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LIBRARY
Michigan State
University

 

 

This is to certify that the

dissertation entitled

'I‘i-IEROIEOF'I‘HEPOLYCMAEMIANCERREGIONIN

REPLICATION OF THE VIRUS IN-VITRD AND IN-

VIVO
presented by

Andrea Amalfitano

has been accepted towards fulfillment
of the requirements for

PhD Microbiology

degree in

 

 

l‘MCL \L n W

Maior professor

6/ 14/ 89

Date

 

 

 

PLACE IN RETURN BOX to remove this checkout from your record.
TO AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

m

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU Is An Affirmetlve Action/Equal Opportunity Institution

 

a

4-.- v.

-o- C

THE ROLE OF THE POLYOMA ENHANCER REGION IN REPLICATION OF
THE VIRUS IN-VITRO AND IN-VIVO

BY
Andrea Amalfitano

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Microbiology and Public Health

1989

 

070(9 IQVZI

ABSTRACT
THE ROLE OF THE POLYOMA ENHANCER REGION IN REPLICATION OF
THE VIRUS IN-VITRO AND IN-VIVO

BY

Andrea Amalfitano

The enhancer region of the mouse polyomavirus has been
extensively studied in tissue culture systems. The
enhancer region is required not only for early gene
expression, but also for replication of the viral genome.

I have studied the role of the enhancer region in directing
replication of the virus in tissue culture systems and in
infections of the mouse. Variant viruses were constructed
which only differed in their rearranged enhancer regions.
Two of these variants, A2(A8) and A2(NG-23), had
rearrangements which included loss of DNA sequences between
nucleotides 5150-5180 (the omega domain) of the viral
genome, a region which has never been reported to be
deleted in viable viruses. These two viruses were shown to
replicate to wild type levels in mouse NIH-3T3 cells,
demonstrating that loss of these sequences is not
absolutely required for viral replication. The enhancer
rearrangements of these two viruses are similar to those

found in polyoma host range mutants capable of growing in

 

the mouse PCC4 embryonal carcinoma cell line. It was found
that these viruses cannot replicate to high levels in PCC4
cells, suggesting that the omega domain is required for
high levels of viral replication in this cell line. The
ability-of the variant viruses to replicate and persist in
mouse infections was also investigated utilizing whole body
in-situ hybridization and tissue DNA isolation techniques.
These experiments revealed that the nucleotides 5141-5264
of the viral genome are required for high levels of viral
replication in the early stages of infection of mice. The
enhancer was also found to play a role in the ability of
the virus to persist in mouse tissues later in infection.
Overall, these results demonstrate that the polyoma
enhancer region plays a pivotal role in replication of the
virus in infections of the mouse, as well as in tissue

culture systems.

 

To my wife, Susan, and our families, for without

their support, my goals would be out of reach

iv

 

ACKNOWLEDGEMENTS

I would like to thank Dr. Michele Fluck, not only for
her guidance and support , but also for her friendship and
caring throughout my graduate education.

I thank the members of my guidance committee, Drs.
Jerry Dodgson, Leland Velicer, Justin McCormick, Susan
Conrad, Karen Friderici, and Sandra Haslam, for their
advice, and interest in my project.

I thank all of the members of the Fluck lab,
specifically Sue K (and family), David, Karen, Ming—Chu,
Hong-Hwa, Larry, Amy, Julie, Li Jyun, and Jyothie, because
they are great people to work with.

 

TABLE OF CONTENTS

List of Tables........ ..... ............. ........ ..... viii

List of Figures.... ......... .. ....... ........ ........ ix

Chapter 1:

Chapter 2:

Chapter 3:

Literature Review

Introduction......... ......... ............ 1
Infectivity of Py in mouse tissues........ 4
Functional mapping of the Py

enhancer region........................ 6
Enhancer region function and altered
host range....... ................... 12

Factors which interact with the Py
enhancer............................... 18

Summary........ ..... .... ..... ............. 25

References.... ....... ..................... 27

Polyoma variants with altered enhancer
regions redefine subdomains required
for replication in tissue culture

systems.
Abstract.................................. 35
Introduction............ ..... ............. 36
Materials and Methods.. ....... ............ 38

Results................................... 43
Discussion................................ 52
References................................ 63

Defining subdomains of the polyoma enhancer
region required for high levels of viral
replication and persistence in mouse

tissues.
Abstract.................................. 68
Introduction................ ..... ......... 69
Materials and Methods..................... 72
Results................. ...... ............ 75
Discussion................................ 94
References......... ..... .................. 105

Summary and Conclusions............................... 110

 

Appendix:

Low probability of double integration
in transformation of nonpermissive

cells by polyoma virus
Abstract.................
Introduction.............
Materials and Methods....
Results..................
Discussion.. ..... ........
References.. ........ .....

vii

000.00....-

112
113
114
116
119
150
157

 

LIST OF TABLES

Appendix: Table 1. Integration of an unselected
viral genome.... ...... ............120

viii

 

Chapter 1:

Chapter 2:

Chapter 3.

LIST OF FIGURES

Figure 1. Physical map of the polyoma
virus genome.............................
Figure 2. Physical map of the
transcriptional amd replicational
enhancer subdomains......................
Figure 3. Physical map of the enhancer
regions of some natural variant and
host range mutant Py viruses.............
Figure 4. Factors which bind to the

enhancer region..................... .....
Figure 1. BclI- -BglI size analysis of
A2(X) viruses.......... ...............

Figure 2. Replication of the A2(X)

viruses in comparison to A2, or in

competition with B-2.....................
Figure 3. Sequence analysis of the

A2(X) viruses............................
Figure 4. Comparison of A2(A8) and

A2(NG-23) to the PCC4 EC mutants.........
Figure 5. Replication of the A2(X)

viruses in PCC4 cells....................
Figure 6. The transcriptional/

replicational enhancer elements, and

the factors which bind to this region....
Figure 1. In-situ hybridization to

control sections.........................
Figure 2. Time course of A2 replication

in mouse tissues visualized by

in-situ hybridization....................
Figure 3. Replication of the A2(X)

viruses in mouse tissues, utilizing

in-situ hybridization....................
Figure 4. Analysis of polyoma DNA

sequences extracted from specific

organs......... . ...................
Figure 5. Persistence of the A2(X)

viruses in mouse tissues, utilizing

in-situ hybridization....................
Figure 6. Analysis of polyoma DNA

sequences extracted from kidneys of

4 w. p. i. mice............................
Figure 7. MspI size analysis of viral

DNA isolated from 4 w.p.i. kidney

tissue...................................

ix

2

9

15

20

41

44

47

79

83

86

89

92

95

 

Chapter 3.

Appendix:

Figure 8. Physical map of the A2(X)
viruses............ ............... ....... 98
Figure 9. The transcriptional/
replicational enhancer elements, and
the factors which bind to this region....101

Figure 1. Integration patterns in F-lll

cells....................................123
Figure 2. Low frequency of integration of

the unselected parent among the

integrated viral sequences in rat

transformants.......... ..... .......... .126
Figure 3. Ratio of wild type and mutant

genomes in the infection mix.............129
Figure 4. Underrepresentation of the

unselected parent among mixed

transformants............................132
Figure 5. Persistence of the viral

genome in the infected cell population...135
Figure 6. Recovery of an unselected viral

genome from transformants obtained from

nondividing or dividing infected F-111

rat cells................................139
Figure 7. Underrepresentation of an

unselelected viral genome in hamster

transformants.............. ... ...........144
Figure 8. Low probability of double

integration events in transformants

derived from mixed infections with two

transforming strains.....................147

 

Chapter 1
Literature review
Introduction

The mouse polyoma virus (Py) is the prototype of the
polyoma family of viruses. It is a double stranded DNA
virus whose genome consists of 5297 bp utilizing the
numbering system of Salzman,1 (which will be utilized
throughout this study), organized as seen in Figure 1. The
genome is divided into an early region and a late region,
separated by approximately 350 base pairs of a non-coding
regulatory region. The early region codes for one mRNA I
precursor, which is differentially spliced to generate
three transcripts coding for the proteins known as small,
middle, and large T antigens. These proteins are essential
for establishment of the lytic cycle as well as for the
transformation properties of the virus. The late region
also codes for one mRNA precursor, which is differentially
spliced to generate the three transcripts coding for the
viral capsid proteins.77

This literature review will focus on the non-coding
region of Py, the effects this region has on viral host
range and tropism in tissue culture systems, and its role
in regulating infections of polyoma’s natural host, the

mouse .

 

 

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Fig. 1 Physical map of the polyoma virus genome.

The inner circle represents the location of the eight
fragments released when the polyoma genome is cut with the
restriction enzyme HpaII. The early and late region
precursor RNAs are spliced as indicated (jagged lines
represent introns) to generate the mRNAs for the T-antigens
or the capsid proteins, respectively. The non-coding

region flanks the origin of replication. The figure is

taken from Soeda gt _;.77

 

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Fig. 1 Physical map of the polyoma virus genome.

 

 

Infectivity of 21 in mouse tissues.

Py was initially discovered as the source of parotid
tumors in Ak mouse leukemic cell extracts. Mouse embryo
tissue culture systems allowed high titer growth of the
agent.2'3'4 The agent was found to elicit a plethora of
tumors when neonatal mice were inoculated. These include
salivary gland tumors, osteogenic sarcomas of the spine,
mammary tumors, epithelial thymomas, and kidney sarcomas,
to name but a few.3'5'6'7'8 Indeed, this variety was the
reason for naming the agent ’Polyoma virus’.

Growth studies measuring infectious viral titers and
hemagglutination demonstrated that in neonatal mice, Py
replicated to the highest levels in kidney tissue.

