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This is to certify that the

dissertation entitled

POLYOMAVIRUS HR-ZTNMUTANTS INDUCE A GZ/M CELL CYCLE
ARREST AND SHOW PREFERENTIAL GENE EXPRESSION AND
REPLICATION IN COMPETITION WITH AZ-DERIVED VIRUSES

presented by

Kathryn Marie Spink

has been accepted towards fulfillment
of the requirements for

 

 

Ph . D . degree in Microbiology and

Molecular Genetics

 

12-14-2000
Date

 

MS U i: an Affirmative Action/Equal Opportunity Institution 0-12771

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

6/01 cJCIRC/DateDuepGS—sz

POLYOMJ
l‘iD SH(

POLYOMAVIRUS HR-T MUTANTS INDUCE A GZ/M CELL CYCLE ARREST
AND SHOW PREFERENTIAL GENE EXPRESSION AND REPLICATION IN
COMPETITION WITH A2-DERIV ED VIRUSES

By

Kathryn Marie Spink

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

2000

POLYOMA‘
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ABSTRACT

POLYOMAVIRUS HR-T MUTANTS INDUCE A GZ/M CELL CYCLE ARREST
AND SHOW PREFERENTIAL GENE EXPRESSION AND REPLICATION IN
COMPETITION WITH A2-DERIV ED VIRUSES

By

Kathryn Marie Spink

Polyomavirus hr-t mutants were isolated in an attempt to identify the viral gene
products involved in transformation. The mutants were selected for their ability to grow
on polyoma-transformed but not on untransformed Swiss 3T3 cells. All the mutants
isolated have a defect in small T and middle T function that results in the inability to
transform all cell types. On the other hand, the host-range property of these mutants is
very cell dependent. In this thesis, I have studied the effect these mutants have on the
NIH3T3 cell cycle. Infection of NIH3T3 cells with the hr-t mutant, B2, results in a GZ/M
cell cycle arrest that is serum independent and multiplicity dependent. The cell cycle
arrest induced by 82 is also seen in semi-permissive FR3T3 cells. The expression of
small T and/or middle T does not abrogate the cell cycle arrest induced by BZ.
Furthermore, the cell cycle arrest appears to be mitotic and may be caused by the over-
expression of VP]. The basis for VPl over-expression is presented below.

The effect of the cell cycle arrest on transformation was also investigated in cis
and intrans. Transformation of FR3T3 with a newly constructed wild type in the BZ
genetic background, thZ, was decreased and exhibited a long delay in the development

of transformed foci compared to wild type A2-infected F R3T3 cells. This delay is

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presumably due to the induced G2/M cell cycle arrest seen in thZ-infected FR3T3 cells.
The ability of wild type A2 to induce transformation was also affected by the presence of
the 82 mutant in mixed infections. While transformed foci of A2+B2-infected FR3T3
cells developed at a similar rate as those in A2-infected cells, the number was slightly
reduced. Transformed foci were selected and expanded following A2-, th2-, and
A2+B2-infections. The th2-transformed cell lines differed from the other two sets of
transformed cell lines. Few of the thZ-transformed cell lines expressed a normal-sized
large T. In accordance with the absence of LT, fewer genome copies were present in the
th2-transformed cell lines. Furthermore, the B2 genome was absent from cell lines
derived from A2+B2 transformed foci. All transformed cell lines did exhibit a few
common features expected for transformed cells: the expression of small T and middle
T, the absence of WI expression, and the presence of wild type Msp I fiagment 4. The
data from thZ- and A2+BZ-transformed cell lines suggest that ‘poisonous’ sequences
on the BZ genome must be eliminated for successfiil transformation.

I have also mapped the replication potential of the B2 mutant and investigated the
‘dominant-negative’ effect previously defined when hr-t mutants are co-infected with A2-
derived viruses. In single infections, the 32 mutant has the capacity to express viral
proteins and amplify viral DNA to high levels despite the absence of small T and middle
T. In mixed infections, the B2 mutant genome is preferentially expressed and replicated
compared to the wild type A2 genome. Both properties of the BZ mutant map to
sequences between nucleotides 1079-2225. Further definition of this region demonstrates
that the replication potential of the B2 mutant is located in the EcoR I/Nsi I fragment,

nucleotides 1562-1912. Two alternate models for the B2 phenotype are presented.

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ACKNOWLEDGEMENTS

I would like to thank my mentor Dr. Michele Fluck for all her guidance,
understanding, support, and friendship throughout my graduate experience. I would like
to thank the members of my guidance committee, Dr. Susan Conrad, Dr. Jerry Dodgson,
Dr. Walter Esselman, and Dr. Jon Kaguni for their interest and expertise. I would like to
thank Dr. Louis King, who runs the F ACS machine, for all his help, technical advise, and
for being accommodating to the demands of my experiments. I would also like to thank
Dr. Steven Heidemann for materials, methods, and expertise on studies of mitotic cells.
Although too numerous to mention, the friends I have made here in the department and
especially in Michele’s lab have been an enormous source of encouragement and support.
Finally, I would like to thank Scott, my husband, for his understanding and patience, and

for always making me laugh.

 

.15! of Tables
L‘sr of Figures

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List of Tables
List of Figures

Chapter 1:

Chapter 2:

Chapter 3:

TABLE OF CONTENTS

Literature Review

Introduction

The cell cycle and cell cycle checkpoints

The polyomavirus lytic cycle and neoplastic transformation
Properties of the early and late genes

The hr-t mutants

Summary

Appendix 1: Tables and Figures for Chapter 1
References

Polyomavirus hr-t mutants induce a mitotic cell cycle
arrest that is not abrogated by the expression of
small T and/or middle T

Abstract

Introduction

Materials and Methods

Results

Discussion

Appendix 2: Tables and Figures for Chapter 2
References

The polyomavirus hr-t mutant, B2, inhibits transformation
by middle T in a cis- and trans-dominant manner
Abstract

Introduction

Materials and Methods

Results

Discussion

Appendix 3: Tables and Figures for Chapter 3
References

PAGE

viii

ix

59

59
59
61
64
76
83
l 12

115
115
116
117
119
125
130
161

1

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Chapter 4:

Mapping the mutation(s) in the BZ mutant that contribute
to its replication potential and its preferential gene
expression and amplification in mixed infections
with wild type A2

Abstract

Introduction

Materials and Methods

Results

Discussion

Appendix 4: Tables and Figures for Chapter 4
References

vii

162

162
162
163
166
169
173
184

Ct

FA

FE

:SU

r31

Table 1-1:

Table 2-1:

Table 2-II:

Table 2-III:

Table 2-IV:

Table 2-V:

Table 3-1:

Table 3-II:

Table 3-III:

Table 4-1:

LIST OF TABLES

Mammalian cyclins and associated cdk proteins.

Effect of the multiplicity of infection on the accumulation
of GZ/M cells.

Cell cycle analysis of BZ-infected NIH3T3 cell lines
expressing sT, mT, and sT/mT.

Cell cycle analysis of A2 and BZ mixed infections and
infections with the thZ virus.

Cell cycle analysis of A2- and B2-infected cells treated
with aphidicolin and mimosine.

Comparison of the ability of several hr-t mutants to induce
a G2/M cell cycle arrest in NIHBT3 cells.

FACS Analysis of A2- and thZ-infected FR3T3 cells.

FACS Analysis of A2-, B2-, and A2+BZ-infected
FR3T3 cells.

Summary of the wild type A2 to mutant BZ genome
ratio in A2+BZ-infected FR3T3 cells.

82 Virus Exchanges.

viii

Page
31

91

93

93

97

105

132

146

154

175

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Figure 1-1:
Figure 1-2:
Figure 1-3:
Figure 1-4:
Figure 1-5:
Figure l-6:

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 2-9:

Figure 2-10:

LIST OF FIGURES

Physical map of the polyomavirus genome.
Restriction point control through Rb.

Regulation of cdc2 activity in the mitotic cell cycle.
Organization of the domains of polyomavirus large T.
Middle T signaling pathways.

Isolation of the polyomavirus hr-t mutants.

Cell cycle profiles of uninfected and wild type A2-,
mutant A185-, and mutant BZ-infected NIH3T3 cells.

Cell cycle analysis of BZ-infected NIH3T3 cells in the
presence or absence of serum factors.

Comparison of the accumulation of early and late viral
proteins and viral DNA replication in wild type A2-,
mutant A185- and mutant BZ-infected NIH3T3 cells.

Comparison of the accumulation of early and late viral
proteins in A2, 82, A2+BZ and thZ infections.

The effect of DNA synthesis inhibitors on viral DNA
replication.

Cell cycle profiles of BZ-infected FR3T3 cells at 48 hours
post release.

Comparison of early and late viral proteins and genome
levels in sub-populations of F R3T3 cells infected with
the B2 mutant.

DAPI staining of uninfected and A2-infected NIH3T3 cells.

DAPI staining of BZ-infected NIH3T3 cells.

VPl associates with B-tubulin.

ix

Page
29
33
35
37
39
41

85

87

89

95

99

101

103

107

109

111

W

de

 

 

1n

 

Figure 3-1:

Figure 3-2:

Figure 3-3:

Figure 3-4:

Figure 3-5:

Figure 3-6:

Figure 3-7:

Figure 3-8:

Figure 3-9:

Figure 3-10:

Figure 3-13:

Figure 3-14:

Figure 4-1:

Figure 4-2:

Figure 4-3:

A2-infected F R3T3 cells develop transformed foci by two
weeks post infection.

Infection of F R3T3 cells with the thZ virus results in the
development of transformed foci by three to four weeks
post infection.

Analysis of viral proteins from A2-transformed cell lines.

Analysis the integrity of integrated viral genomes in
A2-transformed cell lines.

Analysis of viral proteins from thZ-transformed cell lines.

Analysis of the integrity of integrated viral genomes in
th2-transformed cell lines.

Protein analysis of A2-, 32-, and A2+BZ-infected FR3T3
cells.

Analysis of viral genomes in A2-, B2-, and A2+BZ-
infected FR3T3 cells.

Analysis of viral genomes in A2-, 82-, and A2+BZ-
infected FR3T3 cells.

A2-infected and A2+BZ-infected FR3T3 cells develop
transformed foci by two weeks post infection.

Analysis of viral proteins from A2+BZ-transformed
cell lines.

Analysis of the integrity of integrated viral genomes
in A2+B2-transformed cell lines.

Mapping analysis of viral gene expression and replication
of the 32 mutant.

Analysis of early proteins expressed in mixed infections
of A2 with A185 and BZ-derived mutants.

Analysis of wild type and mutant genome amplification
in mixed infections.

134

136

138

140

142

144

148

150

152

156

158

160

177

179

181

Figure 4-41 The
and

 

Figure 4-4: The ability of the BZ mutant to express viral proteins 183
and amplify its genome maps to the EcoR I/Nsi I fragment.

xi

Introduction

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Chapter 1

Literature Review

Introduction

Polyoma is a small DNA tumor virus with 5.3kb of double stranded DNA
packaged into an icosahedral capsid. The genome encodes for six proteins. The three
early proteins, small T (22kDa), middle T (55kDa), and large T (lOOkDa), are translated
from three differentially spliced transcripts. The three late proteins, VPl (45kDa), VP2
(35kDa), and VP3 (23kDa), are also translated from three mRNAs made by differential
splicing of a precursor transcript. Early and late coding regions are separated by the
origin of bi-directional replication and a regulatory region, or enhancer (see Figure 1-1)
(80, 84).

This thesis will focus on the effects of specific polyomavirus mutants, hr-t
mutants, on the host cell cycle and on viruses of wild type A2 origin. Therefore, this
literature review will introduce the following topics: the cell cycle and cell cycle
checkpoints, the polyomavirus lytic cycle and neoplastic transformation, properties of the

early and late genes, and the hr-t mutants.

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The cell cycle and cell cycle checkpoints

The mammalian cell cycle is divided into four distinct phases: G1, S, G2, and M.
A state of quiescence, G0, can also occur when the cell exits the cell cycle. G0 cells have
a decrease in size due to a decrease in protein production. The G1 phase of the cell cycle
has been defined as the time interval between mitosis and DNA synthesis. The length of
G1 varies between cell types but is usually between 5-10 hours. Because of extra
metabolic requirements, cells released from G0 by the addition of growth factors have an
extended Gl phase (143). DNA synthesis occurs in the S phase of the cell cycle where
the entire genome is replicated once. The G2 phase is a second gap phase in which the
cell prepares to undergo mitosis. The two genomes are separated into separate nuclei
during mitosis, M phase, and the cells undergo cytokinesis to yield two daughter cells
each with a full genome equivalent identical to that of the parent cell.

The predominant factors controlling progression through the cell cycle are the
cyclins, cyclin dependent kinases (cdks), and cdk inhibitors. At least 16 cyclins: A, B1,
32, C, D1, D2, D3, E, F, G1, 62, H, I, K, T1, and T2, and nine cdks have been identified
in mammalian cells to date (see Table 1-1). Cyclins are regulatory proteins that are
periodically accumulated and degraded during the cell cycle. All cyclins contain a
conserved ‘cyclin box’ that is important for binding to cdks (98). Binding of cyclins to
cdks results in conformational changes in the cdk that activate the kinase activity and
affect substrate binding (129). However, full activation of the kinase activity requires not
only cyclin binding but also phophorylation and dephosphorylation. Phosphorylation of

conserved threonine residues in the active site predominately affects the affinity of the

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cyclin/cdk complex for protein substrates (129). This phosphorylation is canied out by
the cyclin dependent kinase activating kinases, or CAK (167). Inhibitory phosphates in
the catalytic domain must also be removed for full activation (109). Furthermore, the
binding of cyclin dependent kinase inhibitors, cki, inhibits cyclin/cdk complexes by
disrupting the active site (129). Together the cyclin/cdk complexes regulate cell cycle
progression, transcription, DNA replication, DNA damage responses, apoptosis, and
differentiation (98). In addition, checkpoints ensure that cells do not enter the next phase
of the cell cycle until the previous phase is completed; furthermore, they make certain
that any damage is repaired before cell cycle progression continues. Alteration of
checkpoint control mechanisms ofien results in cell death, genome instability, and/or

transformation (87).
G1 Progression

While many investigators have attempted to divide G1 into several sub-phases by
the requirements of various growth factors such as PDGF and EGF (143, 151), the point
in G1 that has received the most study is the restriction point, R. R was defined at the
point in the G1 phase when the cell commits to entering S phase (132). The
retinoblastoma (Rb) gene product has been implicated in the control of passage through
the restriction point (see Figure 1-2). Rb binds and regulates a variety of cellular proteins
including the EZF family of transcription factors. When Rb is in its hypophosphorylated
state it binds to E2F and inhibits E2Fs transcriptional activation capacity. Cyclin D/odk

complexes phosphorylate Rb resulting in release of E2F as a transcriptional activator.

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E2F then induces the expression of proteins needed for entry into S phase including
cyclins E and A, which together with their associated cdks can maintain Rb in its
hyperphosphorylated state (6). Release of E2F also results in the induction of genes
needed for DNA synthesis including dihydrofolate reductase, thymidine kinase, and DNA
polymerase or (171).

The major checkpoint at the Gl/S border is activated in response to DNA damage
(58). Following DNA damage, the tumor suppressor gene p53 binds and transactivates
p53-responsive genes including the cdk inhibitor p21, GADD45, cyclin G, mdm2, and
bax (102). p21/WAFl/Cipl inhibits cell cycle progression by inactivating G1
cyclin/cdks complexes responsible for progression into S phase such as cyclin Dl/Cdk4,
cyclin E/Cdk2, cyclin A/Cdk2, and cyclin A/cdc2 (108). Inhibition of these complexes
results in the inactivation of the Rb pathway described above. p21 has also been found to
associate with PCNA and inhibit its role in DNA replication but not DNA repair.
GADD45 also interacts with PCNA to inhibit DNA replication. Cyclin G expression is
elevated in the response to DNA damage, but its firnction is not clear at this time (102).
The mdm2 gene can interact with and inhibit the transcriptional activation activity of p53
and thus provides an auto regulatory feedback loop on p53 activity (205). The bax
protein binds bcl-2 and inhibits its ability to prevent apoptosis (102). Therefore, one
possible outcome of induction of the p53 DNA damage response is the induction of
apoptosis. Whether cells undergo apoptosis or not depends largely on the cell type, the

availability of growth factors, and the status of Rb and E2F (108).

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S phase and control of DNA replication

S phase is the period in which the cell replicates its DNA. The length of S phase
varies from species to species; however, the duration of S phase in a given cell type is
relatively constant. This suggests a pattern of regulated events in S phase. Recent
studies in mammalian systems have shown that specific regions of the chromosome,
which contain clusters of replication units, replicate at different times; furthermore, it
appears that transcriptionally active genes replicate early in S phase. This pattern is
referred to as the spatial and temporal order of S and may vary between species (107).

In S. cerevisiae, some origins were first defined as autonomously replicating
sequences, ARS, which were found to bind a six-subunit protein complex called the
origin recognition complex (ORC). ORC is found associated with ARS elements
throughout the cell cycle but only functions to initiate replication in S phase. Changes in
the DNase I footprint of the origin in various stages of the cell cycle suggest that
additional factors are needed for initiation of replication. Candidates for regulating the
activity of ORC include cdc6, cdc7, and the MCM (mimichromosome maintenance)
proteins. The group of six MCM proteins has been implicated as the licensing factor (see
below) (40). Furthermore, an ATP-dependent, 3’->5’ helicase activity has been found
associated with MCM4, 6, and 7 proteins (96).

Blow and Laskey (15) first proposed the concept of licensing factor in
experiments with Xenopus eggs. In these experiments, nuclei functioned as independent
units and could not reinitiate DNA replication without an intervening mitosis. However,

permeabilization of the nuclear membrane, in the presence of cellular extracts, allowed

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re-replication without mitosis. This suggested that a cytoplasmic factor ‘licensed’ the
DNA for replication and this factor was destroyed or inactivated during replication. Only
during mitosis, when the nuclear envelope breaks down, would the licensing factor
become available again. The model of licensing explains how cells prevent re-initiation
of DNA replication (177).

While origins of replication have been defined in yeast systems, identification of
origins in mammalian cells has been more difficult. One of the best-characterized
mammalian origins is that of the DHFR gene, aided by a cell line (CHOC400), which
contains 1000 copies of the DHFR gene. Mapping studies have resulted in defining a
clear initiation zone in which preferred origins are used (107). Recent studies carried out
by D. Gilbert suggested that the decision to use a particular origin over another in the
DHFR initiation zone may be set early after mitosis, within 3-4 hours, and was referred to
as the origin decision point (ODP) (202). Before the ODP, initiation of replication was
seen throughout the DHFR region, while after the ODP initiation was localized to a
single origin of replication. The ODP appears to be controlled by a mitogen-independent
protein kinase that is activated after licensing but prior to the restriction point (203).
Furthermore, transformation by SV4O disrupts the normal pattern of initiation sites at the
DHFR locus (204).

Two other checkpoints have been defined at the transition of S to G2. These are
the S-phase checkpoint that ensures the completion of DNA synthesis and the DNA
damage checkpoint that repairs DNA damage before entry into mitosis. Many of the
genes involved in these two checkpoints are conserved between both yeast species, and

some human homologues have been identified. In S. pombe, the checkpoint kinase,

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Rad3, is the target for both checkpoints; however the downstream targets differ
depending on whether the S-phase arrest or the DNA damage checkpoint is being
activated. On the other hand, the activated checkpoint kinase in S. cerevisiae, Mecl ,
targets Rad53 while the upstream effectors differ between the two pathways (176). The
human homologue of the central checkpoint kinase of both yeasts is the ATM gene. One
downstream target of ATM is p53 suggesting a role for p53 in the GZ/M point as well as

the G1 DNA damage checkpoint (58).

G2/M transition and the spindle checkpoint

The G2/M transition is largely controlled by the status of cyclinB/p34“lcz (see
Figure 1-3) (136). Entry into mitosis requires association of cyclin B with p34 and the
subsequent activation of the p34 serine/threonine kinase activity. Cyclin B levels rise
throughout the S and G2 phases and peak during mitosis. Prior to the onset of mitosis,
the cyclinB/p34 complex is in its phosphorylated inactive state. Dephosphorylation of
T14 and Y15 by cdc25 phosphatase activates the kinase activity of p34 and promotes
initiation of mitosis. Nuclear localization of the Cyclin B/p34 complex also accompanies
entry into mitosis (97). When DNA replication is incomplete or DNA is damaged the
CDC25 phosphatase is inhibited leaving cdc2 in its phosphorylated inactive state;
furthermore, stabilization of cytoplasmic cyclin B also occurs. Finally, ubiquitin
mediated degradation of cyclin B by the anaphase-promoting complex, APC, is required

for the onset of anaphase (101).

