not involved in the detemiination of the age- and
organ-specific viral replication pattern. This pattern is more likely detemiined by host

transcription factors present at different stages of mouse development.

Polyomavirus host range transforming (hr-t) mutants [3] encoding defective MT
and ST have been shown to have defects in VPl phosphorylation, which affects virion
assembly [10, 11, 13]. Casein kinase II (CKII) is responsible for the phosphorylation of
serine 66 on VP] and is often activated by serum, epidemial growth factor, and insulin-
like growth factor [1. 5, 12, 22]. Therefore, it is possible that MT mimics growth factors
to activate CKII, resulting in VPl phosphorylation. I examined the Y6F for defect in
virion assembly by measuring the production of infectious virus particles in the infected
skins from both neonatal and young adult mice. The higher ratios of A2-RE/Y6F in live
virus production compared to those average ratios in genome levels suggests Y6F has
mild defect in virus maturation. This result is also supported by the observation that the
ratios of viral genome level in tissues distal from the site ofinjection are relatively higher

than those close to the site ofinjection.

SUMMARY

In this study, 1 demonstrated the role of six MT C-temiinal tyrosines in viral

replication in mice, with Y6F having a large defect in genome accumulation following

139

- #3.":-

 

 

acute and persistent infection as well as a mild defect in virus maturation. In future
studies, it would be interesting to examine the two triple mutants Y3FM and Y3Fm for
genome accumulation following persistent infection of neonatal mice so that minor

defects could be uncovered.

140

 

 

APPENDIX 4: FIGURES AND TABLES FOR CHAPTER 4

 

 

141

Table 4-1 Properties of polyomaviruses used in mouse studies

This table lists the polyomaviruses and the properties of viral early proteins. WT

= wild type; Mut = mutant; - = not expressed.

Notes:
a. x stands for the number of tyrosines that are mutated.

b. Single nucleotide (A to T) change in MT coding sequence does not affect LT amino

acid sequence.

c. MT-ter expresses wild type ST as well as a truncated MT fragment which has the same

amino acid sequence as ST, except that ST has 4 extra amino acids at the C-terminus.

142

 

 

 

 

 

 

 

 

Mutant LT MT ST
A2 WT WT WT
YxF“ wrb Mut WT
MT-ter WT — WTC
A1 85 WT — __

 

 

 

 

 

143

 

Figure 4-1 Polyomavirus genome accumulation in the skin close to the site of

injection in neonatal mice following a 6-day acute infection

One to three litters of neonatal mice were infected as described in Materials and
Methods. After 6-day infection, skins (close to the site of injection) from each litter were
pooled and processed as described in Materials and Methods. Total DNA (10 pg) was
digested with EcoRI, processed for Southern blotting, and hybridized to a 32P-labeled
polyomavirus genomic probe. To normalize for loading, the blots were striped and re-
probed with a probe hybridizing to the immunoglobulin gene uj region. The ratio of Py to

uj counts was calculated and compared to wild type A2.

144

 

 

 

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Table 4-2 A summary of viral genome accumulation in organs from neonatal mice

following a 6-day acute infection

Neonatal mice were infected and polyomavirus genomes were analyzed as

described in the legend to Figure 4-1. The Py/uj ratio of A2 was normalized to 1 and the

Py/uj ratios of other viruses were compared to A2.

Notes:

a. The viral genome in lungs was relatively low even after 3 weeks of exposure, therefore

the result was not included.

b. Results from single (YlF) and double (Y2F) mutants were similar to that of Y3FM,

therefore only Y3FM data was shown.

c. When the level of genome was too low to be detected, the comparison was made

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Figure 4-2 Genome accumulation of polyomaviruses A2-RE and Y6F in neonatal
mice following a 6-day acute infection

Five litters of neonatal mice were infected with a mixture of AZ-RE and Y6F as
described in Materials and Methods. After 6-day infection, organs from each litter (2
pups per litter) were pooled and processed as described in Materials and Methods. Total
DNA (10 ug) were digested with EcoRI and Nhel, which cuts A2-RE genome into 2
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148

 

 

 

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Figure 4-3A Comparison of different mutants in neonatal mice following a 6-day acute
infection

Neonatal mice were infected with a mixture of A2-RE and a distinct wild type or
mutant virus as described in the Materials and Methods. At 6-day postinfection, skin
(close to the site of injection) from individual mice was processed as described in
Materials and Methods. Viral DNA was analyzed by Southern blotting as described in the
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Figure 4-3B A summary of viral genome accumulation in skin (close to the site of injection)
from neonatal mice following a mixed infection

This chart summarizes the defect of each mutant virus in genome accumulation described
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deviations is presented as error bars.
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153

Figure 4-4 Genome accumulation of polyomaviruses A2-RE and Y6F in neonatal
mice following a 4—week persistent infection

One or two neonatally infected mice from each litter (described in Figure 4-2)
were kept alive for 4 weeks as described in Materials and Methods. Organ DNA from
each mouse was analyzed by Southern blotting as described in the legend to Figure 4-2.

The ratio of A2-RE to Y6F genome was calculated.

154

 

 

 

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Figure 4-5 Detemtination of adult mouse infection period

4-week old mice were infected with A2, A185 or a mixture of both viruses as
described in Materials and Methods. At the indicated time, Total DNA from ribs of two
mice were digested with Mspl and analyzed by Southern blotting. The Mspl fragment 4

of each virus is indicated by arrows.

156

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157

Figure 4-6 Genome accumulation of polyomaviruses A2-RE and Y6F in 4-week old
mice following a 5-day acute infection

Three 4-week old mice were infected as described in Materials and Methods.
Organ DNA was analyzed by Southern blotting as described in the legend to Figure 4-2
The ratio of AZ-RE to Y6F genome was calculated.

* 3 week exposure

 

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159

Figure 4-7 Genome analysis of live virus production of polyomavirus A2-RE and
Y6F

Plaque assay was performed using organ homogenates. (A) Viral DNA from
plaques were used as templates to amplify a fragment of viral genome spanning the XhoI
site on AZ-RE. X1101 digestion cuts A2-RE DNA into two fragments (555 and 619 bp),
whereas Y6F DNA remains undigested (1174 bp). (B) Number of plaques containing

each genome was counted.

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