Salivary gland, liver, lung and spleen tissues supported
intermediate levels of virus replication, with brain,
blood, and thymic tissues having the least ability to
generate virus.9 The highest levels of virus production is
always in neonatally infected mice, which also have the
highest excretion rates of infectious virus in urine,
suggesting that inhalation of infectious urine may be the
mechanism by which Py spreads itself through a colony of
mice.9 Transplacental transmission of virus from mother to
offspring has also been demonstrated to be a possible route
of viral propagation through a colony.10

Py is known to be present in kidney tissue of

inoculated neonate mice for long periods of time, well

 

beyond clearance by the immune system, implicating the
kidney as a major target site for viral replication,
persistence, and source of infectious virus.8'11'12
Interestingly, increased levels of virus could be detected
in the kidneys of persistently infected females once they
became pregnant, specifically during late gestation.13

More recently, utilizing in-situ hybridization and DNA
isolation techniques similiar to the ones utilized in this
study, patterns of Py infection in neonatally infected mice
were more clearly elucidated. Intra-peritoneal infection
of newborn Balb/c mice with the Py wild type A2 strain
resulted in a systemic infection including the lungs,
liver, spleen, and kidney tissues. Virus replication
peaked at eight days post-infection, then decreased as
antibody titres to the virus increased. As expected,
kidney tissue could still be demonstrated to have
detectable Py DNA persisting well beyond the initial immune
response of the mouse.14'15 It was noted that if neonatal
mice were inoculated intra-nasally with the Py A2 strain ,
.the same initial infection pattern was observed, however
now the virus could also be demonstrated to also persist in
lung tissue, a previously unknown site of Py persistence.15

It has been demonstrated that Py virulence in renal
tissue can be changed due to strain differences, which have

yet to be fully defined, or by using deletion mutants of

 

 

the early coding regions, which result in decreased ability
of viruses to grow in kidney tissue.12'16

This study is the first to demonstrate that the Py
non-coding region, (specifically the transcriptional and
replicational enhancer described in the following
sections), has a major role in affecting the replication of

Py in infections of the mouse.

Functional mapping of the By enhancer regign

Over the years Py has proven to be a convenient system
to study DNA replication and transcription in mammalian
cells. It was shown that large T antigen was required in
trans to initiate rounds of replication, by interacting I
with a short region of dyad symmetry at the origin of

7 However, studies demonstrated that deletion

replication.1
mutants which span the PvuII restriction enzyme site at
nucleotide #5130 of the Py genome resulted in decreased
early mRNA expression, even though the early mRNA cap site
is greater than 300 base pairs away18 (see Figure l).
Coincidentally, deletion of this region also eliminated the
ability of Py to replicate, even when large-T antigen is
supplied intrans.18 The 244 base pair BclI-PvuII
restriction fragment of Py DNA (nt 5021-5262) which
encompasses the PvuII site at nucleotide number 5130, was
later found to contain a classical enhancer region.20 This

enhancer region was able to increase transcription of a

 

 

promoter up to three kilobase pairs away, in an orientation

independent manner.19

It was further demonstrated that Py
replication requires an enhancer function per se, and that
this is not specific to the enhancer utilized.21 For
example, if other known enhancer sequences (from SV—40 or
the immunoglobulin enhancer) replace the Py enhancer region
in plasmid constructs, replication of these plasmids can be
restored. The authors suggested using Py replication as an
"enhancer trap", to implicate random DNA sequences which
may have transcriptional enhancer activity.20 These
studies indicated that the Py transcriptional enhancer was
also required for replication, and in order to study one
function or the other, these functions had to be separated,
otherwise blocks in transcription would always result in
blocks to replication.

To study the transcriptional activity of the Py
enhancer region it was linked upstream of a beta globin
promoter driving transcription of the bacterial CAT
(chloramphenicol acetyltransferase) gene. These studies
demonstrated that the 244 base pair enhancer region could
be separated into two distinct transcriptional ’enhancer
elements’, called A (nt 5021-5131) and B (nt 5132-5265),
each of which had different levels of enhancement in
different cell lines.21 Finer deletional analysis revealed
that these two elements were actually composed of three

separate transcriptional enhancer elements, any two of

 

 

which in combination can restore 100% transcriptional
activity of reporter genes in mouse 3T3 cells. These three
elements were called 1,2, and 3, and were mapped to
nucleotides 5039-5073, 5073-5130, and 5131-5229,
respectively (see Figure 2).22

Sequences responsible for enhancement of replication
were also scrutinized. Initially, the replicational
enhancer was separated into an alpha core element (nt 5097-
5126) and a beta core element (nt 5172-5202),23'24 which
are functionally redundant, and in concert with the ori
core in cis and large T antigen supplied in trans, allow
replication of the viral genome. These elements could not
enhance replication when either was placed 500 base pairs
from the ori core (unlike transcriptional enhancement,
which can be detected when the elements are placed up to 3
kb away from the early region promoter) suggesting that for
Py both transcription and replication may require the same
factors initially, but then the pathways diverge such that
replication is sensitive to distances between the ori core
and the replicational enhancer elements. Further studies
utilizing deletional analysis lead to a finer breakdown of
the replicational enhancer into four domains, (see Figure
2) each contributing to replicational enhancement, and
these domains are called A,B,C, and D (located at nt 5108-

5130, 5179-5214, 5148-5179, and 5020-5093, respectively).25

 

 

 

 

 

 

      

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Figure 2. Physical map of the transcriptional and
replicational enhancer subdomains.

The boundaries of each of the functional subdomains of
the polyoma enhancer region are compared in relation to one
another, and to a restriction enzyme map of the area. The
top figure compares the transcriptional enhancer domains,
the bottom figure compares the replicational enhancer

domains, utilizing the boundaries defined in the text.

 

10

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ENHANCER ELEMENTS

“alga" “beta ..

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Figure 2. Physical map of the transcriptional and

replicational enhancer subdomains.

 

 

 

11

Use of linker-scanning mutants in addition to deletional
analysis revealed a functional difference in these domains,
in which each of the two redundant elements is composed of
essential "core" and "auxillary" regions. Thus, the alpha
core sequence (5108-5126) represents an enhancer of
replication which in itself can activate replication, while
an alpha core auxiliary sequence (5073-5102, part of ’D’
above) can only augment alpha core directed replication,
without being able to activate replication by itself.
Similarly, a beta core element was defined, (5172-5202, or
B of above) with two auxiliary sequences (at nt. 5130-5172
and 5202-5218, the latter representing the C domain from
above) which also augment replication from the beta core,
without themselves having any replicational enhancer
activity,26 (see Figure 2).

The above studies also demonstrate that Py utilizes
the same DNA sequences for replicational and
transcriptional enhancement. Studies utilizing point
mutations scattered throughout the enhancer region also
demonstrated that the replicational and transcriptional
enhancer activities cannot be separated, at least in mouse
3T6 fibroblasts.27

It is clear that the enhancer region of Py has adapted
to allow for interactions with a variety of host cellular
factors required for its transcriptional and replicational

activities. The recombinant viruses constructed in this

 

12

study implicate these same regions, and further refine
their roles in regards to replication of the virus in

tissue culture systems and in infections of the mouse.

Enhancer region function and altered host-range

Early investigations of Py demonstrated that mutants
could be isolated which have an altered host range.
Initially, viruses from mutagenized stocks of virus were
selected which had lost the ability to grow in mouse 3T3
cells, while retaining the ability to grow in Py
transformed cells.28 Eighteen mutant viruses were isolated
by this selection procedure, and all were also found to be
deficient in their ability to transform rat cells. These
mutants were designated hr-t, for host range and
transformation defective.28 The non-coding regions of some
recombinant viruses constructed in this study are derived
from the hr-t mutants. Py hr-t mutants A8 and A9 were
obtained by mutagenizing stocks with the chemical ICR-191
and isolating infectious virus, hr-t mutant II-5 was
selected from a wild-type lysate after passage in Py-3T3
cells, and the hr-t mutant 80-15 was selected based upon
its decreased density when banded in CsCl2 gradients.29

Marker rescue experiments with some of these mutants
mapped their host range and transformation defects to the
fourth largest fragment generated by digestion of the Py

genome with the restriction enzyme HpaII. However,

 

 

13

restriction enzyme analysis of these mutants also revealed
changes in other parts of the genome, namely HpaII
fragments #3 and #5 (see Fig. 1) which harbor the non-

30 Later studies

coding regulatory elements of the virus.
revealed that similar size differences in HpaII-fragment #3
actually represent tandem sequence duplications. For
example, tandem duplications have been observed in the
viral strains P16, Toronto, Ts48, MV, and NGS9R.31I67
These duplications all centered around the Py enhancer
region and mimick a repeat in the SV-40 enhancer, (the 72
base pair repeat region) which is required for efficient
SV-40 early gene expression.32 It was suggested that Py,
and possibly papova viruses in general, accumulate these
duplications and thereby facilitate interactions with
various host cells and or tissue types.65
The observation that various strains of Py have been
found with duplications of regions important in viral
expression and replication became even more striking when
examining mutants of Py selected to grow on normally non—
permissive cells (see Figure 3). Mouse embryonal carcinoma
(EC) stem cell lines normally cannot support papova virus
replication unless they are first induced to
differentiate.33 Mutants of Py were isolated which could
grow on the undifferentiated form of the PCC4 EC cell line,
and this new host range was found to correlate with a

duplication of sequences in the A enhancer element coupled

 

14

with a deletion of sequences in the B enhancer element of
the mutant viruses' genomes.34 F9-1 EC cell lines yielded
other Py host range mutants, with a single change at
nucleotide #5235 (or duplication of the region encompassing
this single bp change) responsible for their new tissue
tropism.35'36'37'38 It was demonstrated that a block in Py
RNA expression in EC cells was removed by these altered
enhancer regions.39I40'41 Interestingly, the F9 EC mutant
with a single point mutation could be demonstrated to have
an altered conformation of its naked viral DNA, based upon
sensitivity to the enzyme DNase I.42

Eventually, other Py host range mutants capable of
growth in normally non-permissive cell lines were isolated.
These included mutants which could grow in mouse
trophoblastic, neuroblastic, and Friend leukemic cell
lines.43'44'45 All of these mutants also have rearranged
enhancer regions, (see Figure 3), mainly duplications
coupled with deletions, responsible for their altered host
ranges. Rearrangements of enhancer regions with
coincidental alterations of host range have also been
observed for other polyoma viruses. For example, JC virus,
the human polyomavirus, exhibits rearrangements of its
enhancer which are responsible for determining the tissue
tropism of various strains of the virus in human

infections.46'47

 

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Figure 3. Physical map of the enhancer regions of
some natural variant and host range mutant Py viruses.