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Mitosis can be divided into 5 distinct phases: prophase, prometaphase,
metaphase, anaphase, and telophase. Chromosomes are condensed during prophase,
positioned during prometaphase, moved to a midplane to form a metaphase plate in
metaphase, and separated during anaphase. The reformation of nuclei occurs in
telophase. Finally, cell division (cytokinesis) follows mitosis (123).

The spindle checkpoint insures proper segregation of chromosomes by the mitotic
spindle apparatus. Proper segregation of chromosomes requires that the two kinetochores
of each sister chromatid become attached to opposite spindle poles (134). When the
spindle apparatus is attached properly, the appropriate amount of tension is present and
anaphase progresses. If the kinetochore-microtubule interactions are incorrect, the
spindle checkpoint becomes activated (85). Proteins suspected to be involved in this
process in S. cerevisiae include protein phosphatase 2A (199), BUB (budding uninhibited
by benomyl) (94), and MAD (mitotic arrest defective) (200). p53 has also been
implicated (48). In some cases, mitotic arrests are not indefinite, and cells will eventually
undergo mitosis or apoptosis. Cells that undergo mitosis may result in aneuploidy and

transformation (156).

The polyomavirus lm’c cycle and neoplastic transformation

The polyomavirus lytic cycle

The virus life cycle can be divided into several distinct phases. These include
attachment, penetration, and uncoating of the virus, the early phase, and the late phase

(1). The mechanism by which polyomavirus enters the cell remains to be elucidated;

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however, it is known that the virus recognizes specific sugar residues on the host surface
(69), specifically N-acetyl neuraminic acid, or sialic acid (175). After entry the virus is
seen in monopinocytotic vesicles (82, 83); however, the exact nature of how the virus
penetrates the cell is not known. A recent publication out of Tom Benjamin’s lab (73),
reports that agents which block caveolae- and clathrin- mediated entry, mechanisms by
which SV40 and the related I C virus enter cells, have no effect on polyomavirus
infectivity. Therefore, how polyomavirus is directed to the nucleus remains to be
determined. Experiments with SV40 (41 , 206) have shown that the nuclear pore complex
is involved in the nuclear targeting of SV40 virions. Furthermore, the size differential
between the SV40 virion and the nuclear pore suggests that partial uncoating of the virion
may occur before nuclear entry. Virions are not completely uncoated until they reach the
nucleus (1). After infection, nuclear accumulation of the virus is seen within 15 minutes,
and virus continues to accumulate for up to 10 hours with a peak at 4 hours post infection
(118, 201). The timing of entry of the virus into the nucleus appears to be the same
whether permissive or non-permissive hosts are infected ( l 18).

The early phase, when only non-structural proteins are synthesized, of the lytic
cycle precedes DNA synthesis, and the late phase begins with the initiation of viral DNA
synthesis and continues until cell death occurs (1). Early attempts to investigate the lytic
cycle in relation to the host cell cycle focused mainly on the time in which the host cell
was most susceptible to polyomavirus infection and when viral DNA synthesis occurred
(113, 188, 189). In these experiments, it was found that infection of GI cells resulted in
the maximum yield of virus output (188, 189). Viral DNA synthesis occurred during the

first S phase when infected in GO or early 61; however, when cells were infected in late

 

 

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G1 or S phase, viral DNA synthesis was delayed until the following S phase (113). A
few experiments (86, 195) have shown that infection with polyomavirus can induce
cellular DNA synthesis during a productive infection, but few experiments have
investigated the effects of polyomavirus infection on the host cell cycle in detail.

Recent work in our lab (37) has further characterized the timing of the events of
the lytic cycle in synchronized cells. In these experiments, large T mRNA was seen as
early as 6 hours post infection and protein was detected by western blot by 8 hours post
infection. Viral DNA replication did not initiate until mid-S phase, about 16 hours post
infection. Shortly after the onset of viral DNA replication a large increase was seen in
both early and late gene transcription. VPl protein was detected by 22 hours post
infection. Of note was the delay of both small T and middle T to the late phase of the
infection. Progeny virions were detected by 24 hours post infection, which occurs shortly
after the expression of WI. The precise mechanism by which virions are released from
infected cells is not known; furthermore, many of the produced virions remain cell
associated even after cell death (1).

Experiments with a small T and middle T defective virus indicated four distinct
roles for these proteins in the virus life cycle (38). First the absence of small T and
middle T resulted in a large defect in large T transcription and expression during the early
phase, and a delay of 4 to 6 hours compared to wild type. Second, the absence of small T
and middle T resulted in a decrease in the accumulation of viral genomes, anywhere from
5 to 100 fold. Third, the increase in transcription of both early and late transcripts at the

early to late switch was delayed and dramatically decreased. Finally, a lower yield of

10

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virions was observed. These results not only help define the kinetics of the lytic infection

but also to further define the role of small T and middle T in the viral life cycle.

Neoplastic Transformation

While polyoma infection of permissive cells generally leads to a lytic infection
and cell lysis, infection of non-permissive cells, with little or no virus production, or
semi-permissive cells, with limited numbers of the population producing virions, results
in transformation. Virally transformed cells express the early proteins but do not amplify
viral DNA to the same extent as in lyrically infected cells or produce capsid proteins
(190). Recent work from our lab has shown that in situ amplification of integrated
polyomavirus genomes does occur and requires the presence of both the large T protein
and reiteration of viral DNA sequences at the integration site (182). In polyoma, the
transformation of established cell lines only requires the expression of the middle T
protein (191). However, for primary cells, the expression of large T may also be required
(149). After the initial steps in transformation, it appears that the expression of large T is
no longer necessary while continued expression of middle T is needed for the stable
maintenance of the transformed phenotype (105).

Transformed cells exhibit a variety of properties that are not seen in their
untransformed counterparts and which aid in their selection. For example, they exhibit a
loss of contact inhibition that allows for selection of dense clones growing on top of the
monolayer. They show a decrease in requirement for exogenous serum, presumably by

an increased ability in nutrient uptake, which allows selection of colonies growing in

11

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serum-depleted medium. They also acquire the ability to grow in semisolid medium,
which is the basis of agar assays. Other properties of transformed cells include a loss of a
defined actin cable network, altered cell morphology, an increase in protease production,
and the ability to form solid tumors in animals (81, 190).

Early experiments demonstrated that the efficiency of transformation with SV40
was higher when cells were infected in quiescent cells versus the proliferative state (121).
Experiments carried out in our lab with polyoma have demonstrated similar findings (36).
In these experiments Fischer rat (F R3T3) cells were found to be most susceptible to
polyomavirus infection following release from GO. Infecting in G0 maximized viral gene
transcription and DNA replication, which was delayed to the second G1. Furthermore,
the transformation frequency was greatly increased, an average of 20-fold, when cells
were infected in the quiescent state compared to the proliferative state. In these

experiments, no alteration of the host cell cycle was observed by polyomavirus infection.

Properties of the Early and Late Genes

Early genes

Large T (LT) is a multifunctional protein (see Figure l-4). It plays a role in
initiation of viral DNA replication, regulation of viral transcription, integration, and
transformation. It has also been shown to induce cellular DNA synthesis and
immortalization. Furthermore, unlike middle T, LT is largely nuclear (161, 186). Two

nuclear localization signals have been identified, one at residues 189-195 and the other at

12

residues 280-2
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residues 280-286, and are important for LT function (93). These two sites may act
cooperatively as mutation of one does not completely prevent nuclear translocation but
only diminishes it (152).

Like SV40, polyoma large T protein has several properties that contribute to its
replication function. First, it binds the polyoma origin (ori) in a sequence specific
manner, and the DNA binding domain of LT maps to amino acid residues 282 to 398
(181). Mutation of the palindrome in the origin to which LT binds results in a defect in
replication; however, this defect can be overcome by mutation of aspartate 286 to
asparagine in the DNA binding domain of LT (185). Mutants that are defective for DNA
binding still exhibit other LT firnctions such as imrnortalization (46, 92). Second, large T
also has an ATP dependent helicase in the 3’ -> 5’ direction that results in SSB-
dependent unwinding in a polyoma ori-specific manner (168, 198). The ATP binding
domain of LT maps to amino acids 565-675 (20). ATP binding also induces hexamer
formation, which is required for DNA replication (197). Finally, LT has been shown to
be associated with zinc, and the zinc-binding motif is located around amino acid 452
(1 l). Mutants in the zinc-binding motif show a decreased capacity to bind and replicate
DNA and show defects in multimerization and phosphorylation (154). While zinc
mutants exhibit normal transactivation of both early and late genes, they are defective for
inhibition of early transcription (13).

Initiation of replication from the polyomavirus origin by large T is thought to
occur in a similar manner to that of SV40. First, large T binds as a double hexamer to the
origin and catalyzes the local unwinding of the viral genome in an ATP dependent

Inantler. Initiation of replication proceeds afier large T recruits DNA polymerase a-

13

 

pinnase and RE
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LT als
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primase and RP-A to the initiation site. The helicase activity of large T unwinds the viral
genome ahead of replication fork movement in the 3 ’->5’ direction (59).

LT also has two main effects on the host cell: induction of S phase and
immortalization. Like SV40, polyoma LT binds to Rb (56). LT’s association with Rb
results in the up-regulation of a variety of E2F responsive genes including dihydrofolate
reductase, thymidine kinase, thymidylate synthetase, DNA polymerase alpha, and PCN A,
which results in the induction of S phase (130). Furthermore, smaller doses of LT are
sufficient for induction of S phase than the amount required for origin-mediated viral
DNA replication and transactivation (138). Reports from Brian Schauffhausen’s lab have
separated LT into two discrete portions: the C-terminus, CT, and the N-terminus, NT.
The CT was able to support viral DNA replication but only in the presence of 10% serum
medium. NT could complement viral replication in low serum by its ability to drive cells
into S phase (74). The Rb binding domain localizes to the N-terminal domain of LT (92).
The DNA J domain, which is also located in the N-terminus, is required for regulation of
Rb function. Mutants that no longer bind DNA J are still able to bind Rb but lose their
ability to transactivate E2F responsive genes and induce S phase (170). Mutants
defective for Rb-binding fail to grow in cells that express wild-type Rb because of a
delay in viral DNA and protein synthesis, presumably because of a failure to induce S
phase (66). Recently, an association of LT with she has also been reported which may
lead to activation of S phase in an Rb independent manner (79).

The association of Rb is also implicated in the ability of LT to immortalize
primary cells (150). The N-terminal portion of LT was shown to bind Rb and p107;

firrthermore, it was sufficient to induce immortalization (92). Mutants that fail to

14

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associate with Rb are defective in immortalization; furthermore, the ability of LT to
induce immortalization is serum independent but requires continuous LT expression
(106). This is unlike the role in transformation where LT increases the efficiency of
transformation, presumably by playing a role in viral integration (52), but is not needed
for the stable maintenance of the transformed phenotype (63). However, in some cases,
Rb mutants were found to be normal for transformation despite being defective for
immortalization (67).

LT is phosphorylated on variety of residues (162). Two regions of
phosphorylation were identified: one at the N-terminus (up to amino acid 260) and one
in the C-terminus (16). This phosphorylation is cell cycle dependent; resting cells are not
capable of phosphorylating LT; furthermore, the hr-t mutants have a slightly lower level
of phosphorylation (17). The S phase cyclin A/cdk2 and the G2/M cyclin B/cdc2 but not
the G1 cyclin E/cdk2 are able to phosphorylate LT on tyrosine 278 (110). Mutants at
tyr278 are no longer able to bind and unwind DNA, therefore affecting viral DNA
replication, while induction of S phase is normal. Other phosphorylation sites include
serine residues 271, 274, and possibly 267 (35). Studies on the effect of phosphorylation
of sites in the N-terrninus demonstrated that while the removal of some phosphates
inhibited viral DNA replication, removal of other phosphates actually increased
stimulation of viral DNA replication (196). Furthermore, the increase in phosphorylation
of LT increases the affinity of LT for the nuclear matrix possibly defining the importance
of LT phosphorylation in DNA replication (23, 95). The nuclear matrix has been
implicated as a site of viral DNA replication (23). Similar results were seen with SV40

LT (31, 124, 125, 147, 165, 173). Phosphorylation of threonine 124 promotes binding to

15

the origin an:
123 inhibitsr

finall
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the origin and stimulates viral DNA replication while phosphorylation of serines 120 and
123 inhibits viral DNA replication.

Finally, an association with the p300 transcriptional co—activator, which has
several activities including activation of transcription, activation of histone
acetyltransferase, and, possibly, chromatin remodeling, was recently reported (133). This
interaction occurred via the C-terminal portion of the LT protein. However, these

experiments were done in differentiated cells and may not be extrapolated to other cell

types.

The polyomavirus middle T protein induces a signal transduction cascade similar
to that of the activated PDGF receptor (see Figure 1-5). The mT protein becomes
associated with the cytosolic face of the plasma membrane, and the carboxyl terminus of
mT is essential for this membrane localization (29). At the membrane surface, mT
interacts with pp60°'s‘° and other members fiom the c-src tyrosine kinase family, and this
interaction is responsible for the observed increase in the specific activity of these
proteins (19, 42, 44, 45). Associations with c-fyn (39) and c-yes (103) have also been
reported and may contribute to the transformation process (187). The increase in kinase
activity is associated with an increase in phosphorylation of the pp60°"“‘ protein (30).
Activation of the pp60°'SIC tyrosine kinase results in phosphorylation on tyrosine residues
250, 315, and 322 of the middle T protein (88). These phosphorylated sites can then
serve as docking sites for other cellular proteins that have SH2 domains (Figure 1-5) and

thus transmit the signal to different branches of the signal transduction pathway.

16

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One of the proteins that bind the mT-pp60c’snc complex is she. This association is
mediated by the NPTY (asn-pro-thr-tyr) motif of the mT protein, which includes Tyr-
250, and an SH2 domain of the she protein; furthermore, this interaction results in
phosphorylation of the shc protein (28, 54). Phosphorylation of shc stimulates its
interaction with Grb2 via SH2 domains (114, 155). The SH3 domains of Grb2 recruit
sosl to the activation complex; subsequently, the sosl protein converts inactive GDP-
bound ras to its active GTP-bound state (122). Stimulation of ras activity results in
activation of a kinase cascade, which begins with rafl, by recruitment to the cytoplasmic
membrane, and ends with MAP kinase which targets nuclear transcription factors (144).
Middle T mutants that no longer associate with shc are defective for transformation (54).

The mT-pp60°'s"° complex interacts with and activates a phosphatidylinositol
kinase as well. A role in transformation for this association has been implicated, and is
becoming better characterized. Phosphorylation of mT on tyrosine 315 causes increased
binding of the 80K subunit of phosphatidylinositol 3-kinase (PI3K) (4, 43, 99, 140, 184),
and cells transformed with mT show increased levels of several inositol phosphates such
as inositol 3-phosphate, inositol 3,4-bisphosphate and inositol 1,3,4-triphosphate (193).
While the role of these intermediates in the transformation process is not clear, the
interaction between mT-pp60°'s“’ and phosphatidylinositol 3-kinase is required for
induction of the transformed state (184).

The downstream targets of the mT-pp60c'sm-PI3K complex are becoming
elucidated. Like she, PI3K was shown to activate MAP kinase and induce its
translocation to the nucleus (194). Association of middle T with PI3K also leads to

activation of AKT, another potent oncogene, via phosphorylation of AKT on S473 (180).

17

mnkThE
independent
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Middle T has been shown to activate the ribosomal S6 kinase by src-dependent and src-
independent means (183). The phosphorylation of ribosomal S6 in middle T expressing
cells was later shown to occur through activation of PI3K (50).

Recently, it has been suggested that the she and PI3K pathways act in a
cooperative manner to induce transformation by middle T. Mutants defective for binding
she have a decreased ability to form transformed foci and do not support growth in soft
agar, but exhibit a normal tumor profile in mice. Mutants defective for association with
P13K exhibit a decreased ability to form transformed foci and support growth in sofi agar;
furthermore, they also show a decrease in tumor frequency, and an increase in latency, in
mice. Double mutants are defective for both focus formation and growth in agar;
furthermore, they exhibit a reduced frequency and increased latency in tumor formation
in mice (22). The requirement for PI3K, via AKT, in tumor formation may relate to its
ability to inhibit apoptosis in transformed cells (51). This anti-apoptotic property of
middle T is independent of p53.

Other studies have shown that the mT protein can increase cellular levels of [Pg
(78). This phenomenon is most consistent with an activation of phospholipase C gamma
(PLC'y) by the mT-pp60c's"c complex. Further support that mT activates PLCY comes
fiom studies that show an increase in protein kinase C activity in response to mT
expression (119). Recently, this interaction was shown to be mediated by
phosphorylation of tyrosine 322 of the mT protein (179). Activation of PLCy results in
cleavage of inositol 4,5-bisphosphate to yield diacylglycerol and inositol triphosphate
(1P3). 1P3 is responsible for an increase of intracellular calcium released from internal

stores, and the increase in calcium together with the increase in diacylglycerol (DG),

18

levels results in the activation of protein kinase C (PKC). PKC also leads to activation of
nuclear transcription factors such as AP-l and c-ets (100) and may provide one possible
mechanism by which middle T can activate these nuclear transcription factors.

Phosphorylation of serine 257 of the middle T protein has also been reported
(49). The phosphorylation of serine 257 results in association of middle T with 14-3-3
proteins. Mutants at S257 have a slight defect in complex formation (169) but have a
fairly normal phenotype with the exception of a defect in the ability to induce salivary
tumors. Slight differences in the ability of middle T to act as a transcriptional activator in
transient assays also show some differences (49, 141). The 14-3-3 proteins are a diverse
set of proteins whose downstream targets are not clear. It has been shown that 14-3 -3
may regulate rafl by stimulating its kinase activity (60). 14-3-3 proteins have also been
implicated in PKC regulation (2). Some members are required for the DNA damage
checkpoint in yeast and have been implicated in the control of mitotic progression (65,
91). Phosphorylation of middle T by cdc2 on threonine residues 160 and 291 has been
demonstrated during mitosis and has been shown to be important for transformation
(145). It would be interesting to see if the association of middle T with the 14-3-3
proteins affects the mitotic phosphorylation of middle T.

Oc-src

Finally, a stable complex between the mT-pp6 and protein phosphatase 2A
(PP2A) has been demonstrated which results in the association of phosphatase activity
with the complex (142). Amino-terminal regions of the mT protein appear to be essential
for the binding of PP2A (27, 75). Recent evidence suggests that PP2A may be required

for pp60°‘"° binding and activation; however, the role of the PP2A association is unclear

(76) as catalytically inactive mutants are still capable of supporting association with src

l9

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(1 37). On the other hand, mutants that are defective for association with PP2A are also
defective for association with src; however, some src mutants are still capable of
association with PP2A (21, 27, 7 5). Activation of the JNK kinase by middle T has been I

shown to be dependent on association of middle T with PP2A. Interestingly, small T,

which also associates with PP2A (see below), does not activate the JNK kinase (131).

Recently, a great deal of effort has been expended in investigating whether middle

T targets the activity of p53. This stems from tumor incidence studies and the fact that

other DNA tumor viruses alter p53 function (102, 108); furthermore, while the SV40 LT

antigen targets p53, polyoma LT does not (146). However, expression of polyoma large
T was able to overcome a p53 dependent growth arrest of MEF cells in an Rb dependent
manner (55). In polyoma middle T transformed REF52 cells, the p53 DNA damage
Checkpoint was found to be intact ( 127). Recently, it was reported that abrogation of p53
induced cell cycle arrest and apoptosis required coordinated effects of both polyoma
middle T and small T. The middle T effect appeared to be mediated through AKT, but

the role of small T is unknown (148).

The third early protein produced by polyomavirus is small T. It has been shown
to augment viral DNA replication in 3T6 fibroblasts (12). However, results obtained in
on!“ lab in which both small T and large T antigen levels were measured demonstrated
little, if any, effect of small T on replication initiated at a polyomavirus origin of
replication (24). Small T has also been shown to contribute to the lytic infection (192)
and transformation (120). Cells lines expressing small T have been shown to grow to a

higher density and exhibit some anchorage independent growth (135); furthermore, small

lhas also been

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T has also been shown to protect against tumor necrosis factor induced apoptosis in
mouse cells (14). These properties, while not sufficient for full transformation, probably
augment transformation by middle T. Its only known interaction is with protein
phosphatase 2A (27, 142). Small T replaces the B regulatory subunit in the PP2A
complex (157, 158). Similarly, SV40 small T also interacts with PP2A. This interaction
results in the inhibition of phosphatase activity and results in stimulation of the MAP
kinase pathway (70, 164, 172). Expression of small T has been shown to augment the
capacity of large T to induce S phase (139) and is required for elimination of p27 in
support of this S phase induction in a PP2A dependent manner (166). However, in these
experiments, little S phase induction was seen by the expression of large T alone,
probably due to the harsh method used to induce quiescence. In other experiments, the
interaction with PP2A was shown to be important for activation of the c-fos promoter and
subsequent induction of S phase in quiescent cells (131). In these experiments, middle T
was also shown to induce S phase, but this activity was dependent on phosphorylated
tyrosine signaling and not PP2A. The ability of small T to enhance viral DNA replication

probably relates to its ability to augment S phase induction.

Finally, recently a fourth early protein, tiny T was isolated. Its function in the
virus life cycle is yet unknown, but it does increase the ATPase activity of hsc70. It has
only the DNA] binding domain and has a short half-life, which may explain why it has

gone undetected (153).

21

Late Genes

The majc'.
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implicated in \‘ir
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Late Genes

The major capsid protein, W1, is composed of six distinct species with different
isoelectric focusing points, A-F (18). The differences are generated by post-translational
modifications including methylation, tyrosine sulfuration, calcium binding, hydroxylation
of proline, and phosphorylation (3, 61). Methylation occurs on lysine and arginine
residues (25). Tyrosine sulfuration is found on species B and F, which have been
implicated in virus attachment (117). All six species have been found to bind calcium
(116). The calcium-binding site is in the C-terminus, which has been implicated in
receptor recognition and assembly. Mutations in the calcium-binding domain have been
found to have a defect in capsid assembly (90). Inhibition of hydroxylation of proline
resulted in decreased nuclear transport of WI and decreased production of viral progeny
(115). Three species, D, E, and F, have been found to be phosphorylated on serine and
threonine with the majority phosphorylated on threonine. The threonine sites were
mapped to residues 63 and 156 with Thr63 being the major site of phosphorylation (111).
Mutation of threonine 63 to glycine did not affect virus assembly while mutation of
threonine 156 to alanine was defective in the production of stable virions. The hr-t
mutants are defective in phosphorylation of both residues and imply a role for middle T
in capsid phosphorylation (71). Phosphorylation of serine occurs on residue 66. Mutants
of serine 66 were not viable, but serine 66 has been shown to be phosphorylated by

Casein Kinase II in vitro (112).

22

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After synthesis VP1 is transported to the nucleus where viral assembly takes
place. The nuclear localization sequence of VP1 has been mapped to the N-terminus.
Truncation of the first 11 amino acids, or mutation of this region results in predominantly
cytoplasmic localization of VP1 (34, 128). A role for hsc70 in nuclear transport has also
been suggested (47). The DNA binding properties of VP1 also map to the extreme N-
terminus. DNA binding by VP1 does not appear to be sequence specific (32).

Some virulence determinants are also located in VP1. Mutation of glutamine to
glycine at amino acid 92 results in a small plaque virus (versus a large plaque virus) and
renders the virus defective in replication and tumor induction in the animal (68).
Mutation of valine to alanine at residue 296 results in a more virulent virus. This virus
kills infected mice within 2 weeks of infection. Both residues have been linked with
receptor binding affinity, specifically the ability to bind different forms of sialic acid (7).

Purified VP1 has been shown to assemble into capsid-like particles in vitro and in
viva (126, 160). However, complete virions also contain (the minor capsid proteins VP2
and VP3; firrthermore, productive infection requires the expression of both VP2 and VP3.
Modification of VP2 has also been reported (178). Myristylation of the N—terminus on
glycine plays a role in the efficiency of infection. Mutants blocked in VP2 myristylation
exhibit a defect in the early stages of infection, possibly decapsidation (104).
Replication, spread, and tumor induction in the mouse are also defective in myristylation
mutants (159). The nuclear localization sequence of VP2 maps to the 12 C-terminal
amino acids. Since VP3 shares this sequence, it is inferred that this sequence also
functions as a nuclear localization signal for VP3 (33). However, expression of VP1

facilitates nuclear transport of both VP2 and VP3 (53). Interactions between VP1 and

23

m3 occur be"

 

 

interactions ochl
cith either VP1 -
specific roles 11"

chromosome hat

The hr-t mutant

 

 

Host ran-i
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VP2/3 occur between the C-terminus of VP2/3 and the N-terminus of VPl (5). These
interactions occur before nuclear entry (26). No DNA binding activity has been seen
with either VP2 or VP3; however, VP2 has been shown to associate with histones. The
specific roles for all three viral proteins in appropriate packaging of the viral mini-

chromosome have yet to be elucidated except that all three are required (32).

The hr-t mutants

Host range mutants, called hr-t mutants, were originally isolated in an attempt to
identify viral gene products responsible for transformation. The assumption was made
that the same gene product would be necessary for the viral life cycle. The isolation
involved selection of mutant viruses that could grow on polyoma-transformed cells
(Py3T3) but not on normal 3T3 cells (see Figure 1-6). The premise was that the wild
type function expressed in the transformed cells would complement the mutant gene. Out
of one thousand isolated plaques, only four displayed the properties of interest (9). This
set of four was later expanded with 15 additional host range mutants (8). Furthermore,
another set of mutants was isolated using mouse embryo fibroblasts as the permissive
host and 3T3 cells as the non-permissive host. The five mutants isolated in this screen
are indistinguishable from those isolated previously (62). The complete set of 25 exhibits
the following properties: 1) they are unable to induce transformation in cell culture and
mice (8), 2) they form a single complementation group (174), which is distinguishable
from the ts-a mutants (57, 64), and 3) they all have mutations in the proximal portion of
the early region (89). Cells other than Py3T3, which are permissive, include primary

baby mouse kidney epithelial cells, primary mouse embryo fibroblasts, and Rous

24

Sarcoma vir
BALB cell 1
permissive
Furherrnore
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Sarcoma virus transformed cells. Cells that are not permissive include 3T3 cells, 3T3-
BALB cell lines, and SV40 transformed mouse cell lines. These results indicate that the
permissive state for these mutants relied heavily on the state of the host cell (77).
Furthermore, it suggests that the transformation property of the virus is merely a by-
product of the gene needed to induce a permissive state for viral growth.

Most of the hr-t mutants have deletions in the intron of the LT gene sequence
which result in a lack of expression of both middle T and small T due to an out of frame
deletion (163). NG59 has an insertion of three nucleotides followed by a G->A transition
which results in the absence of small T and the expression of a normal sized middle T
(10). The NG59 mutant was found to lack associated protein kinase activity (8). All
mutants express a normal sized large tumor antigen (163). The defect in the hr-t virus
life cycle in the non-permissive host was found to be a defect in the maturation of the
virus. No differences in the synthesis or accumulation of capsid proteins are seen in hr-t
versus wild-type infections. Modest decreases in viral DNA synthesis are observed, but
this decrease is not enough to explain the defect in production of infectious progeny. The
major defect was found to be in the encapsidation of the viral minichromosome (72).
This defect was found to correlate with the inability of hr-t mutants to phosphorylate

threonine of VP1 (71).

25

Summm

’

This literl
rhe effects of th
study began witl

Bl. resulted in a

 

led to assigning

observations mat

Summary!

This literature review has focused on the progression of the normal cell cycle and
the effects of the polyomavirus on some of the events in cell cycle progression. This
study began with a simple observation that cells infected with a particular hr-t mutant,
BZ, resulted in an accumulation of cells in G2/M. Examination of this phenomenon has
led to assigning a new property to VP1 and has provided explanations of some early

observations made with the hr-t mutants.

26

APPENDIX 1: TABLES AND FIGURES FOR CHAPTER 1

27

Figure 1-1: Physical map of the polyomavirus genome.

The inner circle represents the restriction endonuclease sites of Hpa II/Msp I on
the polyomavirus genome. The early and late coding regions are shown in the outer
circles. The early regions are differentially spliced from a single precursor transcript to
yield small T, middle T, and large T. VP1, VP2, and VP3 are similarly produced. The
splice sites are indicated and jagged lines represent the introns. The non-coding region,
located on the late side of the origin of replication, is also depicted. This figure is a

modification of figure lb from Soeda eta]. 1980.

28

5'10 ”K

 

 

Table l-I: Mammalian cyclins and associated cdk proteins.

The 16 identified mammalian cyclins are listed. The cyclin dependent kinases

(cdks) found in association with the various cyclins along with their functions in the

regulation of the cell cycle are also noted. This table is taken from Johnson and Walker

1999.

30

A c

Cyclins

81.82

(‘2

 

 

 

01, 03

 

Cyclins Associated cdk Function

A cdkl(cdc2), cdk2 S phase entry and transition,
Anchorage-dependent growth

Bl, BZ cdkl GZ exit, mitosis

C cdk8 Transcriptional regulation,
GO-to-S-phase transition

D1, D2, D3 cdk4, cdk6 G0—to-S-phase transition

E cdk2 Gl-to S-phase transition

F ? G2-to-M-phase transition

G1, GZ cdkS DNA damage response

J cdk7 cdk activation, transcriptional regulation,
DNA repair

I ?

K ? Transcriptional regulation,
cdk activation

Tl , T2 Cdk9 Transcriptional regulation

 

31

Figure 1-2: Restriction point control through Rb.

Phosphorylation of Rh by cyclin D/cdk complexes results in the release of E2F
family members. The release of E2F results in its activation as a transcriptional activator
and genes such as cyclin A, cyclin B, dihidrofolate reductase (DHF R), thymidine kinase
(TK), and DNA polymerase a are induced. The induction of these genes leads to the

progression from 61 into S phase.

 

GD

 

 

 

 

 

 

 

 

 

Rb E2F
Cyclin D/cdks
® ®
Rb
Cyclin A
DHFR

33

DNA pol 0L

Figure 1-3: Regulation of cdc2 activity in the mitotic cell cycle.

Cyclin B associates with cdc2 during interphase. The cdc2 kinase does not
become active until residues T14 and Y15 become dephosphorylated. This occurs during
the transition from 02 into mitosis. During anaphase, cyclin B is degraded resulting in

the deactivation of the cdc2 kinase. This figure is taken from Norbury and Nurse 1992.

 

 
  
 
  
  
 
 
  
    

 

PP2A? , ’
/ /
’

INACTWE

and, runs
p34

 

 

 

 

T161

cyan 8
degradation

ACTIVE

 

 

 

 

35

Figure 1-4: Organization of the domains of polyomavirus large T.

Polyomavirus large T landmarks include two nuclear localization signals, the Rb,
zinc, ATP, and DNA binding domains, and two regions of phosphorylation. The N-
terminal domain and the C-terminal domain are defined by limited proteolysis. This

figure is taken from GjQrup et a1 1994.

36

 

 

16 Rb!

he N-

C-TEIW DOMAIN (CT)
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37

Figure 1-5: Middle T signaling pathways.

Depicted are the various domains and residues of polyomavirus middle T. The

signaling cascades induced by middle T are shown.

Christopher Ontiveros.

38

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39

Figure 1-6: Isolation of the polyomavirus hr-t mutants.
A schematic representation of the isolation of polyomavirus hr-t mutants is
shown. A wild-type virus was mutagenized, and the hr-t mutants were isolated based on

their ability to grow on polyoma-transformed cells but not normal 3T3 cells.

40

transformation

 

3T3 # Py3T3
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specific sialyligosaccharide receptors on host cells Virology. 109:188-92.

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protein VP1 before viral DNA encapsidation J Virol. 54:311-6.

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7.

47

74.

75.

76.

30.

Gilbq
cells

Gjor
1994.
replic

Glen]
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Tharne, H. V. 1973. An investigation of factors influencing the susceptibility of
Balb-c 3T3 cells to polyoma virus J Gen Virol. 18:153-62.

Tapp, W. C., D. Lane, and R. Pollack 1981. Transformation by SV40 and
Plyoma Virus, p. 297-338. In J. Tooze (ed), DNA Tumor Viruses, Second
Edition ed. Cold Spring Harbor Laboratories, Cold Spring Harbor.

Treisman, R., U. Navak, J. Favalara, and R. Kamen 1981. Transformation of
rat cells by an altered polyoma virus genome expressing only the middle-T
protein Nature. 292:595-600.

Turler, H., and C. Salomon 1985. Small and middle T antigens contribute to
lytic and abortive polyomavirus infection J Viral. 53:579-86.

Ulug, E. T., P. T. Hawkins, M. R. Hanley, and S. A. Caurtneidge 1990.
Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T
antigen J Virol. 64:3895-904.

Urich, M., M. Y. e1 Shemerly, D. Besser, Y. N agamine, and K. Ballmer-Hafer
1995. Activation and nuclear translocation of mitogen-activated protein kinases
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Biol Chem. 270:29286-92.

Vagt, M., R. Dulbecco, and B. Smith 1966. Induction of cellular DNA synthesis
by polyoma virus. 3. Induction in productively infected cells Proc Natl Acad Sci
U S A. 55:956-60.

Wang, E. H., S. Bhattacharyya, and C. Prives 1993. The replication functions

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57

197.

198.

200.

201.

202.

203.

:64.

Wang, 1
tumor ar

Wang.
unwindi
similarit

Wang.

protein 1
kinetocl‘
17:62 0-

Waters
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detectio

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11101186 1

“h.J.
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111110 g9
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197.

198.

199.

200.

201.

202.

203.

204.

205.

206.

Wang, E. H., and C. Prives 1991. ATP induces the assembly of polyoma large
tumor antigen into hexamers Virology. 184:399-403.

Wang, E. H., and C. Prives 1991. DNA helicase and duplex DNA fiagment
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similarities and differences J Biol Chem. 266: 12668-75.

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Waters, J. C., R. H. Chen, A. W. Murray, G. J. Garbsky, E. D. Salmon, and
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virus 40 nuclear targeting J Virol. 67 :1 19-30.

58

POLYO
ARREST

“e11
hnetics of N
hI-t mutants.
With the 82 n
dcMdent b
91017111 cell ),
Expression of
Cycle arrest.
DNA Sl'Tlthesi
arrest tracks
FWBUHore,

INTRODEC

 

 

 

CHAPTER 2

POLYOMAVIRUS HR-T MUTANTS INDUCE A MITOTIC CELL CYCLE
ARREST THAT IS NOT ABROGATED BY THE EXPRESSIN OF SMALL T
AND/OR MIDDLE T

ABSTRACT

We have been studying the effect of polyomavirus infection on the cell cycle
kinetics of NIH3T3 cells. Here we describe the effect of the small T/middle T defective
hr-t mutants, in particular B2, on the cell cycle of NIH3T3 and FR3T3 cells. Infection
with the BZ mutant results in an accumulation of cells with G2/M DNA content in a dose-
dependent but serum-independent manner. At high multiplicities of infection
(>10pfu/cell), 50-80% of the population becomes arrested with G2/M DNA content.
Expression of small T and/or middle T either in cis or in trans does not abrogate this cell
cycle arrest. We show that the cell cycle arrest induced by B2 relates neither to viral
DNA synthesis out of S phase nor to the amplification of viral genomes. The cell cycle
arrest tracks with the over-expression of VP1 and appears to be mitotic in nature.
Furthermore, the ability to induce a G2/M cell cycle arrest appears to be a common

feature of several hr-t mutants.

INTRODUCTION

The polyomavirus hr-t mutants were originally isolated in an attempt to define the

transforming gene of the virus. They were selected for their ability to grow on polyoma-

59

 

Most o

S9Valence Whit
0111mm dele‘
flamition tha
However, \he
121. All mu}
£1:in t0 ha
capsid Proti
the defect i
amenP’sldat

transformed cells but not untransformed 3T3 cells. The assumption made was that the
gene functions of the virus that were important for the ability of the virus to induce
neoplastic transformation would also be important for virus growth. While all the
mutants isolated are completely defective for their ability to transform cells, the growth
properties of the viruses isolated depends largely on the host cell. For example, some
primary and established cells are capable of supporting virus grth while others are not;
furthermore, the same is true for transformed cells (2).

Most of the hr-t mutants have deletions in the intron of the large T (LT) gene
sequence which results in a lack of expression of both small T and middle T due to an out
of frame deletion (21). NG59 has an insertion of three nucleotides followed by a G->A
transition that results in the expression of a normal sized small T and middle T (4).
However, the NG59 middle T protein was found to lack associated protein kinase activity
(2). All mutants express a normal sized large tumor antigen (21). The hr-t mutants were
found to have a maturation defect. Modest decreases in viral DNA synthesis and viral
capsid protein synthesis were observed, but these decreases were not enough to explain
the defect in production of infectious progeny. The major defect was found to be in the
encapsidation of the viral nrinichromosome (13). This defect was found to correlate with
the inability of hr-t mutants to phosphorylate threonine and serine of VP1 (12).

We have been investigating the effect of polyomavirus infection on the kinetics of
cell cycle progression in NIH3T3 cells. Infection with a particular hr-t mutant, BZ,
induces a G2M cell cycle arrest. Here we characterize this arrest in both NIH3T3 and rat
FR3T3 cells and describe the possible causes of the arrest in both cells. We also expand

the data to include other members of the hr-t class of virus mutants.

60

MATERIALS

Wild tj
been descnber
base pair dele

deletion of 24

 

 

MATERIALS AND METHODS

Yirusea

Wild type A2 (24) and the A2 derived sT/mT defective A185 mutant (17) have
been described previously. The B2 mutant used in this study contains a repair of the 11
base pair deletion in the early region from the original isolate of 82 (16), which has a
deletion of 241 base pairs in the large T intron. The thZ strain was constructed by
replacement of nucleotides 400 to 1079 of BZ with the corresponding A2 DNA
sequences. The other hr-t mutants NG18, NG59, and their rescued wild-types 18R4 and
59RA, and NG23 have also been described (1, 10). Virus stocks were prepared in
NIH3T3 or 3T6 cells and checked for the absence of defectives. Titers were calculated
using standard plaque assay techniques and by comparison of input DNA.

Cells and infections.

NIH3T3 and FR3T3 (Fischer Rat) cells were cultured in Dulbecco’s modified
eagle medium (DMEM; GIBCO) supplemented with 10% heat inactivated bovine calf
serum (GIBCO, SIGMA) at 37°C and in 5% CO;. For induction of quiescence and
release from G0, two protocols were used. Protocol 1: Cells were grown to 90%
confluence followed by 24 hours starvation with 0.5% serum medium. Cells were
released fiom G0 by re-plating at a lower density in 10% serum medium. Adsorption of
virus was carried out after reattachment (4 hours) for one hour. Protocol 2: Cells were
seeded at sub-confluent levels and incubated in 0.5% serum medium for 24 hours,

infected for one hour, and released from G0. Unless otherwise indicated all infections

61

were carried on

 

cell. and the int
FAC S Analvsis

At the II
of phospho-bi;
80% ethanol t1
out Cells We

Iimmature v.

65ml EDTA

 

96“ assaired f
(31.8. and Q:
ham

infect.
sulfate (SDs

manage K

were carried out at a multiplicity of infection (MOI) of 10 plaque-forming units (pfiJ) per
cell, and the infection lysate was removed prior to the addition of 10% serum medium.
FACS Analjgis.

At the time of sampling, cells were treated with trypsin and re-suspended in 1 ml
of phospho-buffered saline (1XPBS) supplemented with 2% serum and fixed in 10ml
80% ethanol for 30 minutes on ice. Samples were stored at 4°C until analysis was carried
out. Cells were then washed once with 1XPBS and stained for 30 minutes at room
temperature with propidium iodide solution: 0.1mg/ml RNaseA, 0.1% triton-x-lOO,
0.5mM EDTA, and 0.05mg/ml propidium iodide in 1XPBS. A total of 5000 cells were
then assayed for DNA content using CellQuest by Beckton-Dickenson. Percentages of
G1 , S, and G2/M were calculated using Multiplus or WinCycle.

Preparation and analysis of ml DNA

Infected cells were lysed in lOmM Tris, 10mM EDTA, and 0.2% sodium dodecyl
sulfate (SDS) (pH7.6). Protein digestion was done overnight at 37°C with SOug/ml
proteinase K (Sigma). Total DNA was extracted with phenol-chloroform and treated
with RNaseA by standard procedures. Samples were digested with EcoR 1 (GIBCO
BRL) to linearize polyoma DNA, electrophoresed in 1% agarose, stained with ethidium
bromide to confirm equivalent loading, and blotted to Hybond membranes (Amersham).
Hybridization was carried out at 65°C in 1X Denhardt’s solution, SXSSPE, and 0.5%
SDS with a 32P-radiolabeled probe, consisting of the polyomavirus A2 genome.
Hybridization probes were labeled to a specific activity of 1-2 x 109 chug with (32F)

dCTP (3,000 uCi/mmol; New England Nuclear) using a multiprime DNA-labeling kit

62

(.tmershar
X-ray film

.