This figure is taken from Amati (ref. 45) and compares
the enhancer region modifications of natural variant
polyoma viruses CSP, MV, TOR, and P16, to the
rearrangements seen in host range mutants capable of
growing in embryonal carcinoma (PCC4-204, PCC4 97, F9-5,and
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u. 20‘
__‘_—. __________ PCC4
J 31
n—“ ‘_*
13:3.“ rm
J TI“
‘30-:
—-—f,-'-
' " ' ml
.._—.>
. 5L1!
_
mun
h ' J HID/I

 

Figure 3. Physical map of the enhancer regions of some

wild type and host range mutant Py viruses.

 

 

17

The ability of the non-coding region to influence Py
replication in mouse tissues has also been investigated.
Mice inoculated with a variant of Py which has a 40 base—
pair duplication proximal to the Bgl I restriction enzyme
site, (see Figure 1) was demonstrated to increase the
incidence of thymic epitheliomas in mice inoculated as
neonates.8'48 However, nude mice inoculated with either
wild type or EC mutant strains of Py did not differ in the
spectrum of tumors evoked.49 A reconstructed Py strain
which had the B transcriptional enhancer element replaced
by the Moloney murine leukemia virus LTR (long terminal
repeat) was found to replicate in pancreatic tissue, and
not kidney tissue, the normal site of high levels of viral
replication.50 The results of this study suggest that the
possible deletion of the B enhancer element may have
allowed for detection of the new pancreatic tissue tropism.
Finally, transgenic mice with the enhancer of the F9-1 Py
EC mutant driving the CAT gene did so much more effectively
than when the wild-type enhancer region was used, even
though both CAT gene constructs were demonstrated to be
active in the same tissues.51

This study establishes the importance of the Py
enhancer region in directing high levels of replication and
persistence in mouse tissues. The recombinant viruses
constructed in this study have rearrangements similar to

those described for host range variants generated in tissue

 

 

 

18

culture systems, underscoring their importance in the

biology of Py.

Factors which interact with the By enhancer

The isolation of various host-range mutants of Py
demonstrated that the regulatory activity of enhancer
sequences themselves could account for altered host range.
This specificity of activity was shown to be due to
interactions with trans-acting factors of the host. Base
line activity of certain enhancers could be modified by
competition with other enhancer sequences present in the
same cell, demonstrating that trans-acting factors were
responsible for enhancer activation and or repression, and
also for the altered host range.52'53

Studies utilizing gel shift, DNase I footprinting, and
methylation interference-protection assays attempted to map
the exact location of specific host-protein and viral-DNA
interactions. The Py B enhancer element was the first
portion of the Py enhancer to be investigated. The B
enhancer element as defined by previous functional
analysiszz, was shown to specifically interact with nuclear
protein factors from murine cellular extracts. More
precisely, nucleotides 5174-5229 of enhancer element B were
implicated as the major target site for a factor
subsequently called PEB-l, (for Py enhancer binding factor
1). The binding of FEB-1 absolutely required the 25 base

 

 

19

pairs encompassing the early proximal portion of the GC
rich palindrome sequence and the SV-40 enhancer core
homology contained within nucleotides 5174-5229 (see Figure
4).54,55,56

Other studies demonstrated that the C enhancer domain
of Py, (as defined in ref. 26), was interacting with a
factor in differentiated or undifferentiated F9 EC cells,
as well as in mouse 3T6 cells. This factor was called EF—C,
(for enhancer binding factor to Py element-C).57'58 The
EF-C binding domain is conserved in all Py viruses to date,
implicating it as important in the biology of the virus.
In this study, a number of recombinant viruses were
sequenced and found to be lacking the EF-C binding domain,
and their host range in tissue culture as well as in
infections of the mouse is described. Whether or not EF-C
and EBP-l interact with each other has not been
investigated. Others have described a 20,000 kD nuclear
protein isolated from rat crude liver extracts which was
purified based upon its strong interaction with the Py B
element, (specifically, nucleotides 5211-5233, see Figure
4), and called it EBP-20, (for enhancer binding protein of
20 kD.).59'6o Finally, it was demonstrated that a Py EC
mutant, PyF441, which had a single nucleotide mutation in
its B element at nt. #5235 (responsible for its ability to
grow in undifferentiated F9 EC cells), could now interact

with a cellular factor in undifferentiated F9 EC cells,

 

 

 
 
 

  
  

 

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20

Figure 4. Factors which bind to the enhancer region.
The binding sites for some of the factors described in
the text are compared to the restriction enzyme map of the

polyoma virus enhancer region.

21

 

I a.” Pgull mu 39g:
PEA-h 2 FEB"
'PEA- _ "
P§A3 .ELQ E 563
FACTORS 1

Figure 4. Factors which bind to the enhancer region.

 

 

 

22

whereas a wild-type sequence of this same region could not,
suggesting a newly created positive acting sequence motif
was affecting the new host range of the virus.61 Together,
these studies suggest that the functional B enhancer
element of Py is the site of interaction with numerous
cellular factors. In this study, the B element has been
implicated as a region critical to Py not only in
establishing an early infection of mice, but also in
allowing the virus to be able to persist at detectable
levels in kidney tissue well beyond clearance by the immune
system. This suggests that a possible lack of interaction
with B enhancer element binding factors present in neonatal
mouse tissues, (most likely equivalent to EBP-20), results
in a serious impediment to the replicative potential of Py.
The A enhancer element of Py has an even more
complicated array of factors interacting with it.
Utilizing a modified DNase I footprinting technique, the 22
base pair core of the alpha domain, (as defined by ref.26,
nucleotides 5108-5130), was found to be the recognition
site for two different proteins with overlapping binding
sites. They were called PEAl and PEAZ, (for Py enhancer A
binding factors 1 and 2).62 PEAl binds to nucleotides
5115-5125 and PEAZ to nucleotides 5122-5130 (see Figure 4).
This finding, along with other studies63, suggeSted that
two recognition sites for trans acting proteins could

evolve from a common recognition sequence.62l63 The

 

 

23

sequence rearrangements of the recombinant viruses examined
in this study also support this hypothesis. It was also
demonstrated that PEAl and PEA2 binding was cooperative,
and that this association could induce DNase I
hypersensitive sites in the viral minichromosome.69 These
DNase I hypersensitive sites were suggested to be
facilitating viral DNA replication. These "core
recognition sequence motifs" can be duplicated to create
functional enhancer elements in SV40 as well.64

PEAl and PEA2 were not found to bind to their
respective recognition sites in undifferentiated PCC4 or F9
EC cells, but when these same non-permissive cells were
induced to differentiate, PEAl and PEA2 binding was
restored, correlating binding of these factors with the
ability of the virus to replicate in previously non-
permissive cells.65 In agreement with the above inference
is the finding that Py PCC4 EC mutants accumulate
duplications of their alpha domains to allow growth in
undifferentiated PCC4 EC cells, which may allow the virus a
better opportunity to interact with PEAl and PEA2.66 A
third protein, PEA3, was also found to bind the Py A
enhancer element at nucleotides 5108-5113, and possibly to
B element sequences at nucleotides 5203-5208 (see Fig.4),
and this binding allowed viral transcription and

replication in mouse fibroblasts.67

 

24

Negatively acting factors which interact with the Py
enhancer have also been investigated.54'55'68 The clearest
example of negative regulation involves the adenovirus Ela
protein. When introduced into mouse cells along with a
reporter gene linked either to the Py or SV40 enhancer, Ela
is able to repress transcription of the reporter gene.69
The observation that an adenovirus mutant with a defective
E1a protein can grow in undifferentiated EC cells suggested
that an endogenous ’Ela like’ activity is present in these
cells, and that this may be the reason why Py and SV40
cannot replicate in undifferentiated EC cells.70 For
example, a labile protein was implicated as preventing Py
replication in undifferentiated PCC4 cells.71 Previously
discussed positively acting factors which were correlated
with Py host range may in fact be modulated by these ’Ela
like’ negatively acting factors, and thus these
observations are not mutually exclusive. Indeed, the
observations underscore the fact that the Py enhancer has
evolved in response to the intricate cellular control
mechanisms responsible for modulating gene expression.

other interactions with the Py enhancer region have
also been noted. It was observed that the tumor promoter
TPA (12-0-tetra-decanoylphorbol—13-acetate), can stimulate
Py enhancer-linked expression, and that this expression was
different depending on the differentiation state of the

72

host cells. The c-Ha-ras proto-oncogene in and of itself

 

25

can stimulate Py transcriptional enhancer function, and a
mutated c-Ha-ras that is oncogenic can further stimulate
the Py enhancer, via a trans-acting factor.73'74 The
sequence in the enhancer region responding to these various
transforming oncogenes, including the middle T protein of
Py, TPA, or even serum, maps to the PEAl binding domain,
indicating that the pathway of transformation is
exquisitely linked to the pathway for Py enhancer

activation.75'76

Possibly Py utilizes its middle T protein
to facilitate interactions with PEAl, allowing its own
genome to be preferentially expressed and replicated, thus
giving a "transforming protein" an actual role in the lytic
cycle of the virus.

The complexity of the factor interactions within the
Py enhancer region underscores the region’s importance in
directing transcription, replication, and the tissue
specificity of the virus. The factors which have been
demonstrated to bind within this region are thus implicated
as being important participants in gene expression and DNA

replication.

umma

This literature review has focused upon the ability
of the mouse polyoma virus to interact with a number of
mouse tissues and various tissue culture cell lines, and

demonstrated how the enhancer region of polyoma has been

 

26

implicated as a major determinant in these interactions.
This complexity allows investigators to investigate virus-
host interactions utilizing a number of various model
systems. This study will describe the construction of new
polyoma viruses with unique rearrangements of their
enhancer regions. These reconstructed viruses allow us to
ask how these rearrangements may alter the ability of the
virus to grow in various tissue culture cell lines, and in

tissues of the mouse, the virus’s natural host.

 

 

27

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Soeda, E., Arrand, J.R., Smolar, N., Walsh, J.B.,
Griffin, B.E. (1980). Coding potential and regulatory
signals of the polyoma virus genome. Nature (London)
283:445:453.

35

Chapter 2

Polyoma variants with altered enhancer regions redefine
subdomains required for replication in tissue

culture systems

ABSTRACT

We have constructed five new polyoma viruses which
differ only in their enhancer regions, and describe the DNA
sequence of their enhancer containing non-coding regions.
Two of the viruses, A2(A8), and A2(NG-23), have extensive
duplications coupled with a deletion of 123 bp which
removes a functional subdomain of the enhancer previously
suggested to be required for viral replicatiOn. These
viruses are shown to replicate to wild type levels upon
infection of mouse NIH-3T3 cells, suggesting that extensive
duplications of enhancer subdomains can functionally
replace domains thought to be indispensable. The viruses
constructed resemble previously described host range
mutants of polyoma which can grow in the PCC4 embryonal
carcinoma cell line. Thus the mutant viruses allowed us to
define which regions of the enhancer are required for

growth in PCC4 cells.

36

lEIBQDQQIIQfl

Polyoma virus has been used extensively as a probe to
understand mammalian gene expression and DNA replication.
In particular, the polyoma virus transcriptional and
replicational enhancer region has been shown to demonstrate
a functional complexity which allows for multiple
interactions with various features of the host cell
machinery involved in gene expression and
replication.22'26'37 Indeed, the 242 base pair BclI-PvuII
fragment between nucleotides 5023 and 5265 (using the
numbering system of Salzman35) has been exhaustively
dissected into a number of subdomains, whose boundaries are
compared in Figure 6. This fragment was initially
described as containing two functionally redundant
replicational enhancer elements, either of which positioned
in cis with the ori core region allows for efficient viral
replication.26'28 This region was further subdivided into
' four functionally redundant subdomains named A,B,C, and D,
based upon deletional analysis.48 Later, deletional
analysis coupled with linker scanning analysis divided the
replicational enhancer region into two functionally
redundant 'enhancer core’ sequences, (which are named alpha
and beta) able to activate replication by themselves (in

cis with the ori core) and ’auxiliary domains’ (one for the

37

the alpha core, two flanking the beta core) which augment
core directed replication, without having any intrinsic
replication enhancer activity of their own.27
- Mapping to this same region are three functionally
redundant transcriptional enhancer elements, any two of
which when present restore full gene expression to linked
reporter genes.12 Polyoma is unique in that a
transcriptional enhancer function per se is required for
replication of the viral genome, suggesting that both
transcriptional and replicational activities of the virus
are sharing similar factors, and or pathways.9'12'42"”'48
A plethora of factors have been demonstrated to interact
with the polyoma enhancer region 3'17'29'30'32,(see Figure
6).

Finally, a number of mutations in polyoma viruses have
been isolated consisting of rearranged enhancer regions
which are responsible for altered tropism of the virus with
regard to replication in various cell lines.1'6'7'1°'11'18'
19'23'41'45'46 These mutations implicate viral
interactions with enhancer binding host factors as one
determinant of the viral host range.2°'21'29 In this study
we have constructed five new viable polyoma virus strains
which have altered enhancer regions, (similar to previously

described PCC4 EC host range polyoma mutations). These

alterations shed light on the role of subdomains of the

38

enhancer in replication, and in determining the host range

of the virus.

Materials and Methods
ggnsttngtign of teconninant vitnsesz

Viral DNA from five hr-t mutants was isolated from
infected NIH-3T3 cells via the method of Hirt.16 For
mutants NG-23 and II-5, viral DNA was digested with the
restriction enzymes BclI and BglI. The two DNA fragments
were separated by electrophoresis in a low-melting
temperature agarose gel, and the small Bcl-BglI fragment of
each mutant was excised. For hr-t mutants A8, A9, and SD-
15, purified DNA was cleaved with BamHI, ligated to BamHI
cleaved pBR322 plasmid, and used to transform dam'dcm' E.
Coli using established techniques. These plasmids are
referred to as pA8, pA9, and pSD-lS, respectively. Plasmid
DNA was isolated, cut with BclI and BglI, and the BclI-BglI
small fragment of each mutant was isolated as above. For
all five mutants, the small BclI-BglI fragment was ligated
to the low melting temperature agarose purified BclI-BglI
large fragment of Hirt isolated A2 strain DNA using the
ligation conditions described by Struhl.40

. The ligated semi-solid reaction mixtures were heated
to 65°C, diluted with an equivalent volume of DEAE-Dextran
(1mg/ml), and used to transfect 1x104 NIH-3T3 mouse cells

on a 35mm dish. Virus production was visualized by cell

39

lysis. High titer viral stocks were grown from individual
plaque isolates of each recombinant virus by infection of
NIH-3T3 mouse fibroblasts. The BclI-BglI source for each
recombinant virus was confirmed by MspI (HpaII) size
analysis of Hirt isolated DNA. Each recombinant virus is
thus denoted as A2(X), where 'X' designates the viral

source of the BclI-BglI small fragments.

Sggngncg nnnlysis of tng tggombinant vitnsgsg

Hirt DNA from each of the recombinant viruses was
isolated from infected NIH-3T3 mouse cells and digested
with BamHI and EcoRI in the cases of the viruses A2(A8),
A2(A9), A2(SD-15), and A2(NG-23), while A2(II-5) Hirt DNA
was cleaved with HindIII and BamHI (since the BclI-BglI
small fragment of the hr-t virus II-5 contains an
additional EcoRI site, data not shown).

The approximately 2200 bp fragments were purified by
low melting temperature agarose electrophoresis and ligated
to the bacterial plasmid pAT153 digested with the
appropriate enzymes to allow directional subcloning.
Ligated DNA's were used to transform competant DH5 dam'dcm‘
E.Coli, and ampicillin resistant, tetracycline sensitive
clones were isolated. 'Clones containing proper inserts
were confirmed by restriction enzyme analysis of miniprep

DNA, and large scale DNA preparations were made. These

40

plasmids are called pAT:A2(X), where ’A2(X)' designates the
source of the polyoma DNA in each plasmid.

Each pAT:A2(X) plasmid was digested with Bell and PstI
to release the enhancer containing 600-700 bp fragments.
These fragments were purified by low melting temperature
agarose electrophoresis and ligated into the polylinker
region of either of the bacteriophage M13mp18 or M13mp19 RF
DNA’s digested with BamHI and PstI. Insert containing
clones were isolated, single stranded DNA of each was
isolated, and both strands of each recombinant virus were
subjected to Sanger dideoxy sequencing from opposite
directions by using the M13 universal primer or a 17mer on

the early side of the polyoma BglI site.

es of h 'c t' o ert'es 0 th recomb'

NIH-3T3 cells were seeded onto 60mm dishes at a
density of 2x105 cells and coinfected with the hr-t mutant
virus B-2 at an MOI of 5, and each A2(X) virus at a MOI of
0.5. Individual infections were also carried out with each
A2(X) virus alone at an MOI of 0.5. Input ratios were
verified 4 hours post-infection via Hirt extractions (data
not shown). PCC4 undifferentiated embryonal carcinoma
cells were kindly provided by A. Levine, infected with each
of the A2(X) viruses at an MOI of 10, and DNA was isolated

as above. All cells were grown in Dulbecco’s

41

Figure 1. BclI-BglI size analysis of the A2(X)
viruses.

Approximately three micrograms of each of the
pAT:A2(X) plasmids were digested with BclI and BglI in the
appropriate reaction conditions, electrophoresed through a
2% agarose gel, and the digestion products were visualized
by ethidium bromide staining of the gel. The arrow
indicates where the various BclI-BglI enhancer containing
fragments of each A2(X) virus migrate in relation to strain
A2. The source of each BclI-BglI fragment is as follows:
Lane 2=A2(A8), Lane 3=A2(A9), Lane 4=A2(SD-15), Lane
5=A2(II-5), Lane 6=A2(NG-23), Lane 7=A2. Lane 1 is a size

marker (Lamda DNA digested with Hind III).

42

 

. Figure 1. BclI-BglI size analysis of the A2(X) viruses.

43

modified Eagle’s medium supplemented with 10% calf serum

(fetal calf serum for PCC4 EC cells) and antibiotics.

Bgsults:
Replicative abilities of 52(X) Vituses: A number of

polyoma hr-t mutant viruses maintained in our laboratory
have gross restriction fragment length polymorphisms
(RFLP’s) in their small BclI-BglI fragments. This had been
noted in the original isolation of the hr-t mutants because
size analyses of their DNA had demonstrated RFLP's in MspII
fragments #3 and #5.13'39 To analyze and test the enhancer
containing regions of these hr-t mutants, we subcloned them
into the genomic BclI-BglI fragment of the wild-type A2
strain (see Methods). The only sequence differences
between these reconstructed 'A2(X)' viruses should be
restricted to their non-coding regions.

The various RFLP's in the small BclI-BglI fragments of
the A2(X) viruses are demonstrated in Fig.1. In comparison
to the same region in wild type strain A2, the A2(A8) and
A2(NG-23) viruses have deletions of approximately 20 bp,
A2(SD—15) and A2(II-5) have additions of approximately 40
bp, (with A2(II-5) also possessing an EcoRI site within
this region) and A2(A9) an addition of approximately 100

bp.

44

Figure 2. Replication of the A2(X) viruses in
comparison to A2, or in competition with B-2.

NIH—3T3 cells were infected with each virus alone at
an MOI of 0.5 ("-" lanes) or in a competition assay in
which the MOI ratio of A2(X):B-2 was 0.5:5 ("+" lanes). At
60 hours post infection viral DNA was isolated by Hirt
extraction, 1159 of each sample was digested with the
restriction enzyme PvuII and electrophoresed through a 1.6%
agarose gel. DNA was transferred to nitrocellulose and
hybridized to a probe specific for the polyoma Hpa II-#4
fragment (B-2 contains a 240 bp deletion in this fragment).
Viruses used in each infection are as follows: 1= A2, 2=

A2(A8), 3=A2(A9), 4=A2(SD-15), 5=A2(II-5), 6=A2(NG-23).