Inf-
31100 glyce
electmpho
blotted to
116161011611-
Pfimm 3:
(rabbit ant:
lgoat anti;
anil-rabbit
We used
118ng SUp
{Amershar
imtI'UCtiOr
primary 3

Chemicon

(Amersham). Hybridized blots were washed using stringent conditions and exposed to
X-ray film (Amersham). Band intensities were determined by phosphorimaging.
Preparation and analfiis of proteins and immunoprecipitations.

Infected cells were lysed with sample buffer (5% SDS, 0.03% bromophenol blue,
20% glycerol, 5% B-mercaptoethanol) and boiled at 100°C for 5 minutes. Aliquots were
electrophoresed in 5% stacking/10% resolving polyacrylarnide (BIORAD) and electro-
blotted to PDVF membranes (N EN). Peritoneal or ascites fluid from rats, which had
developed tumors after injection with polyoma-transformed cells, was used as the
primary antibody to detect polyoma T antigens. Primary antibody for detection of VP]
(rabbit anti-VP1) was a gift from Robert Garcea. Primary antibody for detection of actin
(goat anti-actin) was purchased fiom Santa Cruz Biotechnology. Goat anti-rat IgG, goat
anti-rabbit IgG, and sheep anti-goat IgG coupled to horseradish peroxidase (PIERCE)
were used as secondary antibodies. For detection, chemiluminescense was performed
using SuperSignal solutions (PIERCE) and membranes were exposed to X-ray film
(Amersham). Irnmunoprecipitations were carried out according to manufacturers
instructions (Boehringer Mannheim Irnmunoprecipitation Kit-Protein G) using Sug
primary antibody. B-Tubulin (mouse anti-tubulin) antibodies were purchased from

Chemicon.

63

11811.18 1

 

Infection of .‘
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RESULTS

Infection of NIH3T3 cells by the hr-t mutant, B2, results in an accumulation of cells
with 2C DNA content.

The effect of polyomavirus infection on the kinetics of cell cycle progression of
NIH3T3 cells was analyzed over a 48 hour time period. Cells were synchronized by
induction of a quiescent state, infected at a MOI of 10 pfu/cell, and released from G0
with 10% serum medium as described in protocol 2. The analysis of uninfected cells
(Figure 2-1A) revealed a first S peak at 20 hours post release, with approximately 50% of
the cells in S phase. The end of the first round of DNA synthesis occurred by 24 hours
post release. At that time 37% of the population was in G2/M and 40% of the population
was in G1. A second S peak was seen at 36 hours post release with 42% in S phase, and
by 48 hours post release the majority of the population, 70%, had returned to G1.
Infection with wild type A2 (Figure 2-1B) or the small T/middle T defective A185
mutant (Figure 2-1C) did not alter the progression of the cells through the cell cycle.
However, infection with the sT/mT defective, hr-t type, B2 mutant resulted in a cell cycle
arrest at the GZ/M phase of the first cell cycle (Figure 2-1D). The cell population entered
S phase with the same kinetics as uninfected cells, with 50% of the population in S phase
at 20 hours post release. However, a fraction of the cells accumulated with 2C DNA
content, reaching 60% at 24 hours post release, 47% at 36 hours post release, and greater
than 50% at 48 hours post release. This accumulation represents an increase of 20%,

40%, and 30% over uninfected, A2-infected, and A185-infected at similar times. In these

64

experimer
CPE bega
uninfected
experimen
lower den
asmchronc

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Progression
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experiments, no cytopathic effect (CPE) was seen in the A2 or A185 infections; however,
CPE began after 72 hours post release at this MOI. The high percentage of G1 cells in
uninfected, A2-infected and A185-infected populations at 48 hours post release in the
experiment described above was due to contact inhibition. In experiments carried out at a
lower density, the cell cycle profile at 48 hours post release resembled that of an
asynchronous population (data not shown).

Variations in the progression of NIH3T3 cells through the cell cycle have been
seen. Factors that affect cell cycle progression include serum batches, the health of the
cell population when released from G0, and cell density. In all experiments, the
synchrony of the first cycle was better than that of the second. Furthermore, cell cycle
progression was similar regardless of which of the two protocols described in Materials
and Methods was used. However, with some serum batches, G0 could only be induced
by contact inhibition and serum starvation.

While the percentage of accumulated cells with 2C DNA content with the 82
mutant infection varied from 40% to 60% from experiment to experiment, of the three
virus strains tested here, it was the only one which induced this cell cycle arrest. The
experimental variation seen in the B2 mutant induced cell cycle arrest is likely to be due,
at least in part, to the state of the cell at the time of infection and the progression of the
population through the cell cycle phases. Because cells that do not enter G0 are not as
sensitive to polyomavirus infection as those in G0 (5), the actual percentage of infected
cells, and the amount of input viral DNA, was decreased in some experiments. Different
batches of serum affect not only the induction of a G0 state but also the release into the

cell cycle. In some cases, cells would not enter the cell cycle at the same rate or in the

65

dif

int
dc
of

[in

LI 0

1T}

same total numbers. In these cases, the capacity of B2 to induce the cell cycle arrest was
difficult to detect.

To test for the requirement of serum for cell cycle progression following
infection, quiescent cells, obtained using protocol 2, were infected and re-fed with serum-
depleted medium. In these experiments, protocol 2 must be used because of the inability
of serum-starved cells to reattach in the presence of serum-depleted medium. Under
these conditions, approximately 60% of the A2-infected cells entered S phase and
arrested in the second G1. On the other hand, 80% of A2-infected cells re-fed with
serum-rich (10%) medium entered S phase and progressed through two complete cell
cycles (data not shown). In the absence of serum, the BZ-infected population (Figure 2-
2A) entered the cell cycle, and a peak S phase (60%) was seen at 18 hours post release
(data point not shown). At 21 hours post release, 24% of the population was in S and
57% of the population was in G2/M. In the same experiment, the addition of serum-rich
medium resulted in a peak S of greater than 80% at 21 hours post release (Figure 2-ZB).
At 48 hours post release, about 30% of the cells re-fed with serum-depleted medium were
arrested with 2C DNA content compared to 60% re-fed with serum-rich medium. All
other populations had approximately 15% 2C DNA content at 48 hours post release (data
not shown). These results demonstrate that the cell cycle arrest induced by the BZ mutant

is serum independent.

66

The hr-t mu

absence of sr

Anal}
.1185 mutant
51 (not Show
the .1185 an
comparison I
CXanment n
1arge 0\'er.en
genomes at
13193158 (0qu
and the 33
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7.9).

%

 

 

The hr-t mutan B2 ex resses and re licates its ename ta hi levels des ite the

absence of small T and/or middle T.

Analysis of proteins expressed by NIH3T3 cells infected with wild type A2, the
A185 mutant, and the BZ mutant is shown in Figure 2-3. As expected, the LT, mT, and
sT (not shown) proteins were expressed by wild type A2 while only LT was expressed by
the A185 and B2 mutants (Figure 2-3A). However, the BZ mutant over-expressed LT in
comparison to either wild type A2 or the A185 mutant. This trend was consistent from
experiment to experiment. Analysis of VP1 expression in this experiment demonstrated a
large over-expression induced by the B2 mutant (Figure 2-3B). Quantitation of the viral
genomes at 8 hours post release (4 hours post infection, input) and at 48 hours post
release (output) by Southern blotting showed high amplification for both wild type A2
and the B2 mutant, about lOO-fold amplification was seen by 48 hours post release
(Figure 2-3C). Amplification of the A185 mutant, on the other hand, was only about 10-
fold by 48 hours post release. The defect in A185 replication is discussed elsewhere (6,

7, 9).

The cell cycle arrest induced by the B2 mutant is multiplicity demndent.

To investigate the dose dependence of the B2-induced G2/M cell cycle arrest,
experiments were carried out at different multiplicities of infection (MOI) with the BZ
mutant (Table 2-IA). NIH3T3 cells were synchronized, infected with different M015,

and released from G0 using protocol 2. At a multiplicity of 1, no significant arrest was

67

seen. At a multiplicity of 3, a G2/M cell cycle arrest could be detected at 48 hours post
release with 34% of the population in G2/M compared to 17% in the uninfected control.
The accumulation of cells with 2C content increased with the multiplicity of infection
and then leveled off at a MOI of 20 with approximately 60-70% of the population
arrested with 2C DNA content. Furthermore, as the multiplicity of infection increased,
the cell cycle arrest was induced earlier in the course of the experiment. For example, at
a MOI of 20, 37% and 65% of the population was in G2/M at 30 and 36 hours post
release, respectively. In the uninfected control only 11% and 30% of the population was
in G2/M at 30 and 36 hours post release, respectively. The effect of increasing MOI of
wild type A2 was compared to results with the B2 mutant (Table 2-IB). Infection with
wild type A2 up to a multiplicity of 30 pfu/cell did not result in accumulation of cells in
G2/M. At 48 hours post infection only 20% of the population had G2/M DNA content at
all multiplicities. Analysis of viral proteins (data not shown) showed that the BZ mutant
consistently produced more LT and VP1 proteins than wild type A2.

The over-expression of viral proteins and the high level of DNA amplification in
B2 infections suggest several possible causes for the induced cell cycle arrest. These
include over-expression of LT and/or VP1 proteins, expression of mutant LT and/or VP1
proteins, and over-replication of normal or mutant viral genomes. Previous experiments
have shown that viral DNA synthesis is not restricted to S phase (8); therefore, the
accumulation of G2/M cells may reflect the activation of the completion of S phase
checkpoint control. Furthermore, since A2-infected cells do not arrest in G2/M, the cell
cycle arrest is either specific to the BZ mutant or the expression of small T and/or middle

T might overcome the cell cycle arrest.

68

Expression of small T and/or middle T do not abrogate the cell cycle arrest induced
by the B2 mutant.

To test the effect of sT and mT expression on the G2/M cell cycle arrest by 32,
three types of experiments were carried out. First NIH3T3 cells expressing sT, mT and
sT+mT were infected with the B2 mutant using protocol 1, and the FACS profiles were
compared to those obtained with NIH3T3 cells (Table 2-11). Expression of small T did
not decrease the observed cell cycle arrest induced by the 82 virus. It appeared that the
expression of middle T might have reduced the cell cycle arrest induced by BZ.
However, this may be an artifact of the difficulty of arresting these transformed cells in
G0, which would result in a poorer infection. The analysis of input viral DNA showed
that not as many genomes were present at 4 hours post infection in mT or sT+mT
expressing cells as were in NIH3T3 or sT-expressing NII-13T3 cells. Since the percentage
of cells accumulated with 2C content is strongly related to dose, the decrease of viral
input could explain the lower percentage of cells in G2/M cells observed at 48 hours post
release.

A second strategy to express small T and middle T in trans made use of a mixed
infection with wild type A2 and mutant BZ. As seen by FACS (Table 2-III) the A2+BZ
mixed infection resulted in an accumulation of cells with G2/M DNA content similar to
that seen with the B2 mutant alone. Analysis of expressed T antigens (Figure 2-4A)
showed that in the presence of the BZ mutant, small T and middle T expression from the

wild type A2 was not detected. The effect of the B2 mutant in infections with non-hr-t

69

mutants is probably related to the dominant-lethal effect previously described with mixed
infection of hr-t and ts-a mutants (11). This property is being further characterized (see
Chapter 4). Analysis of VP1 protein levels (Figure 2-4B) demonstrated that the amount
of VP1 produced in the mixed infection was similar to that of the B2 mutant, and these
levels were higher than that detected in A2-infected cells.

Thirdly, small T and middle T were also expressed in cis by constructing a new
wild type in the B2 genetic background. The deletion in the LT intron of the BZ mutant
genome was repaired with wild type A2 sequences fiom the Blp I site at nt400 to the
Ber I site at nt1079. This new ‘wild-type’ virus, thZ, was confirmed to express sT and
mT (Figure 2-4A), and the function of mT was confirmed by its ability to induce
transformation of FR3T3 cells, although with a lower efficiency than A2 (see Chapter 3).
FACS analysis showed (Table 2-III) that the th2 infection resulted in a similar
accumulation of cells with G2/M DNA content as that seen with the B2 mutant. VP1
protein levels demonstrated that th2-infected cells produced similar amounts of VP1 as
BZ-infected cells, which were greater than those seen in A2-infected cells (Figure 2-43).
From these data, we conclude that small T and middle T expression have little if any

effect in overcoming the cell cycle arrest induced by the BZ mutant.

DNA synthesis inhibitors do not relieve the cell cycle arrest.

We have shown that viral DNA synthesis, once initiated is not restricted to S
phase (8). To investigate the contribution of this out-of-S-phase viral DNA synthesis to
the cell cycle arrest, DNA synthesis inhibitors aphidicolin and mimosine were used to

terminate out of S viral DNA replication, after the cell cycle arrest was induced. NIH3T3

7O

cells were synchronized in G0 and infected with the wild type A2 or the BZ mutant at a
MOI of 10 using protocol 2. Control experiments, in uninfected cells, indicated that if
the inhibitors were added at the S/G2 boundary, the cell population would undergo a
normal mitosis and arrest at the G1/S border of the second cell cycle (data not shown).
DNA synthesis inhibitors mimosine (400uM) or aphidicolin (30uM) were added at 12
hours, 24 hours, and 30 hours post release. Addition of inhibitors at 12 hours post release
completely inhibited entry into S phase, as expected. Addition of inhibitors at 30 hours
post release, arrested cells in early S phase of the second cell cycle with 75%G1, 23%S,
and 2%G2/M. At 24 hours post release. the distribution of uninfected cells was 53%Gl,
9%S, and 38%G2/M, that of A2-infected cells was 56%Gl, 10%S, and 34%G2/M while
that of B2-infected cells was 33%G1, 8%S, and 58%G2/M. As shown in Table 2-IV, the
addition of the inhibitors at 24 hours did not inhibit the return of A2-infected cells to the
G1 phase of the second cell cycle, where they arrested for the remainder of the
experiment. By 48 hours post release, 24 hours after the addition of inhibitor, the A2
infected population had approximately 80% in G1, 10% in S, and 10% in G2/M.
However, the BZ-infected population remained arrested in G2/M of the first cell cycle.
By 48 hours post release, the B2-infected population had approximately 60% in G1, less
than 10% in S and over 30% in G2/M. In this experiment, a large portion of the
population infected with BZ lifted into the culture medium. For this reason, the
supernatant was collected and analyzed for DNA content. In these populations, the
majority, greater than 70%, of the cells had 2C DNA content. A small percentage
(<10%) also had DNA content less than 1C, indicative some form of cell death. None of

the A2-infected population lifted into the culture medium. Analysis of 82 mutant viral

71

DNA replication (Figure 2-5) showed that the addition of either aphidicolin or mimosine
inhibited continued viral DNA replication. Similar results were seen with wild type A2
(data not shown). These data suggest that while high levels of viral replication may be
required to arrest the cells in G2/M, continued viral DNA synthesis is not necessary to

maintain the block.

Characteristics of the B2-induced cell cycle arrest in nan-permissive rat cells.

Because little viral DNA replication takes place in non-permissive rat cells, they
provide a good system to separate the contribution of elevated levels of viral proteins and
viral genome amplification to the B2-induced cell cycle arrest. For this reason, infections
of semi-permissive FR3T3 cells were carried out. In these experiments, cells were
synchronized using protocol 1, and a MOI of 50 was used. The experiment was carried
out for 96 hours post release since there is a delay in viral protein expression to the
second cell cycle in FR3T3 cells (5). During the course of the experiments a key
observation was made. Two cell populations could be distinguished: one that rounded
and floated into the culture medium, and one that remained adherent as a monolayer.
These populations were either combined to give a total population (T) or separated to
analyze the contribution of the monolayer (M) and the supernatant (S) to the
characteristics of the total population.

The kinetics of cell cycle progression of F R3T3 cells was similar to that observed
for NIH3T3 cells (data not shown). The first S peak was seen at 20 hours post release

when the distribution of the population was 24% in GI, 49% in S, and 27% in G2/M.

72

The end of the first cell cycle occurred by 24 hours post release when 67% of the
population was in G1, 17% was in S, and 16% was in G2/M. A second S peak was seen
at 30 hours post release with 45% of the cells in S phase. At 48 hours post release, cell
cycle distributions were indicative of an asynchronous population with 48% in G1, 39%
in S, and 12 % in G2/M. From 60 hours post release to 96 hours post release an increase
in the percentage of cells in G1, and a decrease in the percentage of cells in S phase, was
seen. At 96 hours post release the distribution of the uninfected population was 82%G1,
4%S, and 14%G2/M. This reflects a confluency-induced return to G0. Infection with the
wild type A2 did not alter progression through the initial cell cycle or the distribution of
the cell population at 96 hours post release. Analysis of the B2-infected cells resulted in
a cell cycle arrest in G2/M (Figure 2-6). At 48 hours post release, the total population (2-
6A) contained 29% of the cells arrested with G2/M DNA content (compared to 12% in
the uninfected control). At this time only 30% of the total population was in the
supernatant. When the populations were separated, 20% of the cells in the monolayer
were in G2/M (2-6B) while 40% of the cells in the supernatant were in G2/M (2-6C).
This trend was seen throughout the course of the experiment and became more
pronounced at later time points. For example, at 96 hours post release, 17 % of the total
papulation was in G2/M; in contrast, 12% of the cells in the monolayer and 50% of the
cells in the supernatant were in G2/M. The cells in the supernatant contained a high
percentage of cells with distinct metaphase plates as observed by light microscopy. In
addition, the supernatant contained a population with less than 1C DNA content,

indicative of cell death, perhaps by apoptosis.

73

Analyses of protein and DNA levels in the different sub-populations are shown in
Figure 2-7A. LT protein was detected in all papulations throughout the experiment.
With increasing time, elevated levels were observed in the supernatant compared to the
total population. On the other hand, VP1 protein was detectable only in the supernatant
at 48 hours post release and was not detectable in the monolayer at 96 hours post release.
Analysis of viral genomes (2-7B) exhibited the same trend as LT protein. However, as
expected, a lower degree of viral DNA amplification (less than lO-fold) was obtained
compared to the levels reached in NIH3T3 cells. Taken together, these data suggest that
the cell cycle arrest induced by the B2 mutant is not related to viral genome
accumulation. Furthermore, the cell cycle arrest appears to be mitotic and tracks with the

over-expression of VP1 protein.

The induction of a G2/M cell cycle arrest is a common feature of hr-t mutants.

To test whether the cell cycle arrest is a unique property of the B2 mutant, or is a
common feature of the hr-t mutants, several other mutants were tested for their ability to
induce a G2/M cell cycle arrest. These included two deletion mutants, NG18 and NG23,
and the insertion/point mutant NG59. Two ‘wild-type’ strains were also tested: 18R4,
the rescued wild type for NG18, and 59RA, the rescued wild type of NG59. NIH3T3
cells were synchronized in G0 and infected according to protocol 1. Infections were

carried out at a multiplicity of 10pfu/cell four hours post release. The progression of the

74

cell cycle was monitored by FACS and the percentage of cells with G2/M DNA content
at 48 hours post release are shown in Table 2-V. In all cases, infection with the hr-t
mutant or the ‘wild type’ derived from it resulted in an accumulation of cells with G2/M

DNA content. Slight variations are seen due to variations of input virus (data not shown).

The cell cycle arrest appears to be mitotic.

To test for the presence of mitotic cells in A2-and BZ-infected NIH3T3 cells,
NIH3T3 cells were released from G0 according to protocol 2 and infected four hours later
at a MOI of 10pfir/cell. At 48 hours post release, uninfected, A2-infected, and B2-
infected cells were resuspended and treated with DAPI. Figure 2-8 shows examples of
uninfected (A-C) and A2-infected (D-F) samples. Most cells in these populations
exhibited a punctate pattern of DAPI staining consistent with interphase nuclei. Results
with B2-infected cells were slightly different (Figure 2-9). While some cells exhibited
the punctate pattern of interphase nuclei, many showed staining consistent with
condensed chromosomes. We conclude from this that the BZ-induced cell cycle arrest

does proceed fi'om GZ into mitosis.

VP1 associates with B-tubulin.

Because B2-infected NIH3T3 and FR3T3 appeared to arrest in mitosis, and this

arrest tracked with the over-expression of VP1, on possible cause of the induced cell

cycle arrest could be interference with spindle function by the VP1 capsid protein. To

75

test whether polyomavirus VP1 could associate with components of the mitotic spindle,
the association of VP1 with B-tubulin was examined. NIH3T3 cells were synchronized
and released from G0 according to protocol 2. Infections were carried out four hours post
release at a MOI of 10pfu/cell. Samples were collected at 48 hours post release and
immunoprecipitated with B-tubulin primary antibodies. Results shown in Figure 2-10
show that a fiaction of the total VP1 in both A2- and B2-infected NIH3T3 cells

associated with B-tubulin in vitro.

DISCUSSION

We have shown that infection of NIH3T3 cells with the hr-t mutant, 32, causes a
large fraction of the population to arrest with G2/M DNA content. This arrest is largely
dependent on the viral dose at the time of infection. At low multiplicities of infection
(<5pfir/cell), little accumulation of cells with 2C DNA content is seen; however, at higher
MOI (5pfu/cell and higher), a time dependent increase in the accumulation of cells with
2C DNA content is seen, which becomes maximal by 48 hours post infection. The
percentage of cells arrested with 2C DNA content by 48 hours can reach 60-70% of the
total population. Similar dose response experiments carried out with wild type A2 did
not result in any accumulation of cells with 2C DNA content. This suggests that the
induced cell cycle arrest is either specific to the B2 mutant or that the expression of small
T and/or middle T abrogates the induced cell cycle arrest. Furthermore, in experiments

carried out with serum-depleted medium, the BZ-infected cells initiated S phase and

76

arrested in the first G2/M, while wild type A2-infected cells arrested in the next G1
phase. This suggests that the cell cycle arrest induced by BZ is serum independent.

The present set of experiments also demonstrate that infection of cells, brought
into the G0 state by serum starvation, with the BZ mutant induces one round of cellular
DNA synthesis. These results are compatible with previous data. Early experiments with
confluent cultures demonstrated that infection with polyomavirus resulted in a 10-fold
increase in cellular DNA synthesis as measured by 3H-thymidine incorporation compared
to mock-infected controls (27). Furthermore, experiments with the hr-t mutant N618 (3)
did not reveal any differences in the ability of the mutant to induce cellular DNA
synthesis in confluent cultures. Other experiments (14) have shown that large T
expression alone is able to drive cells into S phase. However, in the present case, the
contribution of the viral capsids must also be considered. Experiments from Robert
Garcea (28) have suggested that capsid proteins (namely VP1) are capable of inducing
both c-myc and c- 03. However, the ability of empty capsids to induce 3H-thymidine
incorporation was greatly reduced compared to the intact virion. The results presented
here suggest that large T protein expressed by the B2 mutant is able to induce a single
round of DNA synthesis in quiescent NIH3T3 cells; however, at this time a role for
capsid proteins in the induction of S phase cannot be totally ruled out.

The B2-induced cell cycle arrest is not unique to this particular hr-t mutant.
Three other hr-t mutants tested were able to induce an accumulation of cells with 2C
DNA content similar to that seen with the BZ mutant. The slight variations in the

percentage of the population arrested in G2/M observed with different mutants reflect

77

variations in the amount of input genomes. These results suggest that the ability to
induce a G2/M cell cycle arrest in NIH3T3 cells is a common feature of the hr-t mutants.

The cell cycle arrest induced by B2, and the other hr-t mutants, also relates to the
line of NIH3T3 cells used. Some isolated sub-clones respond better than others to
infection with 82. In some instances, this can be traced to the poor cycling of these
clonal populations, and, perhaps related to this, poor infections leading to greatly reduced
production of viral proteins and genomes. This suggests that some cellular factors may
also contribute to the cell cycle arrest seen here, in much the same way that they
contributed to the permissivity to hr-t mutants in early experiments (15).

Previous experiments carried out in our lab (8) have shown that viral DNA
replication is not restricted to S phase. Thus, one possible cause of the induced cell cycle
arrest which was considered was that out-of-S phase viral DNA replication was inducing
the completion of S phase checkpoint and arresting cells in G2. To investigate this
possibility, two DNA synthesis inhibitors, aphidicolin and mimosine, were used to
prevent further viral DNA synthesis after the cell cycle arrest was achieved. The addition
of either inhibitor did not relieve the BZ-induced cell cycle arrest suggesting that while
amplification of viral DNA may contribute to the cell cycle arrest, continued viral DNA
synthesis is not required to maintain cells in the arrested state. However, this possibility
cannot be ruled out at this time because replication intermediates and/or single-stranded
DNA may still be present in the inhibitor-treated B2-infected population. Data from both
S. cerevisiae and S. pombe suggest that an active replication complex is required to

activate the S phase checkpoint, possibly by the creation of single-stranded DNA (25).

78

 

Therefore, it is possible that the S phase checkpoint is still being activated in these
experiments.

The experiments with semi-permissive FR3T3 cells allowed the separation of the
contribution of viral proteins and viral DNA to the B2-induced cell cycle arrest. Similar
to NIH3T3 cells, infection of FR3T3 cells with the BZ mutant resulted in the
accumulation of cells with 2C DNA content in a fraction of the total population. In this
case, the arrested cells lifted off the monolayer. However, the amplification of viral
genomes seen in BZ-infected NII-I3T 3 cells was not seen in BZ-infected FR3T3 cells;
therefore, induction of the cell cycle arrest does not require a high level of viral
replication. Analysis of viral products showed that while large T and viral genomes were
fairly evenly distributed among the various cell populations (monolayer versus
supernatant), the cell cycle arrest induced in these cells tracked with the over-expression
of VP1. Early studies in Fischer rat F111 cells also showed that the hr-t mutants can
induce a single round of S phase, in contrast to the wild type, which can induce multiple
rounds and polyploidy in a fraction of the population (22). Our experiments suggest that
in these experiments the hr-t mutants were causing a G2/M arrest.

Comparison of polyomavirus and SV40-infected cells reveals a key difference
between the two viruses. While SV40 induces a G2 cell cycle arrest in lytic infections
(18, 20), the majority of polyomavirus wild type A2 infected cells pass through mitosis
into the next cell cycle. In SV40-infected populations, activation of the cyclin B/cdc 2
kinase required for entry into mitosis is inhibited (20). Instead, SV40-infected CV-l cells
undergo a second S phase without an intervening mitosis (18). This results in the

accumulation of polyploid cells in SV40 infections. Furthermore, it has been argued that

79

the amplification of the viral genome makes a large contribution to the apparent
polyploidy. Two experimental results argue against a similar phenomenon in our
experiments. First, the A2 genome amplifies its genome to similar levels as B2 but does
not induce a cell cycle arrest. Second, infection of FR3T3 cells results in a similar cell
cycle arrest to that seen in permissive infections, despite the low level of amplification of
viral genomes.

It appears that the B2-induced cell cycle arrest is mitotic in nature and may be
caused by the over-expression of VP1. Experiments with both FR3T3 and NIH3T3
revealed the presence of metaphase plates and condensed chromosomes, respectively.
The idea of VP1 being associated with a block in mitosis has precedence in the analysis
of VP1 by immunohistochemistry in mouse tumors (26). In these experiments VP1 could
be found in association with the mitotic figures and the spindle apparatus in tumor
tissues. Recently, work from J. Forstova (19), showed that expression of VP1 in yeast
cells results in a mitotic arrest. In this case, cells were able to overcome the cell cycle
arrest induced by VP1 expression, but the cells had to generate a new mitotic spindle.
The population then arrested in the G1 phase where the old spindle could be detected.
The fact that the hr-t mutants induce a mitotic arrest and A2 derived viruses do not may
be solely related to dosage of VP1. At equivalent viral doses, the hr-t mutants produce
more VP1 than A2, and earlier in the course of the infection. Secondly, hr-t mutants
show a failure in virus maturation, which has been associated with a defect in
phosphorylation of VP1. Therefore, more free-VP1 may be available for association with
the spindle apparatus. We have found that both A2 and BZ VP1 associates with B-

tubulin, but that since more VP1 is produced in the B2 infection than in the A2 infection,

80

more VP1 is immunoprecipitated with B-tubulin from B2-infected cells than A2-infected
cells.

Several experiments argue against any role of small T and/or middle T in
abrogating the BZ-induced cell cycle arrest. First, infection of NIHBT3 cells lines
expressing small T, middle T, and small T+middle T with the 82 mutant resulted in a
similar accumulation of cells with 2C DNA content as seen in NIH3T3 cells. A second
approach was to supply small T and middle T in cis. For this purpose, a new virus, th2,
was constructed. While infection of thZ-infected NIH3T3 cells did express small T and
middle T, the cell population still accumulated similar levels of cells with 2C DNA
content by 48 hours post infection as seen with the B2-infected population. Furthermore,
the two ‘wild-type’ viruses 18R4 and 59RA also induce a G2/M cell cycle. Taken
together, these experiments suggest that the expression of small T and middle T do not
abro gate the cell cycle arrest induced by the B2 mutant.

An attempt was made to supply small T and middle T in trans by co-infection of
wild type A2 with the B2 mutant. However, in these experiments, small T and middle T
was not expressed from the A2 genome. The effect of the B2 mutant on A2 gene
expression is reminiscent of the ‘dominant-lethal’ effect seen in mixed infections of hr-t
mutants and ts-a mutants (1 1). In these experiments, complementation between the two
mutants only occurred when the input ratio of the ts-a mutant to the hr-t mutant was
greater than fifty-to-one. Furthermore, in mixed infections with the hr-t mutants NG18
and NG59 and wild type strains small T and middle T were not detected (23). The
preferential expression, and replication, of the 32 mutant genome in mixed infections

with wild type A2 is further characterized in Chapter 4.

81

Initial sequencing of the 82 genome has revealed that other mutations, besides the
deletion in the LT intron, exist throughout the genome. These mutations must partially
compensate for the lack of small T and middle T expression as the A185 mutant has a
large defect in both gene expression and viral DNA replication (6, 7). Preliminary results
indicate that the replication potential of the B2 mutant maps to a region of the genome,
which encodes, in part, for the large T protein. The contribution of these mutations to the
cell cycle arrest is currently being investigated.

In conclusion, we have shown that infection of NIH3T3 and F R3T3 cells with the
hr-t mutant, BZ, results in the accumulation of cells with 2C DNA content in a dose-
dependent, serum-independent manner. We show that the expression of small T and/or
middle T does not abrogate the BZ-induced cell cycle arrest. Furthermore, the induction
of a G2/M cell cycle arrest appears to be a common feature of the hr-t class of
polyomavirus mutants. Finally, experimental results are consistent with the hypothesis
that the observed cell cycle arrest is mitotic in nature and may be caused by the over-

expression of VP1.

82

APPENDIX 2: TABLES AND FIGURES FOR CHAPTER 2

83

Figure 2-1. Cell cycle profiles of uninfected and wild type A2-, mutant A185-, and
mutant B2-infected NII-13T3 cells.

NIH3T3 cells were synchronized in G0 by protocol 2. Infections with wild type
A2, mutant A185, and mutant BZ were carried out at a MOI of 10. Progression through
the cell cycle phases was followed for 48 hours. Cells were fixed, stained, and 5000 cells
for each sample were analyzed for DNA content by flow cytometry. Displays of the cell
cycle progression of uninfected (A), A2-infected (B), A185-infected (C), and B2-infected
cells (D) are shown. FACS profiles of samples taken at 12, 20, 24, 36, and 48 hours post
release are shown. In this experiment hours post release, is equivalent to hours post

infection.

84

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

85

Figure 2-2. Cell cycle analysis of BZ-infected NIH3T3 cells in the presence or absence
of serum factors.

NIH3T3 cells were synchronized in G0 by protocol 2. Infections with B2 were
carried out at a MOI of 10 followed by the addition of serum-depleted medium (A) or
serum-rich medium (B). Progression through the cell cycle phases was followed for 48
hours. Cells were fixed, stained, and 5000 cells for each sample were analyzed for DNA
content by flow cytometry. Displays of the cell cycle progression are shown. FACS
profiles of samples taken at 12, 21, 24, 30, 36, and 48 hours post release (equivalent to

hours post infection) are shown.

86

 

 

87

48hpr

 

Figure 2-3. Comparison of the accumulation of early and late viral proteins and viral
DNA replication in wild type A2-, mutant A185-, and mutant BZ-infected NIH3T3 cells.
Infection of NIH3T3 cells at a MOI of 10 was carried out according to protocol 1
(infections were carried out four hours after release from G0). Protein lysates (A and B)
were collected at various times past release and one-fifth of the sample was assayed for
early proteins (A) and late proteins (B) as described in Materials and Methods. Total
DNA (C) was isolated at the times indicated, digested with EcoR I, and blotted to
nitrocellulose membranes. Membranes were hybridized with polyoma A2 genomic DNA

to measure viral amplification.

88

A2-Infected A l 85-Infected B2-Infected
A. T Antigens

hpr 24 30 48 24 3o 48 24 3o 48

 

B. VP1
VP1 —> d .,, ”I“ ‘ ‘
C. Viral Genomes W W B2-Infected
8 hpr —> . .
48hpr —>

 

89

 

Table 2-1: Effect of the multiplicity of infection on the accumulation of G2/M cells.
NIH3T3 cells were synchronized and released from G0 according to protocol 2.
Infections were carried out with different multiplicities of infection (M01) as indicated
(time of infection is equivalent to time of release form G0). Samples were collected at
the times shown; FACS analysis was carried out on 5000 cells for each sample; and the
percentage of cells in G1, S, and G2/M was calculated. The cell cycle distributions for
BZ-infected cells are shown in A. A comparison of A2- and B2-infected cells is shown in

B. Only the percentage cells in G2/M cells is shown in this case.

90

Table 2-1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

M9915 MQI 1 M91 3 MQI 5
%G1 %8 %62 %Gt %S %62 %G1 %8 %G2 %G1 %S %62
0hr 89 4 7
12hr 65 24 11 86 4 10 88 5 7 88 5
20m 6 45 491 6 37 6 39 55 7 36
24hr 61 8 31 64 5 31 57 6 37 57 6 3
30hr 61 28 11 55 32 1 53 33 141 48 34 1
36hr 53 17 30 49 19 33 41 19 40 35 21
48h 60 24 17 51 28 21 31 35 341 22 33
MQI 1Q _MQI 29 M9139
%G1 %S %62 %G1 %S 9662 %G1 %S 9662
12hr 86 6 83 6 1 88 6
20hr 4 36 2 46 5 4 39
24hr 47 9 45 39 7 36 9
30hr 48 30 33 30 3 39 33
36hr 23 23 17 18 20 17
48h 13 25 61, 9 21 7 11 29
A2-Infected Bz-Intoctad
M011 5 10 20 30 M01 5 10 20 30
0hr 10 10 10 10 0hr 10 10 10 10
12hr 8 8 8 81 12hr 8 8 8
20hn 1 32 0 24 20hr 0 38 0
24hr 38 31 45 36 24hr 57 41 62 5
30hr 15 13 17 6 30hr 38 36 52 4
36hr 28 23 31 29 36hr 48 53 67 7
48hr 23 24 20 17 48h 39 66 69 6 .

 

 

 

 

 

91

 

 

Table 2-11. Cell cycle analysis of B2-infected NIH3T3 cells lines expressing sT, mT, and
sT+mT.

Cells were synchronized and infected using protocol 1 (infections four hours post
release), and progression through the cell cycle was followed for 48 hours. At various
times post infection samples were fixed, stained, and 5000 cells were analyzed for DNA
content by flow cytometry. The percentage of cells with 2C DNA content at 48 hours

post release is shown.

Table 2-III. Cell cycle analysis of A2 and B2 mixed infections and infections with the
th2 virus.

Cells were synchronized and infected using protocol 1 (infections four hours post
release), and the progression through the cell cycle was followed for 48 hours. At
various times post infection samples were fixed, stained, and 5000 cells were analyzed
for DNA content “by flow cytometry. The percentages of cells in G1, S, and GZ/M were

calculated by Wincycle for Windows. Profiles at 48 hours post infection are shown.

92

Table 2-11

CELL LINE UNINFECTED BZ INFECTED
NIH3T3 13 74

sT 20 76

mT 10 42
20 42

 

Table 2-III

 

%Gl °_/o_S_ %G2/M

 

Uninfected 34 48 20

 

BZ-Infected 22 3 1 47

 

A2-Infected 27 48 25

 

A2+B2 19 21 6O

 

thZ-Infected l 7 29 54

 

 

 

 

 

 

93

 

Figure 2-4. Comparison of the accumulation of early and late viral proteins in A2, B2,
A2+B2, and th2 infections.

Infection of NIH3T3 cells at a MOI of 10 per virus strain was carried out
according to protocol 1. Protein lysates were collected at various times and one-fifth of
the sample was assayed for early proteins (A) and late proteins (B) as described in
Materials and Methods. The times indicated are hours post release from GO (infections

were carried out four hours after GO release).

94

& B_2 MB;- fl

 

mT—> a. m ‘

hpr 30 48 30 48 30 48 30 48

95

Table 2-IV. Cell cycle analysis of A2- and BZ-infected cells treated with aphidicolin and
mimosine.

Cells were synchronized and infected using protocol 2 (infection time at the time
of release from GO), and progression through the cell cycle was followed for 48 hours.
Aphidicolin and mimosine were added at 24 hours post release (hpr). At times shown
samples were collected, fixed, stained, and 5000 cells were analyzed for DNA content by
flow cytometry. The cell cycle profiles for B2-infected cells are shown in the top panel.
At 48 hours post infection a large portion of the population was found in the supernatant
and was analyzed separately (48 hpr Supp). The cell cycle profiles for A2 infected cells

are shown in the bottom panel.

96

 

 

 

 

 

e m B 2 o 8 2 mm 8 3
o m a 3 m 8 8 8 mm 8
e s 8 o o. 8 e 9‘ cm on ~<
3 2 mm 3 2 mm 3 o. 8 4m
2. m mm 2. m m: 8 m S .83 3
mm o 8 8 m 8 2. mm m. 3
8 u 8 8 9 m4 8 8 3 8 mm
5 m 5 3 o 8 «a 9 mm 8
on m 8 3 m 8 an m 8 a
0.x. was 5.x. a. Q. IF 0. 0N .Ijlflolo Es
3; S... a @5853 an em a 5.8622 862.5 ms:

 

 

 

 

 

 

arm 03.5.

97

Figure 2-5: The effect of DNA synthesis inhibitors on viral DNA replication.

DNA fi'om B2-infected cells was isolated and analyzed as described in the
Materials and Methods. The time of collection in hours post release (equivalent to hours
post infection) and the time of addition of either BOuM aphidicolin (A) or 400uM
mimosine (M) are indicated. The average counts per minute as calculated by

phosphorimaging of duplicate samples are given.

98

oov

 

cog

 

 

mv

xv

 

E M: 2%
22+ ~_<+ o
vm em em

3

  

Q

o—

Ego

aflogwob

a:

99

Figure 2-6. Cell cycle profiles of B2-infected FR3T3 cells at 48 hours post release.

F R3T3 cells were synchronized in G0 by protocol 1. Infections with A2 and B2
were carried out at a MOI of 50 four hours post release. Progression through the cell
cycle phases was followed for 96 hours. Cells were fixed, stained, and 5000 cells for
each sample were analyzed for DNA content by flow cytometry. FACS profiles at 48
hours post release are shown. The two populations, supernatant and monolayer, were
combined (A) or analyzed separately to determine the contribution of the monolayer (B)

and the supernatant (C) to the overall profile seen in the total population.

100

 

l1 Ill

101

 

Figure 2-7. Comparison of early and late viral proteins and viral genome levels in sub-
populations of F R3T3 cells infected with the B2 mutant.

The populations of B2-infected cells were pooled to yield a total population (T) or
separated into the monolayer (M) or the supernatant (S) and analyzed separately.
Because approximately 30% of the total population was in the supernatant, the
supernatant from two plates were also combined and analyzed (8x2). Protein lysates (A)
were collected at various times and one-fifth of the sample was run on polyacrylamide
gels, transferred to PDVF membrane and assayed for LT, VP1, and actin as described in
Materials and Methods. Total DNA (B) was isolated and approximately 1x105 cells was
digested with EcoR I, and blotted to nitrocellulose membranes. Membranes were

hybridized with polyoma A2 genomic DNA to measure viral amplification.

102

@383 0380.5 83ch 32...

 

 

 

 

. , . ... . ‘.—. , , . . I; . t . .3. _ .
a 13.2. ... . ._ ,. flow. a hFI... in.“ ......J.. 2.12... k... ‘11 Gmw0<

‘- P. 7:00.11. A1 :3

 

  

. . . 4 . .. .. ......‘1 I; ..,..;r M.,...Ufm
.p.

vv.

fifim—ZHQmmEmemm—ZH

 

 

a; 3 a: 2 a; we

~ doom

103

Table 2-V: Comparison of the ability of several hr-t mutants to induce a G2/M cell cycle
arrest in NIH3T3 cells.