45

.mum saws :0wuwumdeou cw so w~< cu

comwumaeoo cw momsuw> Ax1~< one no COwumowHQmm

 

.m musowm

 

46

To assess the ability of each recombinant virus to
replicate in permissive mouse cells, NIH-3T3 cells were
infected with either a recombinant virus alone, or
coinfected with the recombinant virus along with the hr-t
mutant B-2. This competition assay was devised in our lab
to detect subtle differences in the ability of viral
strains to replicate (see Figure 2). It is readily
apparent that all of the recombinant viruses replicate to
levels equivalent to the A2 strain of virus, either in
single infections or when forced to compete with B-2, even
when B-2 was initially introduced in a 10 fold excess over
the A2(X) viruses in the initial infection mix. The
intensity of every band on this blot is within 2.5 fold of
any other based on densitometric scanning, indicating that
the A2(X) viruses all replicate to wild type A2 levels, and
they do not significantly differ in their ability to
replicate in NIH-3T3 cells. These results have been
obtained in three independent experiments using different
multiplicities of infection, and is indirectly verified by
the similar titers of infectious virus obtained when these
viruses are grown in NIH-3T3 cells (data not shown).

Sequence analysis of thg 52(X) vitnses: We sequenced
both strands of the BclI-BglI region of each of the A2(X)
viruses by subcloning into the bacteriophage M13 and
utilizing the Sanger dideoxy chain termination method (see

Methods). The sequencing results are described

47

Figure 3. Sequence analysis of the A2(X) viruses.

The sequence of each of the A2(X) viruses as well as
our A2 strain are compared to a restriction map of the A2
strain of Salzman (35). The strand equivalent to the early
mRNA is presented. Dark parallel lines represent
duplications and dashed lines indicate their relation to
the genome. Nucleotide substitutions are placed directly
above their location, with nucleotide additions placed over
a "+", and nucleotide deletions placed over a "-".
Boundries of the major duplications are placed in relation

to the nucleotide numbers of the A2 schematic diagram.

48

 

 

 

 

 

 

 

 

 

 

"=9"\
\\
& \
\
\\
\
F\- \\
Max:222 ‘Gc\ \\ 1;; écg ._
G
PEI-'\
\\ \\\
\
1m, -\\ 1c; 4 (9
¢
"Fora.
‘\
J:
:ggx. 5 g ICC 4 99
__&§.C_.
1.93—
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I CC
I l I l
5038 90¢ 5\A\ $76 5’15" 52363.2}? 3 R '3
5“»
PvuII PVIJ" Bgll

 

Figure 3. Sequence analysis of the A2(X) viruses.

49

schematically in Figure 3. All the A2(X) viruses contain
at least a single duplication of the previously described A
transcriptional enhancer element14, a point followed up in
the discussion.

Briefly, the recombinant viruses A2(SD-15) and A2(II-
5) demonstrate 44 bp duplications identical to those
described previously for the P16 and Toronto strains of
polyoma, respectively.33'34 However, the three remaining
recombinants have multiple and more extensive duplications
of the region only duplicated once in the A2(SD-15) and
A2(II-5) viruses. In strains A2(A8) and A2(NG-23) this
amplification is coupled with a deletion of 123 bp between
nucleotides 5141 and 5264. Since all of these recombinants
are able to replicate to wild type levels in mouse NIH-3T3
cells, the 123 bp deleted sequences are not absolutely
required for viral replication in these cells.

These sequence rearrangements are particularly
striking when they are compared to the sequence variations
of some host range mutants of polyoma selected to grow on
the normally non-permissive undifferentiated PCC4 embryonal
carcinoma cell 1ine.18'25 The enhancer regions of the PCC4
variants 204 and 97 are shown in comparison to the enhancer
regions of the A2(A8) and A2(NG-23) viruses (see Figure 4).

The viruses compared in Figure 4 all have

50

Figure 4. Comparison of A2(A8) and A2(NG-23) to the
PCC4 EC mutants.

The enhancer region of the viruses A2(A8) and A2(NG-
23) are compared to the same region in the embryonal
carcinoma mutants PCC4-97 and PCC4-204. Regions suggested
to be required of all PCC4 mutants by Blangy, gt nl.25 and

the "omega" element, as described by Amati, gt nl.23 are

schematically presented also.

51

 

 

 

 

 

 

 

 

 

 

518222) ‘GC-.. \\._ I“ + ‘9
.Q-SL
- Gr:;5-1~
\ ‘ \\
;: ““- {Q A
“(A-g) race \1% -.C + co
"' ~~~ “‘
Egg-97 ”'°--;;'-~-
‘=:2_ v-—~.
W ~~~-_— "“_
II—-—I Blongy
01d

Figure 4. Comparison of A2(A8) and A2(NG-23)

EC mutants.

to the PCC4

52

duplications coupled with deletions, however A2(A8) and
A2(NG-23) have a deletion which is larger than that of the
polyoma PCC4 mutants. The larger deletion includes
nucleotides 5173-5200, a region previously suggested to be
required in all polyoma mutants able to grow on
undifferentiated PCC4 cells isolated to date.25 We
infected undifferentiated PCC4 cells with each of the
reconstructed viruses, and assessed their ability to
replicate (see Figure 5). Wild type strain A2 replicated
to very low levels, and none of the constructed viruses
were able to replicate to levels any greater than that
observed for wild type A2 strain, suggesting that the 5173-
5200 bp region of polyoma is required for replication in
undifferentiated PCC4 cells, (in addition to the
duplications and deletions already observed in the PCC4

mutants isolated to date).45

Discnssign

Previous studies have defined the polyoma BclI-PvuII
242 bp fragment as a transcriptional enhancer element by
its ability to increase transcription from a distant
promoter in an orientation independent manner.8 Numerous
studies have dissected the transcriptional enhancer into

12,14,26

functionally redundant subdomains, as summarized in

Figure 6. Those domains, designated as ’transcriptional

53

Figure 5. Replication of the A2(X) viruses in PCC4 EC
cells.

PCC4 cells were infected at an MOI of 10 with each of
the A2(X) viruses or A2. (ill of the Hirt extractable viral
DNA was isolated from each plate at 3 or 72 hours post
infection, digested with the restriction enzyme BamHI, and
electrophoresed in a 0.7% agarose gel. DNA was transferred
to nitrocellulose and hybridized to a probe specific for
the entire polyoma genome. Viruses used are as follows:
a=A2, b=A2(A8), 6=A2(A9), d=A2(SD-15), e=A2(II-5) and

f=A2(NG-23).

54

PCC4- EC
Shpi 72 PM

__1
I
.. -.~~‘~~ .-----

viruszabcdef abode:

Figure 5. Replication of the A2(X) viruses in PCC4 EC

cells.

55

enhancer elements’, have also been demonstrated to be
required for viral DNA replication.12'22'42'44'48 We have
studied rearrangements of the non-coding region of viable
viruses constructed in this study (see Figure 6) and how
they relate to these enhancer elements.

Most striking is the frequency in which polyoma
variants possess duplications of subdomains of the enhancer
region.1 Indeed, all five viruses investigated in this
study have at least a single duplication of sequences
encompassing the ’A’, or ’alpha core' domains. These same
sequences have been demonstrated to be responsive to the
middle T protein of polyoma.so Since these enhancers were
originally isolated from hr-t mutants with defective middle
T antigens,2'39 the duplications may have arisen in order
to increase interactions with middle T responsive trans-
acting transcriptional factors (which bind to middle T
responsive sequences within the duplicated region).4'9'50

It is interesting to note that each rearranged region
contains a G to C transversion at nt 5117 and an A to G
. transition at nucleotide 5123. These changes have been
observed in other previously sequenced strains of polyoma,
notably Toronto and P16.34 The transversion at 5117 is
particularly interesting because it has been demonstrated

that a non-functional enhancer sequence (B122), which

contains multiple point mutations, can return to wild type

56

Figure 6. The transcriptional/replicational enhancer
elements, and the factors which bind to this region.

The boundaries of the transcriptional and
replicational enhancer element boundaries, as described by
others,14'15'27'28'48 are compared. The DNA binding sites
of trans-acting factors which have been demonstrated to
bind to this region are also presented. These features are
all compared to the physical map of A2(NG-23). In the
figure, DH=DNase hypersensitive site, and filled in

triangles=inverted repeat region.

57

 

 

 

 

 

 

 

9‘" {DH Pyull {DH A?" 89”
I “A., “B"
t1 2 3
TRANSCRIPTIONAL ’
ENHANCER ELEMENTS
“alga“ “beta.
D As. ..A— tC BM
am- :2: be: (9::- In;
REPLICATIONAL ’
ENHANCER ELEMENTS
3ch (99" mu 39”
b I
PEA FEB-l
w "’9‘" Les; mu
sggm
FACTORS "
—§\

 

58

levels of function with only a single reversion at nt
5117.42 In addition, the positive trans-acting factor
PEAl, binds to nucleotides 5115-5125,“:31 a binding which
can be specifically blocked by mutations at nucleotides
5116 and 5118.32 Possibly, these duplicated regions
increase the ability of polyomaviruses to interact with
PEAl, or PEAl like factors.

Interestingly, there is an inverted repeat sequence in
the polyoma genome at nucleotides 5120-5127 and 5099-5106
(see filled in triangles, Figure 6). In fact, this
sequence is part of the recognition sites for two different
factors, PEAl and PEA2.32 The nucleotides making up the
5099-5106 bp portion of the inverted repeat are duplicated
in all of the reconstructed viruses, (without the A to G
transition present at nucleotide 5123, within the right
hand portion of the inverted repeat), up to four times in
the enhancers of A2(A8), A2(A9), and A2(NG-23), suggesting
that the sequences at 5099-5106 may be functionally
important, possibly by binding trans-acting factors similar
to PEAl and PEA2.

A third feature of the reconstructed viruses is that
the location of their duplications always center about one
of the two known DNaseI hypersensitive sites mapped to
nucleotide 5100.15 This region may be more sensitive to
duplication since it is relatively nucleosome free,

suggesting that the naked DNA might present a more

59

susceptible substrate for the recombination machinery of
the host cell. This site is located within the A
transcriptional enhancer, the dominant enhancer element in

4 Together, these features may favor

mouse cells.1
selection of variant genomes which possess these duplicated
regions. In the most extreme examples of amplification,
the rearrangements of A2(A8) and A2(NG-23), a simultaneous
deletion of the less dominant B enhancer element14 has
also occurred.

The results of Figure 2 clearly demonstrate that each
of the reconstructed viruses can replicate to wild type
levels in the mouse NIH-3T3 cell line. A2(A8) and A2(NG-
23) can replicate to wild type levels, despite deletions of
sequences equivalent to the C replicational domain48 or
'Omega’ element,23 (nt 5159-5183, see Figure 5), providing
the first documentation of viable polyoma viruses deficient
in this element. It has been previously suggested the the
’omega element' is absolutely required for viability, since
no viral isolate (normal or host range mutant) has ever

23 Indeed, a factor

been reported to lack this domain.
called EF-C was found to bind this sequence in a number of
cell lines, including mouse L cells, implicating it as a

DNA binding protein pivotal to polyoma replication.29 The
multiple duplications of the A enhancer domain present in

the viruses A2(A8) and A2(NG-23) may have allowed for the

subsequent loss of the C domain.

60

A number of host range mutants have been isolated
based upon their ability to grow in cells normally non-

1 It was shown

permissive for polyomavirus replication.
that these mutants were selected due to their possession of
rearranged enhancer subdomains.1 The most relevant
comparison of the A2(X) enhancer rearrangements is to those
of the host range mutants which grow in the
undifferentiated embryonal carcinoma (EC) cell lines. The
EC cells are non-permissive for polyoma unless they are
first induced to differentiate. Polyoma mutants which
replicate in the F9 EC cell line have at least a single bp
change at nucleotide 5235 (or duplication encompassing this
bp change) responsible for their new host range 10'11'36'43
Polyoma host range mutants selected on the PCC4 EC cell
line have more extensive modifications responsible for
their new host range, most commonly a duplication in the A
transcriptional enhancer domain coupled with a deletion in
the B transcriptional enhancer domain.25'38'45'46 Trans-
acting factors bind only weakly to the A transcriptional
enhancer domain in undifferentiated EC cells, while binding
is easily demonstrated upon differentiation.21 The
existence of polyoma PCC4 EC mutants suggests that
duplication of this region may allow for increased
interaction with these factors, which correlates with the

new host range. However, our results demonstrating lack of

replication in undifferentiated PCC4 cells of the viruses

61

A2(SD-15), A2(II-5), and especially A2(A9) (see Figure 5)
argue against this being the only requirement for growth in
EC cells, as has previously been noted for other viral
strains with duplications of this region.25
Others have suggested that the deletions in the B
transcriptional enhancer domain prevents polyoma host range
mutants from interacting with negatively acting factors
present in the undifferentiated EC cells, which then allows
for viral replication. Labile proteins, as well as Ela
like repressor activities have been suggested to be
responsible for the replicational blockades, possibly by
binding to sequences deleted in the PCC4 host range
mutants"'5'47 However, these deletions have never been .
demonstrated to extend into the C domain of any polyoma
host range mutant to date, suggesting that this domain is
crucial for viral replication in undifferentiated PCC4 EC
cells.25 The viruses A2(A8) and A2(NG-23) have enhancer
region rearrangements which are strikingly similar to the
rearrangements of known PCC4 EC mutants (each possessing
extensive duplications of the A transcriptional enhancer
domain concommitant with deletions in the B transcriptional
enhancer domain) with the exception that they have a larger
deletion in the region which includes the C transcriptidnal
enhancer domain. Their lack of replication in
undifferentiated PCC4 EC cells supports the hypothesis that

the C domain is required for growth of polyoma viruses in

62

PCC4 EC cells, even when duplications of the A
transcriptional enhancer domain coupled with deletions in
the B transcriptional enhancer are present in the same
genome.

In summary, we have constructed a number of viable
polyoma viruses which all have multiple enhancer region
rearrangements. The actual boundaries of these
rearrangements allowed us to conclude that the previously
described omega element is not absolutely required for
replication of polyomavirus, at least in mouse NIH-3T3
cells. However, the results did suggest that the enhancer
sequences between nucleotides 5159-5183 are required for
replication of polyomavirus in PCC4 EC cells (as had been
previously suggestedzs). Finally, we are currently
investigating the ability of each of these viruses to
infect neonatal mice, to ascertain whether the same regions
implicated as important in the tissue culture systems used
in this study are also relevant in actual infections of
polyomas natural host. The complexity of these
interactions underscores the highly regulated nature of

mammalian gene expression and replication.

10.

63

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Herbomel, P., Saragosti, S., Yaniv, M. (1981). Fine
structure of the origin-proximal DNase I
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Hirt, B. (1967). Selective extraction of polyoma DNA
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Johnson, P.F., Landschulz, W.H., Graves, B.J.,
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Katinka, M., Vasseur, M., Montreau, N., Yaniv, M.,
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Katinka, M., Yaniv, M., Vasseur, M., Blangy, D. .
(1980). Expression of polyoma early functions in mouse
embryonal carcinoma cells depends on sequence
rearrangements in the beginning of the late region.
Cell 20:393-399.

Kovesdi, I., Satake, M., Furukawa, K., Reichel, R.,
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65

Kryszke, M.M., Piette, J., Yaniv, M. (1987). Induction
of a factor that binds to the polyoma virus A enhancer
on differentiation of embryonal carcinoma cells.
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Luthman, H., Nilsson, M.G., Magnusson, G. (1982). Non-
contiguous segments of the polyoma genome required in
cis for DNA replication. J. Mol. Biol. 161:533-550.

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P., Augusti-Tocco, G., Amati, P. (1985). Selection of
mouse neuroblastoma cell-specific polyoma virus
mutants with stage differentiative advantages of
replication. EMBO 4: 3215-3221.

Martin, M.E., Piette, J., Yaniv, M. Tang, W.J., Folk,
W.R. (1988). Activation of the polyomavirus enhancer
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85:5839-5843.

Melin, F., Pinon, H., Reiss, C., Kress, C., Montreau,
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Mueller, C.R., Mes-Masson, A.M., Bouvier, M., Hassell,
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586-599. '

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66

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43.

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67

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68

Chapter 3

Defining subdomains of the polyoma enhancer region
required for high levels of viral replication and

persistence in mouse tissues.

ABSTRACT

The ability of polyoma virus variants to replicate in
mouse tissues is described, utilizing a modified whole body
in-situ hybridization technique and extraction of viral DNA
from individual organs. Wild-type A2 strain polyomaviruses
carrying enhancer regions from hr-t mutants were tested.
Specifically, the viruses A2(A8) and A2(NG-23) have
deletions of 123 bp (which contains a subdomain not
required for replication mouse NIH-3T3 fibroblasts),
responsible for a 30-fold decrease in the ability of these
viruses to replicate in neonatal mouse tissues when
compared to a wild type strain. Another virus, A2(A9), can
replicate to wild type levels early in infection but cannot
persist to wild type levels in 4 week old kidney tissues.
This suggests that viral persistence is not linked to high

levels of replication early in infection.

69

Introduction

Polyoma virus was initially discovered as a
contaminating source of parotid tumors in cell-free
extracts of leukemic Ak mice.16 The agent was shown to be
a virus able to elicit a plethora of tumors including
tumors of the salivary glands, mammary tumors, epithelial
thymomas, and hair follicle tumors.2'3'5'18'41 Over the
years well over 30 different types of tumors have been
described.6 This ability to induce tumors in such a vast
number of distinct mouse tissues, epithelial as well as
mesenchmyal, suggests that the virus has been under
selective pressure to interact with ubiquitous
factors/pathways present in all of these tissues.5 Because
of their small size, polyomavirus as well as the closely
related SV40 have been used extensively as probes to
understand mammalian gene expression and replication.

Early studies demonstrated that the highest levels of
infectious virus production is always observed in
neonatally infected mice.37 Infectious virus was isolated
at the highest levels in the kidneys, with salivary gland,
liver, lung, and spleen harboring intermediate levels, and
brain and blood containing the lowest levels of infectious

virus.38

70

More recent work based on a modified tissue transfer
technique and isolation of polyoma DNA from infected
neonate tissues has confirmed and refined these early
findings.1°'11 It was demonstrated that in neonate mice
infected intra-peritoneally (i.p.) with the wild type A2
strain, a systemic infection develops which includes the
lungs, liver, spleen, and kidneys, with replication levels
peaking at 7-8 days post infection (dpi), and then
decreasing as neutralizing antibody titers rise.1°'11 As
had been demonstrated previously,25'28 the virus was found
to persist in kidney tissue well beyond clearance by the
immune response.11 Interestingly, when the neonates were
inoculated inta-nasally, the lungs also become a site of
viral persistence.11

It is not clear, however, what determinants of the
virus actually control tissue tropism in mouse infections.
The wide range of cell types displaying tumors and/or lytic
damage indirectly suggests that the virus receptor and host
factors controlling viral gene expression are ubiquitous.

It might be anticipated that the enhancer region could
play a role in tropism. However, studies with natural
isolates which only differ in the enhancer region have not
demonstrated any significant changes in the tumor pattern
elicited by these viruses.2 In a more artificial

construct, a virus whose B enhancer domain was substituted

with the LTR sequence from the Moloney murine leukemia

71

virus demonstrated a profound alteration in tissue tropism,
as evidenced by replication in pancreatic tissue, a tissue
that is not normally a site of viral replication36
Furthermore, non-coding sequences outside of the enhancer
on the early side of the replication origin have been shown
to specifically control epithelial thymoma
formation.5'12'13
In tissue culture systems it is clear that the
enhancer region is directly involved in modulating the
replicative host range of the virus.1 This region is
composed of multiple, functionally redundant subdomains,
required not only as transcriptional enhancers, but also as
replicational enhancer sequences.9'19'20'26'30'32'43'44f46
A number of host range mutants have been shown to undergo
rearrangements of their enhancer regions to allow
replication in normally non-permissive cell lines.1 Some
of these cell lines include embryonal carcinoma,
trophoblastic, neuroblastic, and Friend leukemic cell
lines.7'3"‘4'22'27'29'42'45 In some studies actual
interaction with cell line specific factors has been
correlated with the new replicative potential.23'24'47
We have undertaken to test a collection of viable
mutant strains which only differ in their enhancer regions.
The derivation and properties of these isolates in tissue

culture systems along with their enhancer rearrangements is

described in Chapter 2. In the present report we will

72

describe an investigation into the replicative abilities of
these viral strains in infections of neonate Balb/c mice
using a modified whole body in-situ hybridization
technique. Our results suggest that various alterations of
the enhancer affect replication in mouse tissues. In
particular, a subdomain of the polyoma enhancer not
required for growth of the virus in mouse fibroblast tissue
culture cell lines is required for maximal replication in

the mouse.

a ' nd t ods
Construction and isolatign Qt polyoma mutants;

We have previously described the construction of the
A2(X) mutants. Briefly, the enhancer containing BclI-BglI
small fragment from the hr-t mutant strains A8, A9, SD-15,
II-5, and NG-23 were subcloned into the BclI-BglI large
fragment of strain A2. These recombinants were all viable
when grown on NIH-3T3 cells. Stocks of equivalent high
titer were produced, as determined by plaque assay on NIH-

3T3 cells.