NH-I3T3 cells were synchronized and released from GO according to protocol 1.
Infections at a MOI of lOpfu/cell were carried out four hours post release fiom GO. At 48
hours post release, cell populations were fixed and analyzed by FACS for DNA content
as described in Materials and Methods. The percentage of GZ/M uninfected (None) and

hr-t mutant-infected cells at 48 hours post release from two experiments are shown.

104

Table 2-V

 

 

 

 

VIRUS NONE E; NGl8 1 R4 NG59 59RA NG23
Exp. 1 23 72 48 ND 42 46 ND
Exp. 2 20 55 58 47 49 ND 44

 

 

 

 

 

 

 

 

105

 

 

Figure 2-8: DAPI staining of uninfected and A2-infected NIH3T3 cells.

NIH3T3 cells were synchronized and released fiom GO according to protocol 2.
Infections were carried out four hours post release at a MOI of lOpfu/cell. Samples were
collected at 48 hours post release, and cells were stained for 10 minutes in 300nM DAPI
at room temperature. Images A-C and D-F are of uninfected and A2-infected NH-I3T3

cells, respectively.

"Images in this thesis/dissertation are presented in color."

106

 

107

Figure 2-9: DAPI staining of BZ-infected NII-13T3 cells.

NH-13T3 cells were synchronized and released from GO according to protocol 2.
Infections were carried out four hours post release at a MOI of lOpfu/cell. Samples were
collected at 48 hours post release, and cells were stained for 10 minutes in 300nM DAPI .

at room temperature.

108

 

109

Figure 2—10: VP1 associates with B-tubulin.

NIH3T3 cells were synchronized and released from GO according to protocol 2.
Infections were carried out at a MOI of lOpfu/cell four hours post release. Samples were
collected and immunoprecipitated with B-tubulin primary antibodies. Samples were then
assayed for the presence of VP1 by western blot analysis and compared to un-

immunoprecipitated samples.

110

81‘. Total Tubulin-IP

 

‘93 W NIH A2 B2

1111111

   

lll

IO-

11.

12.-

13

REFERENCES

Benjamin, T. L. 1970. Host range mutants of polyoma virus Proc Natl Acad Sci
U S A. 67:394-9.

Benjamin, T. L. 1982. The hr-t gene of polyoma virus Biochim Biophys Acta.
695:69-95.

Benjamin, T. L. 1971. Isolation and Characterization of Non-Transforming
Mutants of Polyoma Virus, p. 300-305, The Biology of Oncogenic Viruses.

Benjamin, T. L., G. G. Carmichael, and B. S. Schaffhausen 1980. The hr-t
gene of polyoma virus Cold Spring Harb Symp Quant Biol. 44:263-70.

Chen, H. H., and M. M. Fluck 1993. Cell cycle control of polyomavirus-induced
transformation J Virol. 67 : 1 996-2005.

Chen, L., and M. Fluck 2000. Kinetic Analysis of the Steps of the Polyomavirus
Lytic Cycle: Timing of Middle T + Small T Synthesis and Tanscriptional Control
of the Early -> Late Switch Manuscript submitted.

Chen, L., and M. Fluck 2000. The role of middle T/small T in the lytic cycle of
polyomavirus: Control of the early to late transcriptional switch and viral DNA
replication Manuscript submitted. -

Chen, L., K. Spink, D. Redenius, and M. Fluck Manuscript in preparation.

Chen, M. C., D. Redenius, F. Osati-Ashtiani, and M. M. Fluck 1995.
Enhancer-mediated role for polyomavirus middle T/small T in DNA replication J
Virol. 69:326-33.

Feunteun, J ., L. Sompayrac, M. Fluck, and T. Benjamin 1976. Localization of
gene functions in polyoma virus DNA Proc Natl Acad Sci U S A. 73:4169-73.

Flack, M. M., R. J. Staneloni, and T. L. Benjamin 1977. Hr-t and ts-a: two
early gene functions of polyoma virus Virology. 77 :610-24.

Garcea, R. L., K. Ballmer-Hofer, and T. L. Benjamin 1985. Virion assembly
defect of polyomavirus hr-t mutants: underphosphorylation of major capsid
protein VP1 before viral DNA encapsidation J Virol. 54:31 1-6.

Garcea, R. L., and T. L. Benjamin 1983. Host range transforming gene of

polyoma virus plays a role in virus assembly Proc Natl Acad Sci U S A. 80:3613-
7.

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14.

15.

16.

17.

18.

19-

20-

21-

22-

23-

24-

25

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Gjorup, O. V., P. E. Rose, P. S. Holman, B. J. Bockus, and B. S. Schaffhausen
1994. Protein domains connect cell cycle stimulation directly to initiation of DNA
replication Proc Natl Acad Sci U S A. 91: 12125-9.

Goldman, E., and T. L. Benjamin 1975. Analysis of host range of
nontransforming polyoma virus mutants Virology. 66:372-84.

Hattori, J., G. G. Carmichael, and T. L. Benjamin 1979. DNA sequence
alterations in Hr-t deletion mutants of polyoma virus Cell. 16:505-13.

Lania, L., M. Griffiths, B. Cooke, Y. Ito, and M. Fried 1979. Untransformed
rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t)
mutant Cell. 18:793-802.

Lehman, J. M., J. Laffin, and T. D. Friedrich 2000. Simian virus 40 induces
multiple S phases with the majority of viral DNA replication in the G2 and second
S phase in CV-l cells Exp Cell Res. 258:215-22.

Palkova, Z., T. Adamec, D. Liebl, J. Stokrova, and J. Forstova 2000.
Production of polyomavirus structural protein VP1 in yeast cells and its
interaction with cell structures F EBS Lett. 478:281-9.

Scarano, F. J., J. A. Laffin, J. M. Lehman, and T. D. Friedrich 1994. Simian
virus 40 prevents activation of M-phase-promoting factor during lytic infection J
Virol. 68:2355-61.

Schaffhausen, B. S., J. E. Silver, and T. L. Benjamin 1978. Tumor antigen(s) in
cell productively infected by wild-type polyoma virus and mutant NG-18 Proc
Natl Acad Sci U S A. 75:79-83.

Schlegel, R., and T. L. Benjamin 1978. Cellular alterations dependent upon the
polyoma virus Hr-t function: separation of mitogenic from transforming
capacities Cell. 114:587-99.

Silver, J., B. Schaffhausen, and T. Benjamin 1978. Tumor antigens induced by
nontransforming mutants of polyoma virus Cell. 15:485-96.

Soeda, E., J. R. Arrand, N. Smolar, J. E. Walsh, and B. E. Griffin 1980.
Coding potential and regulatory signals of the polyoma virus genome Nature.
283:445-53.

Stewart, E., and T. Enoch 1996. S-phase and DNA-damage checkpoints: a tale
of two yeasts Curr Opin Cell Biol. 8:781-7.

Talmage, D. A., R. Freund, T. Dnbensky, M. Salcedo, P. Gariglio, L. M.
Range], C. J. Dawe, and T. L. Benjamin 1992. Heterogeneity in state and

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27.

28.

 

expression of viral DNA in polyoma virus- induced tumors of the mouse
Virology. 187:734-47.

Vogt, M., R. Dulbecco, and B. Smith 1966. Induction of cellular DNA synthesis
by polyoma virus. 3. Induction in productively infected cells Proc Natl Acad Sci
U S A. 55:956-60.

Zullo, J., C. D. Stiles, and R. L. Garcea 1987. Regulation of c-myc and c-fos

mRNA levels by polyomavirus: distinct roles for the capsid protein VP1 and the
viral early proteins Proc Natl Acad Sci U S A. 84:1210-4.

114

 

CHAPTER 3

THE POLYOMAVIRUS HR-T MUTANT, B2, INHIBITS TRANSFORMATION
BY MIDDLE T IN A CIS- AND TRANS- DOMINANT MANNER

ABSTRACT

In previous experiments, we have shown that infection of NIH3T3 cells with the
hr-t mutant, B2, results in a G2/M cell cycle arrest; furthermore, the expression of small
T and/or middle T does not abrogate this arrest. Infection of semi-permissive rat cells
(FR3T3) with the B2 mutant also resulted in a G2/M cell cycle arrest in a proportion of
the infected population. Here we report that infection of FR3T3 with the thZ virus
results in a similar cell cycle arrest that likely contributes to a delay in the development of
transformed foci in th2-infected cells. Cell lines derived from th2 transformed foci
were compared to cell lines derived from A2 transformed foci. The th2-transformed
cell lines differed from the A2-transformed cell lines in two distinct ways. First, most
(72%) of the th2-transformed cell lines did not express normal-sized large T antigen.
Second, and probably related, most of the th2-cell lines contained little viral DNA.
Previous reports have also demonstrated that in mixed infections of NIH3T3 cells with
wild type A2 and the B2 mutant, small T and middle T expression is not detected. Here
we report that similar mixed infections of FR3T3 cells resulted in a G2/M cell cycle
arrest in a proportion of the population; furthermore, the expression of small T and
middle T from the A2 genome was inhibited in the presence of the B2 mutant.
Transformed foci developed in the mixed infections by two weeks post infection with

timing similar to that observed for A2-derived transformed foci; however, a decrease in

115

the number of A2+B2 transformed foci was seen. None (0 of 18) of the A2+B2-
transformed cell lines derived from those transformed foci contained the B2 genome.
Furthermore, cell lines developed from A2+B2 transformed foci exhibited properties of

A2-like transformed cell lines.

INTRODUCTION

As described previously, infection of NIH3T3 and FR3T3 cells with the hr-t
mutant, B2, results in a G2/M cell cycle arrest that is not overcome by the expression of
small T and/or middle T. Furthermore, co-infection of the B2 mutant with the wild-type
A2 results in a lack of expression of small T and middle T. The preferential gene
expression of the B2 genome shares some similarity with the ‘dominant-lethal’ effect
previously described. In these experiments, complementation between the hr-t mutants
(small T and middle T defective) and the ts-a mutants (large T defective) would only
occur when the input ratio of ts-azhr-t was greater than 50:1 (3). Furthermore, the
presence of the hr-t mutant also inhibited gene expression from a co-infected wild type
virus (10). The focus of the present study was to investigate the effect of the induced cell
cycle arrest by the B2 mutant on transformation of FR3T3 cells by middle T both in cis
and in trans. We have also created several transformed cell lines from A2-, th2- and
A2+B2-transformed foci. Preliminary analysis of viral proteins and the status of the viral
genomes demonstrate striking differences between th2-derved transformed cell lines

and A2- or A2+B2-derived cell lines.

116

MATERIALS AND METHODS
V_irEs_<:.s_.

Wild type A2 (1 1) has been described previously. The B2 virus used in this study
contains a repair of the 11 base pair deletion in the early region from the original isolate
of B2 (6). The th2 virus was constructed by replacement of the Ber I-Blp I
(nucleotides 400-1079) restriction fragment with A2 sequences. Virus stocks were
prepared in NII-13T3 or 3T6 cells and checked for the absence of defectives. Titers were
calculated using standard plaque assay techniques and by comparison of input DNA.
Cells_and infection;

FR3T3 cells were cultured in Dulbecco’s modified eagle medium (DMEM;
GIBCO) supplemented with 10% heat inactivated bovine calf serum (GIBCO, SIGMA)
at 37°C and 5% C02. Cells were grown to 90% confluence followed by 24 hours
starvation with 0.5% serum medium. Cells were released fi'om GO by re-plating at a
lower density in 10% serum medium. Adsorptions were carried out after reattachment (4
hours) for one hour at 37°C and 5%C02. Unless otherwise indicated all infections were
carried out at a multiplicity of infection (MOI) of 10 plaque—forming units per cell, and
the infection lysate was removed prior to the addition of 10% serum medium.
Transformation assafi.

F R3T3 cells were synchronized and infected using the protocol described above
with the following exception. Following infection, cells were re-fed with 5% serum

medium and replaced as needed. The development of cells growing on top of the

117

monolayer was monitored, and these transformed foci were stained with 1% methylene
blue in 5% acetic acid and 20% isopropanol and photographed.
FACS Analysis.

Cells were treated with trypsin and re-suspended in 1 ml of phospho—buffered
saline (1XPBS) supplemented with 2% serum and fixed in 10ml 80% ethanol for 30
minutes on ice. Samples were stored at 4°C until analysis was done. Cells were then
washed once with 1XPBS and stained for 30 minutes at room temperature with
propidium iodide solution: 0.1mg/ml RNaseA, 0.1% triton-x-lOO, 0.5mM EDTA, and
0.05mg/ml propidium iodide in 1XPBS. A total of 5000 cells were then assayed for
DNA content using CellQuest by Beckton-Dickenson. Percentages of G1, S, and G2/M
were calculated using Multiplus or WinCycle.
Preparation and analysis of viral DNA

Infected cells were lysed in lOmM Tris, lOmM EDTA, and 0.2% sodium dodecyl
sulfate (SDS) (pH7.6). Protein digestion was carried out overnight at 37°C with SOug/ml
proteinase K (Sigma). Total DNA was extracted with phenol-chloroform and treated
with RN aseA by standard procedures. Samples were digested with the restriction
endonuclease Msp I (GIBCO BRL) which cuts the polyoma genome into eight fiagments,
electrophoresed in 2% agarose, stained with ethidium bromide to confirm equivalent
loading, and blotted to Hybond membranes (Amersham). Hybridization was carried out
at 65°C in 1X Denhardt’s solution, SXSSPE, and 0.5% SDS by standard procedures with
a 32P-radiolabelled probe consisting of the A2 genome. Hybridization probes were
labeled to a specific activity of 1-2 x 109 cpm/ug with (32P) dCTP (3,000 uCi/mmol; New

England Nuclear) using a multiprime DNA-labeling kit (Amersham). Hybridized blots

118

were washed using stringent conditions and exposed to X-ray film (Amersham). Band
intensities were determined by phosphorimaging.
Preparation and_a_n_alysis of proteins.

Infected cells were lysed with sample buffer (5% SDS, 0.03% bromophenol blue,
20% glycerol, 5% B-mercaptoethanol) and boiled at 100°C for 5 minutes. Aliquots were
electrophoresed in 5% stacking/10% resolving polyacrylamide (BIORAD) and
electroblotted to PDVF membranes (N EN). Peritoneal or ascites fluid from rats, which
had developed tumors after injection with polyoma-transformed cells, was used as the
primary antibody to detect polyoma T antigens. Primary antibody for detection of VP1
(rabbit anti-VP1) was a gift from Robert Garcea. Goat anti-rat IgG and goat anti-rabbit
coupled to horseradish peroxidase (PIERCE) were used as secondary antibodies. For
detection, chemiluminescense was performed using SuperSignal solutions (PIERCE) and

membranes were exposed to X-ray film (Amersham).

RESULTS

Infection of FR3T3 cells with the th2 virus strain induces a G2/M cell cycle arrest.

As described previously (Chapter 2), infection of FR3T3 cells with the B2 mutant
results in a G2/M cell cycle arrest in a proportion of the total population. A similar
experiment was carried out with the thZ virus. Cells were synchronized in GO by
growth to confluence followed by 24 hours of serum starvation. Cells were released from

GO by re—plating at a lower density and infected with a MOI of SO-pfu/cell four hours

119

post release. The experiment was followed for 96 hours post release. As seen in 32-
infected F R3T3 cells, two populations were distinguished: one that rounded up and
floated into the culture medium, and one that remained adherent as a monolayer. These
populations were either combined to give a total population (T) or separated to analyze
the contribution of the monolayer (M) and the supernatant (S) to the characteristis of the
total population.

Analysis of the kinetics of the progression through the cell cycle demonstrated
that infection with the th2 virus had little effect on progression through the first cell
cycle (data not shown). At 48 hours post release, the uninfected population had 51% in
G1, 38% in S, and 11% in G2/M while the th2-infected population had 45% in G1,
28% in S, and 27% in G2/M. By 60 hours post release the separation of the supernatant
and monolayer became clear and the G2/M cell cycle arrest was evident by analysis of
cells in the supernatant (Table 3-1). At 60 hours post release, 64% of the cells in the
supernatant contained G2/M DNA content versus 16% of the cells in the monolayer. At
this time, the total population contained approximately 32% of the cells with G2/M DNA
content. At 72 hours post release, 60% of the total population and the supernatant
contained cells with G2/M DNA content while cells in the monolayer only had 20% with
G2/M DNA content. At 84 and 96 hours post release, the majority of the cells with
G2/M DNA content were found in the supernatant. Also present in the supernatant was a
population of cells with sub-G1 DNA content similar to that seen in BZ-infected FR3T3
cells (data not shown). From 60 to 96 hours post release, the uninfected population only

contained approximately 10% of the population with GZ/M DNA content.

120

 

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Infection of FR3T3 cells with thZ results in a delay in transformation of th2
compared to infection with wild _t_v_2e A2.

To test the effect of the induced cell cycle arrest by thZ on transformation,
FR3T3 cells were synchronized in GO and infected with either A2 or th2 at a
multiplicity of l or lOpfu/cell as described in Materials and Methods. As seen in Figure
3-1, cells infected with A2 developed transformed foci overgrowing the monolayer by
two weeks post infection in a dose dependent manner. On the other hand, F R3T3 cells
infected with the th2 strain did not develop transformed foci until three to four weeks
post infection (Figure 3-2). Furthermore, the apparent number of transformed foci in the
th2-infected cells was reduced. The actual magnitude of the decrease cannot be
calculated because wild type infected cells were confluent with transformed foci. In
contrast, the transformed foci developed in thZ-infected cells were distinct.

Several transformed foci from A2- and th2-infected F R3T3 cells were selected,
expanded, and tested for the presence of early and late proteins, and for viral DNA. Of
eighteen A2 transformed foci selected, all expressed small T and middle T, twelve
expressed a normal-sized LT, and none expressed VP1 (Figure 3-3). To investigate the
integrity of the integrated genome, specifically the large T and middle T coding region,
digestion of total DNA with Msp I was carried out. Digestion fragments 4, 5, 7, and 8
encompass the middle T coding region while fragments 2 and 6 correspond to the distal
portion of the large T coding region (see Figure 1-1). Because fragments 7 and 8 were
run out of the gel, and fiagments 5 and 6 are difficult to separate, particular attention was

paid to fragments 2 (large T) and 4 (middle T). Figure 3-4 shows results from Msp I

121

digestion of the A2-transformed cell lines. Because of the requirement for middle T
expression by polyoma-transformed cell lines, all cell lines contain a normal-sized
fragment 4, as expected. Cell lines 21, 26, and 32 had apparent reduction in fiagment 2
size that may correspond to the observed smaller LT protein seen in Figure 3-3. Cell
lines 21, 26, 32, 33, 36, 38, and 39 showed a decrease in total signal, which matches the
decrease in LT antigen levels seen by western analysis.

Analysis of cell lines from th2 transformed foci yielded strikingly different
results. As expected, all cell lines expressed small T and middle T (Figure 3-5).
However, only 5 of 18 expressed a normal-sized LT. Similar to the A2-transformed cell
lines, none of the cell lines developed from th2 transformed foci expressed VP1.
Figure 3-6 shows the results from digestion of total DNA with Msp 1. Similar to A2
transformed cell lines, all th2 transformed cell lines exhibited a normal sized fi'agment
4. Unlike the A2-transformed cell lines, few th2-transformed cell lines exhibited
strong hybridization signal. Those that did (2, 10, 11, 12, and 16) correspond to those
cell lines that expressed detectable levels of a normal-sized LT antigen. Cell line #9 also
exhibited a strong hybridization signal despite an apparent small-sized LT antigen. In
this case, it is apparent that the LT expressed in this cell line is functional for in situ
amplification. Cell lines 1, 4, 6, 7, 8, 14, and 17 are missing an apparent fragment 2.
Cell lines 3, 5, 13, 15, and 18 appear to have a normal Msp I fragment 2; however, further
analysis will need to be carried out to determine if these fragments are in fact from the
large T coding region. Western analysis of these particular cell lines showed that they do

not express a normal-sized large T protein.

122

 

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The 32 mutant also affects transformation of wild type A2 in mixed infections.

FR3T3 cells were synchronized in GO and infected with the wild type A2 strain,
the B2 mutant, or both at a multiplicity of l and lOpfii/cell as described in Materials and
Methods. Cell cycle analysis was carried out for the first 72 hours. The cell cycle
profiles at 72 hours post release are shown in Table 3-11. As can be seen, 96% of the
uninfected population contained 1C DNA content. These cells had reached confluency
and returned to G0. The same was observed in A2-infected cells at both multiplicities of
infection. This is expected because the fiaction of FR3T3 cells transformed, or
abortively transformed, is typically less than 0.5% (4, 5). As described previously, a
fraction of the population detached fiom the monolayer in B2-infected FR3T3 cells. A
similar separation was seen in the A2+B2 mixed infection. At 72 hours post release these
populations were analyzed separately. FACS analysis of these cells showed that
approximately 50% of the cells in the supernatant of BZ- and A2+B2-infected cells
contained G2/M DNA content. Less than 10% of the monolayer in all other populations
contained G2/M DNA content.