- ' ' ' at'o w ' use c s:
' Balb/c pups, less than 24 hours old, were each
- injected i.p. with 50 uL of the appropriate virus stock,

(either one of the A2(X) viruses or wild type strain A2)

73

all at 2.4x107 pfu/mL. At the times indicated, mice were
sacrificed by etherization and embedded into a block of 3%
carboxy-methylcellulose by freezing in liquid nitrogen gas.
Blocks could be stored at -70°C for several months without
significant loss of hybridizeable signals. Blocks were
sectioned to an appropriate sagittal plane with a LKB #2258
cryomicrotome, and then 40 micron sections were transferred
to 3M tape #845. Sections were air dried for one minute,
fixed in a 3:1 mixture of ethanol/acetic acid for 20
minutes at room temperature, dehydrated twice in 95%
ethanol for 5 minutes, air dried and stored at room
temperature. The ethanol/acetic acid fixation proved to be
the superior fixative with regards to allowing for maximal
hybridization with minimal loss of tissue morphology.17
Fixed sections could also be stored several months at room
temperature without significant loss of signal.

After fixation and drying, sections were denatured at
65°C in a preheated solution of 95% deionized formamide in
0.1x SSPE for 20 minutes, and then plunged into ice-cold
0.1x SSPE, (1x SSPE is 0.15M NaCl, 0.01M NaH2P04.H20, and
0.1mM EDTA pH=8.0).

Hybridization was carried out as described,40

except
that SSPE was substituted for SSC, which seems to decrease,
but not eliminate, non-specific binding of 32F labelled
probes to bone tissue (especially a problem in older mice).

Tape with sections were placed into Seal-A-Meal bags with 3

74

mL of prehybridization solution per section and incubated
at 37°C for 18-24 hours. Prehybridization solution (P8)
is: 50% deionized formamide, 5x SSPE, 2.5x Denhardt’s
solution (1x Denhardt's is 0.02% bovine serum albumin,
0.02% ficoll, and 0.02% polyvinylpyrrolidine) with 100
ug/ml of boiled, sonicated, salmon sperm DNA.
Hybridization to the denatured sections was carried out at
37°C for 66-72 hours with 1 mL per section of PS and 0.5-
1x106 cpm of polyoma specific whole genome probe, (probe
was random primed, column purified, and always of specific
activities greater than 1x109cpm/ug). Hybridized sections
were washed three times for 30 minutes in 2x SSPE at 37°C,
once for 30 minutes in 2x SSPE at 65°C, and finally for 30
minutes in 0.1x SSPE at 65°C. Sections were then stained
in haemotoxylin for one minute, rinsed in H20, washed in 1%
Lizco3 for one minute, rinsed again in H20, and air dried.
This staining protocol limited, but did not eliminate
dehydration artifacts in the sections. Finally, sections
were adhered to transparent sheets and exposed to Kodak X-
Omat film with intensifier screens for 24-96 hours at -
70°C. Developed films were directly compared to the
stained section to identify the tissues containing viral-
specific signals.

T§§§Qe_gxtragtign§; Mice were sacrificed and the
appropriate organs were removed and either stored in liquid

nitrogen for later processing, or placed directly into 1

75

mL/mg "tissue digestion buffer’ (tissue digestion buffer
is: 100 mM NaCl, 10 mM Tris pH=8, 25 mM EDTA pH=8, 0.5%
SDS, and proteinase K at 0.1 mg/mL). Tissue was
homogenized at 30,000 rpm with a Tekmar tissuemizer and
digested at 50°C for 12-18 hours with shaking. Digested
tissue was extracted with phenol/chloroform, treated with
RNase A (1 ug/mL, at 37°C for 1 hour) reextracted with
phenol/chloroform, then chloroform. Total DNA was
precipitated by addition of 1/10 volume 3M NaAcetate and
two volumes of 95% ethanol, mixed, and stored overnight at
-20°C. DNA was collected by centrifugation at 10,000 rpm
in a Sorvall SA-600 rotor, resuspended in 10 mM Tris, 1 mM
EDTA, and the final DNA concentration was measured by
absorbance at 260 nM. Digestions with restriction
endonucleases were as described in the figure legends using

the appropriate reaction conditions.

B.EéBltJ

In-sitn hybridization of mouse sections: To

facilitate the detection of DNA by hybridization in tissue
sections, we modified a previously described in-situ
hybridization technique originally derived to detect viral
RNA.40 The specificity of the hybridization conditions is
demonstrated in Figure 1. Probes generated from random DNA
sequences did not hybridize to any sections (data not

shown). The fact that even without denaturation

76

Figure 1. In-situ hybridization to control sections.

Adjacent sections were taken from an A2 infected mouse
and fixed as described in Methods. Section numbers 1, 2,
and 7 were not denatured prior to hybridization with the
polyoma specific probe, allowing only for hybridization to
RNA sequences.40 Section 1 was treated with RNase A, and
section 7 was treated with RNase and DNase prior to
hybridization. Thus, the probe hybridizing to the tissues
in sections 1 and 2 is most likely binding to single-
stranded DNA, as even extensive RNase A digestion does not
eliminate this binding. Sections 3-6 were denatured and
hybridized to the polyoma probe. Section 3 was RNase
treated, 5 was DNase treated, and 6 was both RNase and
DNase treated prior to hybridization. Again, double
stranded DNA is being hybridized to, without significant

contributions from viral RNA.

 

77

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Figure 1. In-situ hybridization to

control sections.

78

some polyoma sequences are detected (Figure 1, sections 1 &
2) and the fact that this hybridization is resistant to
extensive RNase A digestion, (Figure 1, section 1) suggests
that the hybridization conditions facilitate some DNA
denaturation, or that some single stranded viral sequences
are present. Figure 1 also demonstrates that DNA is the
predominant signal, and viral RNA does not significantly
hybridize to probe in infected mouse sections. This was
true for all sections analyzed. This suggests that either
the viral RNA is extremely unstable or at a concentration
which is below detectable limits. This protocol allows us
to not only detect viral DNA in tissue, but also viral DNA
circulating as encapsidated particles. Control sections
from non-infected mice of equivalent age were included in
all experiments, especially when studying sections of older
mice.

While non-specific binding of probe is not a problem
for 8 day old mice, (see Fig. 2 and Fig. 3), four week old
mice exhibit some non-specific binding, most likely due to
.increasing mineralization of bone tissue. This problem can
be limited by adding SSPE to all hybridization solutions
and by extensively purifying away unincorporated
radiolabelled nucleotides from the probe (see Methods). As
described previously, the technique facilitates
visualization and analysis of a number of tissues in a

single section.4o

79

Figure 2. Time course of A2 replication in mouse
tissues visualized by in-situ hybridization.

Neonatal mice were infected with 1.2x106 pfu of wild
type A2 virus, sacrificed at the indicated times, and
sections were hybridized to polyoma specific probe as
described in Methods. Input virus can be detected 12 hours
post infection (see arrow) and by 48 hours a substantial
increase in viral DNA sequences is detected throughout the
abdominal viscera when compared to the 12 hour time point.

Two mice per time point are presented.

80

 

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Fig. 2 shows a time course of replication when
neonatal mice are infected i.p. with 1.2x106 pfu of the
wild type A2 strain. The mid-sagittal sections demonstrate
that viral replication is reaching high levels by 5 dpi
(days post infection) throughout the mouse, consistent with
previous studies.1°'11 This experiment also demonstrates
that the viral DNA examined by in-situ hybridization at
times beyond 5 d.p.i reflects high levels of viral
replication, and is not just a reflection of input virus.

Bgnlicntion of viruses witn ynriant ennnnggrg in
neonntnl mice: We have previously described the derivation
of 5 new viable strains of polyoma virus called the A2(X)
viruses. These strains have identical coding regions (that
of wild type A2) and differ in the enhancer regions, a
region known to affect host range of the virus in tissue
culture systems.1 The enhancer mutations were derived from
natural isolates and consist of duplications and deletions
of enhancer subdomains, typical of natural variants.7'22'45
We asked whether these enhancer alterations would result in
changes in viral replication in mouse tissues upon
inoculation of neonatal mice.

Balb/c mice that were less than 24 hours old were
infected intra-peritoneally with approximately 1.2 x 106
pfu of the variants under study, using wild type A2 as a

reference. The results of in-situ hybridization of mid-

82

sagittal sections taken from 7 d.p.i. mice are shown in
Fig. 3.

The A2 strain demonstrates the highest levels of
polyoma sequences in the kidney tissue, salivary glands,
and skin layers, followed by lesser levels in the liver,
intestines, and lungs, with a notable absence of signal in
the brain tissue and lumens of the stomach and bladder.
When infections with enhancer variants are compared to that
seen with A2, the following pattern of replication is seen.
Viruses A2(A9), A2(SD-15), and A2(II-5) all exhibit
patterns of replication in tissues identical to that of A2,
with slight decreases seen in the total amount of sequences
hybridizing in each section. In contrast, viruses A2(A8)
and A2(NG-23) demonstrate a definite lack of replication
when compared to the A2 pattern. This is a quantitative
defect, for when these sectiOns are exposed for longer
periods, the same tissue pattern of replication is seen as
was for the A2 strain, albeit at much lower intensities.

We have observed these patterns of replication in a minumum
of 8 mice for the A2(SD-15) and A2(II-5) viruses, up to 14
mice for the A2 and A2(A8) viruses. We conclude from these
results that the viruses A2(A8) and A2(NG-23) have a defect
in their ability to replicate in neonatal mouse tissues
relative to A2, A2(A9), A2(SD-15), and A2(II-5) infected

mice.