Analysis of viral proteins (Figure 3-7) showed that at 72 hours post release no
expression of small T and middle T could be detected in the mixed infection, presumably
reflecting the fact that the B2 mutant was preferentially expressed over wild type A2.
The expression of small T and middle T in the mixed infection was detectable by one-
week post infection. The expression of VP1 was detectable at a MOI of 10 out to two
weeks post infection in the B2- and A2+B2-infections; however, at two weeks post

infection VP1 was not detected in the A2-infected cells. VP1 was also not detected at the

123

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low multiplicity in A2-infected cells. In contrast, in B2-infected and A2+BZ-infected
cells VP1 could be detected at a MOI of 1 early in the infection.

Analysis of viral genomes in the mixed infections demonstrated that early in the
course of the infection, the B2 mutant was prevalent while later in the infection wild type
A2 became prevalent. Each genome can be tracked by the difference in size of Msp I
fiagments 3 and 4. The BZ mutant contains a duplication of the enhancer region,
resulting in a larger fragment 3, and a deletion in the LT intron, resulting in a smaller
fragment 4. In this experiment, the ratio of wild type A2 to mutant B2 was calculated
using the band intensities of fragment 4. At 24 hours post infection the A2:B2 ratio was
1:3 at both MOIs; however, at 48 hours post infection the A2:B2 ratio was 1:3 at 8 MOI
of l but 1:8 at a MOI of 10 (data not shown). At 72 hours post infection (Figure 3-8), the
ratio of wild type A2 to mutant BZ was 1:3 at a MOI of 1, 1:5 in the monolayer at a MOI
of 10, and 1:6 in the supernatant at 3 MOI of 10. By one-week post infection the ratio of
wild type A2 to mutant B2 was 1:2 and 1:3 at a MOI of 1 and 10, respectively (data not
shown). However, by two weeks post infection (Figure 3-9) the ratio of wild type A2 to
mutant B2 reversed to 3:1 and 2:1 at a MOI of 1 and 10, respectively. These results are
summarized in Table 3-III.

At two weeks post infection, transformed foci were fixed and stained as described
in Materials and Methods (Figure 3-10). At a multiplicity of 1 pfu/cell for each virus, the
number of transformed foci developed in the A2+B2 mixed infection was similar to that
seen in the A2-infection alone. However, at a MOI of 10 pfu/cell, a decrease in the
number of transformed foci was seen in the mixed infection. Several transformed foci

were isolated and expanded to create A2+B2-transformed cell lines. Analysis of viral

124

proteins (Figure 3-11) showed that at least 13, and possibly 15, of 18 cell lines expressed
a normal-sized LT antigen. As expected, all cell lines expressed small T and middle T
and none expressed detectable amounts of VP1. Figure 3-12 showed the results of
digestion of total DNA extracts with Msp 1. None of the cell lines had a detectable B2
fi'agment 4. Most cell lines exhibited a strong hybridization signal and these
corresponded to those that expressed large amounts of normal-sized LT antigen. Cell
lines 50, 57, and 63, which did not express a normal-sized T antigen, were missing a
normal-sized fiagment 2. Cell lines 61 and 67, which initially appeared to express a
normal sized large T, are also missing a normal-sized fragment 2. Based on these
comparisons, it appears that 13 of 18 A2+BZ cell lines exhibit expression of a normal-
sized large T. These results are similar to those seen in A2-transformed cell lines and are

quite different from the results seen in the th2-transformed cell lines.

DISCUSSION

We have previously shown that infection of F R3T3 cells with the hr-t mutant, B2,
results in a G2/M cell cycle arrest in a proportion of the total population (Chapter 2).
Here we report that similar results are seen with a ‘wild-type’ virus constructed in the B2
genetic background, th2. The th2-induced cell cycle arrest probably contributes to
the observed delay in the development of transformed foci compared to wild-type A2-
infected cells. Presumably, cells infected with the th2 virus must overcome the
induced G2/M cell cycle arrest before transformed foci develop. This can be achieved by

integration of the genome such that the sequences that contribute to the cell cycle arrest

125

are no longer present. Because integration is a rare event, along with the requirement for
selective integration, the development of transformed foci in the th2-infected cells is
delayed.

Experiments were also carried out in which FR3T3 cells were infected with both
wild-type A2 and the B2 mutant. In these experiments, a fi'action of the population
arrested with G2/M DNA content by 72 hours post release. Early in the course of the
experiment, the BZ genome was preferentially expressed and small T and middle T were
not detected. Of interest is that VP1 was detected through the first two weeks in B2- and
A2+B2-infected cells at the high dose. In contrast, in A2-infected cells, VP1 expression
was detected until one-week post infection but not at two weeks post infection. This
suggests that the VP1 detected in the mixed infection is being expressed fiom the B2
genome. Furthermore, as the experiment progressed the ratio of wild typezmutant
genomes shifted. Early in the course of the infection, the B2 genome was prevalent. On
the other hand, by two weeks post infection, the A2 genome became prevalent. This is
presumably due to the fact that those cells with a high mutantzwild type ratio arrested in
G2/M, lifted off from the monolayer, and were lost.

Several transformed foci from th2-, A2-, and A2+B2-infected FR3T3 cells were
selected and expanded to create transformed cell lines. Analysis of protein expression
and the integrity of the integrated viral genomes reveal some similarities and some
striking differences. First, all of the cell lines express small T and middle T and none
express VP1. This is expected since middle T is required for transformation. Because
small T is encompassed within the middle T coding region, its expression is also

expected. The requirement for an intact middle T coding region is also seen in the

126

analysis of viral DNA. All cell lines tested contain a relatively normal-sized Msp I
fragment 4.

Second, only 28% of the th2-transformed cell lines express a normal-sized large
T antigen while most (greater than 80%) of the A2- and A2+B2-transformed cell lines do.
The cell lines that do not contain a normal-sized large T often appear to be missing a
normal-sized Msp I fragment 2. Fragment 2 encompasses the distal portion of the large T
coding region. Many of the properties of the B2 mutant appear to map to this region of
the genome (see Chapter 4). Because the th2 genome is ‘BZ-like’ in this region, the
loss, or alteration, of fragment 2 presumably explains how transformed foci are able to
develop in th2-infections. The other thZ-transformed cell lines (e.g. #9-12), that do
express an apparent normal-sized large T protein, may have resolved the ‘toxicity
problem’ of the B2 mutant in other ways. First, it is possible that integration disrupted
the ability of these mutants to express high levels of VP1. Second, mutation of the
sequence that contributes to the cell cycle arrest may have occurred. Third, mutations in
the host cell may compensate for the presence of the B2 mutant sequence. Further
analysis of these cell lines will need to be carried out to resolve this issue. The A2+BZ-
transformed cell lines resolve the issue of ‘toxic’ B2 sequences through elimination of the
BZ genome (see below).

Third, most of the A2- and A2+B2-transformed cell lines exhibited strong
hybridization signals while the th2-transformed cell lines did not. This may reflect the
ability of A2-transformed cell lines to undergo in situ amplification of the integrated
genome (12), which requires a functional large T protein and the reiteration of part of the

viral genome. Because many of the cell lines tested appeared to exhibit the presence of

127

all Msp I fiagments, we expect that they will also contain tandem duplications of at least
part of the viral genome.

Early experiments with A2-transformed rat cells (1, 8) showed the viral genome is
present both as fiee viral DNA and integrated into the cellular genome. The amount of
free virus decreased with passage (1). Most of the integrated genomes are present in
more than one copy and were organized in head-to-tail tandem arrays (l, 8). A few cell
lines contained less than one genome integrated at a single site. These lines did not
express a normal-sized large T antigen (8, 9). In contrast to transformed rat cell lines,
transformed hamster cell lines displayed a lack of tandem repeats and a lack of a portion
of the large T coding region (7). The A2- and th2-transformed cell lines generated in
this study will require further study to ascertain whether free viral genomes are present
and the topology of the integrated genomes. One would predict that few free viral
genomes would be present in the th2-transformed cells because of the induced G2/M
cell cycle arrest induced by this virus. Also of interest would be to determine if fusion of
the th2-transformed cells to NIH3T3 cell would result in production of live virus. The
expectation is that most would not result in the production of progeny virions.

In early experiments with hr-t and ts-a mixed infections (2), transformed clones
were obtained, via complementation. Characterization of several clones revealed that
many contained both parental genomes. Furthermore, firsion of the transformed clones
with permissive NIH3T3 cells resulted in the production of both hr-t and ts-a virus
progeny. It would be interesting to see if fusion of the A2+BZ-transformed cell lines

with NIH3T3 cell would result in the production of both A2 and B2 viral progeny. Since

128

the B2 mutant fi'agments 3 and 4 were not detected in A2+B2 transformed cell lines, the

expectation is that the B2 mutant virus would not be recovered from these cells.

129

APPENDIX 3: TABLES AND FIGURES FOR CHAPTER 3

130

Table 3-1: FACS Analysis of A2- and th2-infected FR3T3 cells.

FR3T3 cell were synchronized, released from G0, and infected with 3 MOI of 50
pfu/cell as described in Materials and Methods. Samples were collected at various times,
FACS analysis was carried out on 5000 cells for each sample, and the percentage of cells
in G1, S, and G2/M were calculated. The results of samples collected from 60 to 96

hours post release are shown.

131

Table 3-1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

UNINFECTED WTB2 INFECTED
hpr %G1 %S %G2/M %G1 %S %GZIM

6O 61 28 11 39 29 32
60 M] 56 28 16
60 S 23 13 64
72 66 23 ll 27 14 60
72 53 27 20
72 S 15 14 61
84 75 13 12 66 15 19
84 M] 64 2O 16
84 S 24 ' 18 58
96 83 7 10 67 16 17
96 M 65 18 17
96 S 29 18 52

 

 

 

 

 

 

 

132

 

Figure 3-1: A2-infected FR3T3 cells develop transformed foci by two weeks post
infection.

A2-infected FR3T3 cells were fixed and stained at two weeks post infection as
described in Materials and Methods. The results fiom infecting at a MOI of l and 10

pfu/cell are shown. Uninfected FR3T3 cells were also stained for comparison.

51“

10113

mdll

FR3T3

A2MOIl

134

A2MOI 10

 

Figure 3-2: Infection of FR3T3 cells with the thZ virus results in the development of
transformed foci by three to four weeks post infection.

FR3T3 cells were infected with a MOI of 1 and 10 pfu/cell as described in
Materials and Methods. Plates were fixed and stained to test for the presence of

transformed foci at two, three, and four weeks post infection.

135

2 Weeks 3 Weeks 4 Weeks

11111111 MOI 1

rlbedi

MOI 10

 

136

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rid-1431'! «a is ..

Figure 3-3: Analysis of viral proteins from A2-transforrned cell lines.
Transformed foci from A2-infected FR3T3 cells were isolated and expanded.
Protein lysates were collected and assayed for the presence of early and late viral

proteins. Also included was an A2-infected NIH3T3 sample for reference (*).

137

    

 

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138

Figure 3-4: Analysis of the integrity of integrated viral genomes in A2-transformed cell
lines.

Total DNA from A2-transformed cell lines was extracted, digested with
restriction endonuclease Msp I, electrophoresed, blotted to nitrocellulose membranes, and

hybridized to polyoma A2 genomic DNA. This is a 48-hour exposure.

139

 

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Figure 3-5: Analysis of viral proteins from thZ-transformed cell lines.
Transformed foci from thZ-infected F R3T3 cells were isolated and expanded.
Protein lysates were collected and assayed for the presence of early and late viral

proteins. Also included was an A2-infected NIH3T3 sample for reference (*).

141

 

w

142

Figure 3-6: Analysis of the integrity of integrated viral genomes in th2-transformed
cell lines.

Total DNA from th2-transformed cell lines was extracted, digested with Msp I,
electrophoresed, blotted to nitrocellulose membranes, and hybridized to polyoma A2

genomic. This is a one-week exposure.

143

LT Status

13 14 15 l6 17

12

ng

 

\o
\
to
O0
LL

Table 3-11: FACS Analysis of A2-, B2-, and A2+B2-infected FR3T3 cells.

FR3T3 cell were synchronized, released from GO, and infected with a MOI of 1
and 10 pfu/cell with each virus as described in Materials and Methods. Samples were
collected at various times, FACS analysis was carried out on 5000 cells for each sample,
and the percentage of cells in G], S, and G2fM were calculated. The results at 72 hours

post release are shown.

145

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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146

.-

Figure 3-7: Protein analysis of A2-, B2-, and A2+B2-infected FR3T3 cells.

FR3T3 cells were synchronized and released from GO and infected with l and 10
pfu/cell with each virus as described in Materials and Methods. Protein lysates were
collected at various times and assayed for the presence of early and late viral proteins.

Results from 72 hours, 7 days, and two weeks post infection are shown.

147

 

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148

Figure 3-8: Analysis of viral genomes in A2-, BZ-, and A2+B2-infected FR3T3 cells.
FR3T3 cells were synchronized and released fiom GO and infected with 1 and 10
pfu/cell with each virus as described in Materials and Methods. Total DNA was
extracted at various times post infection and digested with Msp I. Results from 72 hours
post infection are shown. Fragments l, 2, 3, 5/6, and A2- and B2-fragments 4 are
indicated. The results from separation of the populations into the monolayer (M) and

supernatant (S) are also indicated.

149

133

A149 8

IE N<

413

 

 

 

41%

IE

we 22 _ we 22 _ 2 _ 52
8+? 8 N44

150

Figure 3-9: Analysis of viral genomes in A2-, B2-, and A2+B2-infected FR3T3 cells.

F R3T3 cells were synchronized and released from G0 and infected with 1 and 10
pfu/cell with each virus as described in Materials and Methods. Total DNA was
extracted at various times post infection and digested with Msp I. Results from two
weeks post infection are shown. Fragments 1, 2, 3, 5/6, and A2- and B2-fragments 4 are

indicated.

151

A2 B2 A2+B2

 

M01 1 M01 10 M01 1 M01 10 M01 1 M01 10

 

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11111211111
DNA 1‘1 fg3—>
; 110111111
1161113411

A2 fg4—>

132 fg4—>

 

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152

Table 3-III: Summary of the wild type A2 to mutant B2 genome ratio in A2+B2-infected

FR3T3 cells.

153

 

 

 

 

 

 

 

 

 

 

 

 

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3.. 2 m; w; m; 22 52
E m; m; 2 m; :02

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154

Figure 3-10: A2-infected and A2+BZ-infected FR3T3 cells develop transformed foci by

two weeks post infection.
A2-infected and A2+B2-infected FR3T3 cells were fixed and stained at two
weeks post infection as described in Materials and Methods. The results from infecting

at a MOI of l and 10 pfu/cell are shown.

155

M01 1 M01 10

A2+B2

 

156

Figure 3-11: Analysis of viral proteins fiom A2+B2-transformed cell lines.
Transformed foci from A2+B2-infected FR3T3 cells were isolated and expanded.
Protein lysates were collected and assayed for the presence of early and late viral

proteins. Also included was an A2-infected NIH3T3 sample for reference (*).

157

 

 

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Wm
WM

158

Figure 3-12: Analysis the integrity of integrated viral genomes in A2+B2-transformed
cell lines.

Total DNA from A2+B2-transformed cell lines was extracted, digested with Msp
I, electrophoresed, blotted to nitrocellulose membranes, and hybridized to polyoma A2

genomic DNA. This is a 48-hour exposure.

159

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REFERENCES

Birg, F., R. Dulbecco, M. Fried, and R. Kamen 1979. State and organization of
polyoma virus DNA sequences in transformed rat cell lines J Virol. 29:633-48.

Fluck, M. M., R. Shaikh, and T. L. Benjamin 1983. An analysis of transformed
clones obtained by coinfections with hr-t and ts-a mutants of polyoma virus
Virology. 130:29-43.

Fluck, M. M., R. J. Staneloni, and T. L. Benjamin 1977. Hr-t and ts-a: two
early gene functions of polyoma virus Virology. 77:610-24.

Fogel, M., and L. Sachs 1969. The activation of virus synthesis in polyoma—
transformed cells Virology. 37 :327-34.

Fogel, M., and L. Sachs 1970. Induction of virus synthesis in polyoma
transformed cells by ultraviolet light and mitomycin C Virology. 40: 174-7.

Hattori, J., G. G. Carmichael, and T. L. Benjamin 1979. DNA sequence
alterations in Hr-t deletion mutants of polyoma virus Cell. 16:505-13.

Israel, M. A., D. F. Vanderryn, M. L. Meltzer, and M. A. Martin 1980.
Characterization of polyoma viral DNA sequences in polyoma-induced hamster
tumor cell lines J Biol Chem. 255:3798-805.

Lania, L., D. Gandini-Attardi, M. Griffiths, B. Cooke, D. De Cicco, and M.
Fried 1980. The polyoma virus 100K large T-antigen is not required for the
maintenance of transformation Virology. 101:217-32.

Lania, L., A. Hayday, G. Bjursell, D. Gandini-Attardi, and M. Fried 1980.
Organization and expression of integrated polyoma virus DNA sequences in
transformed rodent cells Cold Spring Harb Symp Quant Biol. 44:597-603.

Silver, J., B. Schaffhausen, and T. Benjamin 1978. Tumor antigens induced by
nontransforming mutants of polyoma virus Cell. 15:485-96.

Soeda, E., J. R. Arrand, N. Smolar, J. E. Walsh, and B. E. Griffin 1980.
Coding potential and regulatory signals of the polyoma virus genome Nature.
283:445-53.

Syu, L. J., and M. M. Fluck 1997. Site-specific in situ amplification of the

integrated polyomavirus genome: a case for a context-specific over-replication
model of gene amplification J Mol Biol. 271:76-99.

161

 

 

 

CHAPTER 4

MAPPING THE MUTATION (S) [N THE BZ MUTANT THAT CONTRIBUTE
TO ITS REPLICATION POTENTIAL AND ITS PREFERENTIAL GENE
EXPRESSION AND AMPLIFICATION IN MIXED INFECTIONS WITH WILD
TYPE A2

ABSTRACT

We reported that the hr-t mutant, B2, amplifies its genome to high levels despite
the absence of small T and middle T expression. Furthermore, in mixed infections with
wild-type A2, the B2 genome is preferentially expressed and amplified, resulting in a
reduced expression of small T and middle T by the A2 genome. Here we report that
these properties of the B2 mutant map to sequences that encompass a portion of the LT

coding region, specifically to the EcoR I/Nsi I fragment (nucleotides 1562-1912).

INTRODUCTION

In previous chapters, several unique properties of the B2 mutant, a member of the
hr-t class of mutants, were described. These include the induction of a G2/M cell cycle
arrest, the capacity to express viral proteins and replicate viral DNA to high levels in the
absence of small T and middle T, and the preferential gene expression and amplification
in mixed infections with wild type A2. Early experiments demonstrated preferential gene
expression and replication of hr-t mutants, specifically NG18, in mixed infections with

wild type viruses (10). Experiments in our lab have also shown that B2 has a 20-fold

162

 

replicative advantage in competition with the A185 strain, a small T/rniddle T defective
virus in the A2 genetic background (4). Sequence analysis has revealed several
mutations throughout the genome when compared to the wild type A2 DNA sequence. In
the present chapter, an attempt to map the mutation(s) in the BZ mutant that contribute to

the replication potential and advantage over A2-derived viruses was carried out.

MATERIALS AND METHODS

XiLusesi

Wild type A2 (11) and the sT/mT defective A185 (8) have been described
previously. The B2 mutant used in this study contains a repair of the 11 base pair
deletion in the early region from the original isolate of B2 (7). Table 4-1 lists five new
viruses in which mutant B2 sequences were replaced with wild type A2 sequences.
Exchanges 1-4 have novel restriction sites in the exchanged fi'agment. These restriction
sites were created by conservative changes in the sequence of A2. Exchange 5 can be
distinguished fi'om B2 by a smaller Msp I fragment 3 due to the absence of a duplication
in the enhancer. The B2 and A2 viral DNA was digested with the restriction
endonucleases indicated and fiagrnents were gel purified. The large genomic fragments
from digestion of the B2 mutant were ligated with the corresponding small marked
fragments from wild type A2. Ligations were transfected into NIH3T3 cells using
lipofectamine (GIBCO) according to manufacturer’s specifications. After CPE was
observed, lysates were collected and analyzed for the introduction of the novel restriction

site or the smaller Msp I fragment 3. The new viruses were plaque purified, re-checked,

163

and virus stocks were prepared in NH-I3T3 or 3T6 cells and checked for the absence of
defectives. Titers were calculated using standard plaque assay techniques and by
comparison of input DNA.

Cells and infections,

NIH3T3 cells were cultured in Dulbecco’s modified Eagle medium (DMEM;
GIBCO) supplemented with 10% heat inactivated bovine calf serum (GIBCO, SIGMA)
at 37°C and 5% C02. For induction of quiescence and release fiom GO cells were grown
to 90% confluence followed by 24 hours starvation with 0.5% serum medium. Cells
were released from 00 by re-plating at a lower density in 10% serum medium.
Adsorptions were canied out after reattachment (4 hours) for one hour at 37 °C and 5%
C02. Infections were carried out at a multiplicity of infection (MOI) of 10 plaque-
forrning units per cell (pfu/cell), and the infection lysate was removed prior to the
addition 10% serum medium. For mixed infections, a MOI of 10 pfu/cell for each virus
was used.

Preparation and analysis of viral DNA

Infected cells were lysed in lOmM Tris, lOmM EDTA, and 0.2% sodium dodecyl
sulfate (SDS) (pH7 .6). Protein digestion was carried out overnight at 37°C with SOug/ml
proteinase K (Sigma). Total DNA was extracted with phenol-chloroform and treated
with RN aseA by standard procedures. Samples were digested with EcoR I (GIBCO
BRL) to linearize polyoma DNA, electrophoresed in 1% agarose, stained with ethidium
bromide to confirm equivalent loading, and blotted to Hybond membranes (Amersham).
Samples from mixed infections were digested with Msp I (GIBCO BRL) which cuts

polyoma into eight fragments, electrophoresed in 2% agarose, stained, and blotted as

164

described above. Hybridization was carried out at 65°C in 1X Denhardt’s solution,
SXSSPE, and 0.5% SDS by standard procedures with a 32P-radiolabelled probe,
consisting of the polyomavirus A2 genome. Hybridization probes were labeled to a
specific activity of 1-2 x 109 cpm/ug with (32P) dCTP (3,000 uCi/mmol; New England
Nuclear) using a multiprime DNA-labeling kit (Amersham). Hybridized blots were
washed using stringent conditions and exposed to X-ray film (Amersham). Band
intensities were determined by phosphorimaging.
Preparation grid analysis of protein;

Infected cells were lysed with sample buffer (5% SDS, 0.03% bromophenol blue,
20% glycerol, 5% B-mercaptoethanol) and boiled at 100°C for 5 minutes. Aliquots were
electrophoresed in 5% stacking/10% resolving polyacrylamide (BIORAD) and
electroblotted to PDVF membranes (N EN). Peritoneal or ascites fluid from rats, which
had developed tumors after injection with polyoma-transformed cells, was used as the
primary antibody to detect polyoma T antigens. Primary antibody for detection of VP1
(rabbit anti-VP1) was a gift from Robert Garcea. Goat anti-rat IgG and goat anti-rabbit
coupled to horseradish peroxidase (PIERCE) were used as secondary antibodies. For
detection, chemiluminescense was performed using SuperSignal solutions (PIERCE) and

membranes were exposed to X-ray film (Amersham).

165

 

RESULTS

The replication mtential of the B2 mutant maps to a seguence encompassing the LT

codin r 'on.

The expression of viral proteins and the accumulation of viral genomes by the B2
mutant, and the five newly constructed virus strains described in Materials and Methods
was compared in NIH3T3 cells. Cells were synchronized in GO and infected four hours
post release with lOpfu/cell. Analysis of LT and VP1 protein levels at 24 and 48 hours
post release (Figure 4-1A) showed that all viruses expressed LT to similar levels;
however, exchange #4 exhibited a large difference in the accumulation of VP1 protein by
48 hours post release. Analysis of viral DNA replication (Figure 4-lB) showed that all
virus constructs amplified .their DNA to similar levels (greater than lOO-fold) except for
exchange #4. Exchange #1, #2, #3, and #5 amplified their genomes 77-, 158-, 140-, and
250-fold, respectively. On the other hand, exchange #4 only amplified its genome 20-

fold by 48 hours post release.

The preferential expression and amplification of the B2 genome in mixed infections
with wild type A2 also maps to a seguence encompassing the LT coding region.

To investigate which region contributes to the preferential expression and

amplification of the B2 genome in mixed infections with wild type A2, mixed infections

166

 

of wild type A2 with the B2 mutant and the five viral exchanges were carried out and
compared to A2+Al85 mutant mixed infections. Cells were infected at a multiplicity of
lOpfir/cell with each virus in GO as described in Materials and Methods. Analysis of
expressed proteins (Figure 4-2) at 24 and 48 hours post release demonstrates that in
mixed infections, expression of small T and middle T by wild type A2 was only seen
when in combination with either A185 or exchange #4. Furthermore, the overproduction
of VP1 was not seen in mixed infections with A2 and A185 or exchange #4 (data not
shown).

When the replication of each genome was measured in the mixed infections, a
similar pattern was seen (Figure 4-3). When A2 was in the presence of BZ, exchange #1,
#2, #3, or #5, the amplification of its genome was impaired compared to the amplification
seen when mixed with either A185 or exchange #4. For each mixed infection, the
amplification of each genome over input was calculated. This fold amplification was
calculated based on band intensities of Msp I fragment 4 for each virus (the difference in
size was compensated for). A wild type:mutant genome ratio was calculated using
fragment 4 band intensities at 48 hours post release. In the mixed infections of wild type
A2 and mutant B2, the A2 genome amplified 40-fold while the B2 genome amplified
300-fold resulting in a wild-type:mutant genome ratio of 1:12. In the wild type A2 and
exchange #1 mixed infection, the A2 genome amplified 50-fold and #1 amplified 130-
fold resulting in a wild-type:mutant ratio of 1:4. In the wild type A2 and exchange #2
mixed infection, the A2 genome amplified 50-fold and the #2 genome amplified BOO-fold
resulting in a wild-type:mutant ratio of 1:10. In the wild type A2 and exchange #3 mixed

infection the A2 genome amplified 30-fold and #3 amplified 300-fold resulting in a wild-

167

‘1

type:mutant ratio of 1:13. In the mixed infection of wild type A2 and #5, the A2 genome
amplified 50-fold and #5 amplified 200-fold resulting in a wild-type:mutant ratio of 1:7.
0n the other hand, in mixed infections of wild type A2 and exchange #4, the A2 genome
amplified 250-fold and #4 amplified lOO-fold resulting in a wild type:mutant ratio of
1:0.5. Similarly, in the mixed infection of wild type A2 and the A185 mutant, the A2
genome amplified ISO-fold and the A185 genome amplified 30-fold resulting in a wild

type:mutant ratio of 1:02.

The high expression and replication mtential of the 82 mutant maps to the EcoR

I/Nsi I sguence.

To further define the mutation(s) in the BZ mutant that contribute to its replication
potential, several new viruses were constructed. Restriction endonuclease sites at 1079
(Blp I), 1562 (EcoR I), 1912 (Nsi I), 2120 (Bsu36 I), and 2225 (Afl H) were used to
generate B2 (A2 Blp-RI), B2 (A2 RI-Nsi), B2 (A2 Nsi-Bsu), and B2 (A2 Bsu-Afl) in a
similar manner to the first five exchanges constructed. Recombinant viruses were created
and purified as described in Materials and Methods. NIHBT3 cells were synchronized in
GO and infected at a multiplicity of lOpfu/cell as described previously. Analysis of viral
protein levels (Figure 4-4A) showed that replacement of the EcoR I/Nsi I fragment of the
BZ mutant with wild type A2 sequences resulted in a dramatic decrease in expression of
both LT and VP1 proteins. Quantitation of viral DNA amplification (Figure 4-4B)
showed that the replication potential of the B2 mutant also mapped to the EcoR I/Nsi I
fragment. The B2(A2 Blp-RI), B2(A2 Nsi-Bsu), and B2(Bsu-Afl) genomes all amplified

approximately ZOO-fold while the B2(A2 RI-Nsi) only amplified S-fold. This represents

168

 

 

 

a 40-fold decrease in viral amplification at 48 hours post release. The expression of viral
proteins and viral DNA replication seen in this construct resembles that of the A185

mutant.
DISCUSSION

The polyomavirus hr-t mutant, B2, has the ability to express viral proteins and
amplify viral genomes to high levels despite the absence of small T and middle T
expression. This property is not evident the small T/middle T mutant A185, which is in
the A2 genetic background (2-4). Preliminary sequence analysis of the BZ genome
revealed mutations throughout its genome. Therefore, a series of virus exchanges were
constructed to map the mutation(s) that contribute to the ability of B2 to exhibit a high
expression/replication phenotype in the absence of small T and middle T expression.
Results described here suggest that this property of B2 maps to sequences that encompass
a portion of the LT coding region. Replacement of nucleotides 1079-2225 results in a
decrease in viral DNA replication similar to that seen when replication of the A185
mutant is compared to that of wild type A2. In the experiment presented here, this
decrease was approximately S-fold. In repeated experiments, the defect in replication of
exchange #4 varies from 5-fold to 20-fold. Further constructs mapped the high
replication potential of the B2 mutant to the EcoR I/Nsi I fi'agment (nucleotides 1562-
1912). Replacement of these sequences in the B2 mutant with wild type A2 sequences

resulted in a 40-fold decrease in viral DNA amplification.

169

l.

“a“

The ability of the B2 mutant to preferentially express and replicate its genome in
competition with wild type A2 also mapped to sequences from nucleotides 1079 to 2225.
Results demonstrate that in mixed infections of wild type A2 with the B2 mutant and B2
exchanges #1, #2, #3 and #5 the expression of small T and middle T from wild type A2
was not detected, and the amplification of A2 viral genomes was decreased. However,
co-infections of wild type A2 and B2 exchange #4 showed expression of small T and
middle T fi'om the A2 genome. Furthermore, the A2 genome was preferentially
amplified, similar to mixed infections of wild type A2 and mutant A185. Based on the
results discussed above, we predict that co-infection of wild type A2 with B2 (A2 RI/Nsi)
will yield similar results. Preliminary experiments have supported this hypothesis.

Several properties can be assigned to the B2 mutant that are not seen in the small
T/middle T defective mutant (A185) in the A2 genetic background. These include the
ability of B2 to express and replicate its genome despite a lack of small T and middle
T expression, the preferential expression and replication of the B2 genome in mixed
infections, the ability to inhibit transformation (Chapter 3), and the ability to induce a
G2/M cell cycle arrest (Chapter 2). The data presented here suggest that the first two of
these phenotypes map to the same sequence on the B2 genome. We predict that the other
phenotypes will map to the same sequence.

The EcoR I/Nsi I sequence encompasses amino acids 334-451 of the LT protein,
which contains part of the DNA binding domain (12). Other experiments in our lab (6)
have mapped a mutation 59RA, another hr-t mutant, which affects viral DNA synthesis in
Rat F-l 11 cells to the EcoR I/Nsi I fragment. The only nucleotide change present in this

region of 59RA is a C-to-G transition at nucleotide 1791, which changes proline to

170

alanine at amino acid 412 of LT. This is part of an amino acid sequence, Pro-Glu-Glu,
which is conserved in SV40 (Brian Schaufflrausen, personal communication). In 59RA,
this sequence is changed to Ala-Glu-Glu. Interestingly, preliminary sequence analysis of
the B2 sequence suggests that this sequence is also altered, possibly to Pro-Lys-Lys.
Both mutant sequences have profound changes in the amino acid composition, which.
could alter the function of the LT protein.

Other classes of mutants in which the mutant virus prevents replication of the
wild type virus have been reported for both polyoma and SV40 (5, 9, 13). In these cases,
the mutants were referred to trans-dominant mutants and were also mutants in the LT
coding region. The expression of mutant LT proteins prevented replication of DNA by
wild type LT protein. However, some key differences exist between these mutants and
the polyoma hr-t class of mutants. First, none of the trans-dominant mutants were viable.
Second, these mutants were unable to replicate their DNA. Finally, the mutations were in
the ATP-binding domain. In the case of the trans-dominant mutants of polyoma and
SV40, it was suggested that the mutant 1LT protein would form non-functional hexamers,
both by itself and in the presence of wild type LT protein (1). The case with the polyoma
hr-t mutants may be quite different. If the hr-t LT protein did not form functional
hexamers, one would not expect to see the replication of viral genomes or viable mutants.
This is clearly not the case. Furthermore, the presence of the BZ mutant does not
completely inhibit replication of the wild type A2 genome, as is the case with the trans-
dominant mutants.

Two alternate models, which are not mutually exclusive, can be envisioned.

Model 1 is the involvement of a cis-acting sequence between nucleotides 1562 and 1912.

171

 

The B2 genome contains a sequence that mediates preferential targeting of the B2
genome to a cellular site (e.g. the nuclear matrix) where both transcription and replication
take place. This same sequence in the A2 genome is weak; therefore, the B2 genome is
preferentially expressed and replicated in mixed infections with wild type A2. Alone, the
B2 genome is able to express proteins and amplify DNA to high levels in the absence of
small T and middle T expression. Model 2 is a large T mediated effect. Sequence
alterations in the B2 genome result in a LT protein that has different properties than that
of wild type A2. In this case the BZ large T cannot repress B2 early gene expression,
whereas the A2 large T can repress early gene expression, resulting in high levels of gene
expression in single infections and preferential gene expression in mixed infections. The
replication advantage of B2 could be mediated by a preferential binding to the B2 origin
or by the higher number of B2 genomes involved in active transcription, which are also
templates for DNA replication. The exact mechanism by which the B2 mutant is

preferentially expressed and replicated remains to be elucidated.

172

 

APPENDIX 4: TABLES AND FIGURES FOR CHAPTER 4

173

Table 4-I: BZ Virus Exhanges.
Five new viruses were constructed in the B2 genetic background. Exchanges #1,
#2, #3, #4, and #5 replaced nucleotides 2225-4108, 4108-5023, 4108-4634, 1079—2225,
and 5023-400 respectively with wild type A2 sequences. The restriction endonuclease
sites used to create these fragments are indicated. Furthermore, the new restriction sites ‘
introduced or the change in the Msp I digestion pattern created in the new virus strain are

also noted.

 

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Table 4-1: B2 virus exchanges

 

 

 

 

 

 

 

 

Exchange # Region New Site

1 EcoR V - A]! H Spe I
4108 — 2225

2 BC] I — EcoR V Nhe I
5023 — 41 O8

3 BamH I — EcoR V Nhe I
4634 — 4108

4 Blp I — Afl II Sac II
1079 — 2225

5 BC] I — Ber I Smaller Msp I

5023 - 400 Fragment 3

 

 

 

175

MEL-m.
. .

Figure 4-1: Mapping analysis of viral gene expression and replication of the B2 mutant.
Infection of NIH3T3 cells at a MOI of 10 pfu/cell was carried out as described in
Materials and Methods. Protein lysates were collected at 24 and 48 horas post r6113ase
and assayed for the presence of LT (A) and VP1 (B) as described in Materials and
Methods. Total DNA was collected at 8 hours post released (not shown) and 48 hours
post release (C), digested with EcoR I, and blotted to nitrocellulose membranes‘

Membranes were hybridized with polyoma A2 genomic DNA to measure viral DNA

amplification.

 

 

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Materials r.“

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: 1118111111111

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#3

#2

#1

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24

48

24

48

24

24 48 24 48

48

24

LT

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177

 

Figure 4-2: Analysis of early proteins expressed in mixed infections of wild type A2
with A185 and B2-derived mutants.

NIH3T3 cells were infected with a MOI of 10 pfu/cell with each virus as
described in Materials and Methods. Protein lysates were collected at 24 and 48 110“”
post release and assayed for the presence of small T, middle T, and large T. Results from

mixed infections of A2 with B2 (A), exchange #1 (B), exchange #2 (C), exchange #3 (D)-

exchange #4 (E), exchange #5 (F) and mutant A185 (G) are shown.

 

A B C .D E F G

01111111111131 __ _ _ __ _ ._ _
hpr 24 48 24 48 24 48 24 48 24 48 24 48 24 48

    

LT —>"
1 each 1111“
24 81148111 mT—> 1
1. 1151115111:
(changefi 111

sT—> a

179

Figure 4-3: Analysis of wild type and mutant genome amplification in mixed infections.
NIH3T3 cells were infected with 10 pfu/cell of each virus as described in
Materials and Methods. Total DNA was isolated, digested with Msp I, electrophoresed,
blotted to nitrocellulose membranes, and hybridized with polyoma A2 genomic DNA.
The Msp I digestion patterns at 48 hours post release of A2 with B2 (A), exchange #1
(B), exchange #2 (C), exchange #3 (D, exchange #4 (E), exchange #5 (F), and mutant
A185 (G) are shown. Msp I fragments 1, 2, and 5/6 are the same for all viruses. The B2
mutant contains a duplication of the enhancer region that results in a larger sized
fragment 3. The differences in Msp I fragment 4 due to deletions in the B2 and A185

mutants are indicated. The wild type:mutant ratio is also noted.

180

 

1 infants
desalted 1
110111111811
10111: D11
ruling! ‘1
and nm
a. 1111
we
1 2,1111

   

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A185 fg4—> . ~ fi.

 

 

ratio 1:12 1:4 1:10 1:13 1:0.5 1:7 1:02

181

 

Figure 4-4: The ability of the B2 mutant to express viral proteins and amplify its genome
maps to the EcoR I/Nsi I fragment.

Infection of NIH3T3 cells at a MOI of 10 pfu/cell with the newly constructed B2
(A2 Blp-RI) (lane A), B2 (A2 RI-Nsi) (lane B), B2 (A2 Nsi-Bsu) (lane C), and BZ (A2
Bsu-Afl) (lane D) were carried out as described in Materials and Methods. Protein
lysates were collected at 24 and 48 hours post release and assayed for the presence of LT
and VP1 (A). Total DNA was isolated at 8 hours post release (not shown) and 48 hours
post release (B), digested with EcoR I, blotted to nitrocellulose membranes, and

hybridized with polyoma A2 genomic DNA to measure viral amplification.

182

 

 

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11.

REFERENCES

Bentivoglio, C. M., J. Zhu, and C. N. Cole 1992. Mechanisms of interference
with simian virus 40 (SV40) DNA replication by trans-dominant mutants of SV40
large T antigen J Virol. 66:4209-19.

Chen, L., and M. Fluck 2000. Kinetic Analysis of the Steps of the Polyomavirus
Lytic Cycle: Timing of Middle T + Small T Synthesis and Tanscriptional Control
of the Early -> Late Switch Manuscript submitted.

Chen, L., and M. Fluck 2000. The role of middle T/small T in the lytic cycle of
polyomavirus: Control of the early to late transcriptional switch and viral DNA
replication Manuscript submitted.

Chen, M. C., D. Redenius, F. Osati-Ashtiani, and M. M. Fluck 1995.
Enhancer-mediated role for polyomavirus middle T/small T in DNA replication J
Virol. 69:326-33.

Farber, J. M., K. W. Peden, and D. Nathans 1987. trans-dominant defective
mutants of simian virus 40 T antigen J Virol. 61:436-45.

Hacker, D. L., K. Friderici, and M. M. Fluck 1989. A nonlethal mutation in
large T antigen of polyomavirus which affects viral DNA synthesis J Virol.
63:776-81.

Hattori, J., G. G. Carmichael, and T. L. Benjamin 1979. DNA sequence
alterations in Hr-t deletion mutants of polyoma virus Cell. 16:505-13.

Lania, L., M. Griffiths, B. Cooke, Y. Ito, and M. Fried 1979. Untransformed
rat cells containing free and integrated DNA of a polyoma nontransforming (Hr-t)
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Nilsson, S. V., and G. Magnussan 1984. Activities of polyomavirus large-T-
antigen proteins expressed by mutant genes J Virol. 51:768-75.

Silver, J., B. Schaffhausen, and T. Benjamin 1978. Tumor antigens induced by
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Soeda, E., J. R. Arrand, N. Smolar, J. E. Walsh, and B. E. Griffin 1980.
Coding potential and regulatory signals of the polyoma virus genome Nature.
283:445-53.

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Sunstrom, N. A., N. H. Acheson, and J. A. Hassell 1991. Determination of the
origin-specific DNA-binding domain of polyomavirus large T antigen J Virol.
65:6998-7003.

Zhu, J. Y., and C. N. Cole 1989. Linker insertion mutants of simian virus 40
large T antigen that show trans-dominant interference with wild-type large T
antigen map to multiple sites within the T-antigen gene J Virol. 63:4777-86.

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