83

Figure 3. Replication of the A2(X) viruses in mouse
tissues, utilizing in-situ hybridization.

Neonatal mice were infected with equivalent titers of
the viruses indicated, and sacrificed 7 days post
infection. Sections were then hybridized to polyoma
specific probes as described in Methods. Mice infected
with A2, A2(SD-15), A2(II-5), and A2(A9) have the highest
levels of polyoma DNA present, while A2(A8) and A2(NG-23)
have very low levels of viral DNA present. Note the

complete lack of hybridization to non-infected sections.

84

 

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To better quantitate the replication levels observed
by in-situ hybridization, replication in specific organs
was analyzed by extracting total DNA from the spleen,
liver, lung, and kidney tissues of infected mice. The
organ DNAs from three to six similarly infected mice were
pooled, equivalent amounts of DNA were digested with BamHI,
electrophoresed, transferred to nitrocellulose, and
hybridized to a polyoma specific probe (Figure 4).
Consistent with the results from in-situ hybridization,
infections with wild type A2 strain generates the highest
levels of polyoma DNA in the kidney, with liver, lung, and
spleen harboring lesser levels. This pattern has been
observed by others.11 With viruses A2(SD-15), A2(A9), and
A2(II-5) a very similar pattern is observed, with amounts
of polyoma DNA nearly equivalent to those of A2 infected
mice. As expected from the in-situ hybridization data, a
very different pattern is observed with mutant viruses
A2(A8) and A2(NG-23). Based upon densitometric scanning
analysis A2(A8) and A2(NG-23) infections demonstrate a
minimum of 30-fold decreases in the amount of viral DNA
present in all four tissues analyzed. We conclude from
both in-situ hybridization and tissue extracted DNA
analysis that the enhancer rearrangements in strains A2(A8)
and A2(NG-23) lead to a systemic defect in replication in

neonatally infected mice.

86

Figure 4. Analysis of polyoma DNA sequences extracted
from specific organs.

Neonatal mice were infected with the variant viruses
being studied, sacrificed at 7 d.p.i., and the spleens,
livers, lungs, and kidneys were dissected and pooled from
equivalently infected mice. DNA was isolated as described
in Methods, a BamHI partial digest of five micrograms of
each sample was electrophoresed in a 0.7% agarose gel,
transferred to nitrocellulose, and hybridized to a polyoma
specific probe. The viruses are numbered as follows:

1=A2(A8), 2=A2(NG-23), 3=A2, 4=A2(A9), 5=A2(SD-15, and

6=A2(II-5).

87

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88

Pergistence studies in kidney tigsng: Previous

studies have demonstrated that in neonatally infected mice
the kidney is a major target site for polyoma persistance
at 4 w.p.i.11'28 In-situ hybridization analyses of
sagittal sections from a total of 38, 4 week old mice (6
per virus, 2 non-infected mice) proved more difficult than
those of 7 day old mice due to an increase in non-specific
binding of the probe coupled with an overall decreased
level of viral DNA (see Methods). We did note, however,
that in A2(A8), A2(NG-23), and possibly A2(A9) infections,
the levels of viral DNA in the kidneys was reduced compared
to that observed in A2 infections, or infections with the
other A2(X) viruses (see Figure 5). While it is known that
polyoma also persists in salivary tissue,1°'28 (as some of
our sections demonstrate), we could not eliminate non-
specific binding of our probe to these same regions in some
non-infected control sections, making interpretation of
hybridization in this region difficult.

The analysis of viral sequences extracted from kidneys
. of individual mice is shown in Figure 6. It demonstrates
that in infections with A2, A2(SD—15), and A2(II-5)
strains, viral sequences persist at nearly equivalent
levels. In contrast, infection with strains A2(A8), A2(NG-
23), and A2(A9) led to a 15 fold decrease in the amount of
viral DNA sequences persisting in the kidney tissue of 4

week old mice.

89

Figure 5. Persistence of the A2(X) viruses in mouse
tissues, utilizing in-situ hybridization.

Neonatal mice were infected with the designated
viruses and sacrificed at 4 weeks post infection. Sections
were hybridized to a polyoma specific probe as described in
Methods. A2, A2(SD—15), and A2(II-5) consistently had
viral DNA persisting in kidney tissue, unlike mice infected
with A2(A8), A2(NG-23), and A2(A9). Polyoma specific probe
is also observed to be hybridizing to salivary tissue in
these sections. Note the non-specific binding of probe to

the spinal column of one of the non-infected mice.

90

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91

As expected from the low levels of primary replication
observed at 7 d.p.i. for strains A2(A8) and A2(NG-23), very
low levels of persistent replication were observed in
kidney tissue at 4 w.p.i. In contrast, the A2(A9) strain,
which was shown to replicate to near wild type levels at 7
d.p.i. (Figure 4) shows a defect in persistent replication
at 4 w.p.i. This result suggests that high levels of viral
accumulation at 7 d.p.i. is not the only requirement for
high levels of polyoma DNA persistence up to 4 w.p.i. (see
Discussion).

To analyze whether infections of the mouse select for
new genetic variants, the viral DNA's recovered at 4 w.p.i.
were compared to the the sequences of the original stocks,
by restriction endonuclease analysis. MspI was chosen, as
digestions with this enzyme gives rise to a fragment (#3)
which contains the enhancer region, and easily reveals
rearrangements around the origin (fragments 3 & 5) as well
as deletions in other regions (Figure 7). The MspI
analysis of the pooled 4 w.p.i. kidney DNA demonstrates
that the size of fragment #3 from each strain, (as well as
all other fragments) has been stable throughout the course
of infection. This analysis will fail to reveal subtle DNA
changes. However, the most common mutations of interest in
the enhancer region usually are duplications and/or

deletions.1

92

Figure 6. Analysis of polyoma DNA sequences extracted
from kidneys of 4 w.p.i. mice.

Neonatal mice were infected with the indicated
viruses, and sacrificed at 4 w.p.i. The DNA from the
kidneys of individual mice was extracted, and 10 micrograms
of each were digested with BamHI. The samples were
electrophoresed through a 0.7% agarose gel, transferred to
nitrocellulose, and hybridized to a polyoma specific probe.
The mice infected with the various viruses are indicated,

and each lane represents DNA extracted from a single mouse.

93

 

4wpi

KIDNEY
A2(II-5) A2(SD-IS)
I 4

;L~§ .

_. A2(A-8) A2(A-9) * A2(NG-23) MOCK
I 4 6 1 2 3 3 I
Q”. ~0 'I' . ' *

Figure 6. Analysis of polyoma DNA sequences from kidneys

of 4 w.p.i. mice.

94

Finally, we have observed that mice which were
infected with viruses which replicate to high levels at 7
d.p.i. (A2, A2(SD-15), A2(II-5), and A2(A9)) were more apt
to display a previously described runting syndrome,5 have
inflammatory reactions of the eyes, and generally look
sicker than mice infected with A2(A8), A2(NG-23), or non-
infected mice. Possibly, the high levels of replication in

25 allowing

the neonatal kidney tissue damages the tubules
accumulation of toxic metabolites in the blood, causing a

generalized runting phenomenon.

Discnssign

We have begun to analyze what role the polyoma virus
enhancer sequences have in modulating the tissue specific
replication of the viral genome in the mouse. This
question has been vigorously investigated in tissue culture
systems, and the enhancer region has been shown to have a
pivotal role in controlling host range.1'14'2°'22'
27'29'42'45 Whether the findings in tissue culture are
also important in infections of the mouse had not been
specifically addressed. We have begun this analysis by
utilizing natural virus isolates, which contain deletions
and/or duplications of enhancer subdomains. These strains
have been shown to replicate normally in mouse NIH-3T3

cells. For the purpose of facilitating this discussion,

these alterations are diagrammed schematically in Figure 8.

95

Figure 7. MspI size analysis of viral DNA isolated
from 4 w.p.i. kidney tissues.

The 4 w.p.i. kidney DNA's from equivalently infected
mice were pooled, digested with MspI, electrophoresed in a
2% agarose gel, transferred to nitrocellulose, and
hybridized to a polyoma specific probe. Only 5-10
micrograms of A2, A2(SD-15), and A2(II-5) pooled DNA were
digested, while nearly 40 micrograms of A2(A8), A2(A9), and
A2(NG-23) DNA had to be digested, (and exposed longer) to
generate images of comparable intensity. The various MspI
fragments are depicted (the enhancer rearrangements are
within fragment #3). The original viruses used in the
infections are as follows: A=A2, B=A2(SD-15), C=A2(II-5),

D=A2(A8), B=A2(A9), and F=A2(NG-23)

96

MSPI Fragment

Number

 

A B C D E F ‘ Virus

Figure 7. MspI size analysis of viral DNA isolated from 4

w.p.i. kidney tissues.

97

Our results demonstrate that enhancer rearrangements
harbored by the viruses A2(A8) and A2(NG-23) lead to a
significant decrease in replication compared to wild type
A2 at early stages of the infection in neonates as measured
at the peak of viremia (7 d.p.i) both by whole body in-situ
hybridization, and extraction of organ DNA. Three types of
differences are detected when comparing the sequence
rearrangements in the enhancer regions of A2(A8) and A2(NG-
23) to the A2 sequence (Figure 8) These suggest three
possibile causes for the replication defect:

1. There are a number of single base pair additions,
substitutions, and deletions in the A2(A8) and A2(NG-23)
viral sequences beyond nucleotide 5265, on either side of
the ori core of replication (denoted as Region A in Figure
8). The fact that other viruses, such as A2(SD-15) and
A2(II-5), have these changes and are able to replicate to
A2 levels at 7 d.p.i. suggests that these changes are not
detrimental to viral replication.

2. The multiple duplications of sequences around
nucleotides 5088-5141 (Region B, Figure 8) found in A2(A8)
and A2(NG-23) may interfere with viral replication,
possibly by increasing interactions with negatively acting
factors present in mouse tissues. It has been demonstrated
in tissue culture systems that the enhancer does interact

with negatively acting factors.4'47 However, the fact that

98

Figure 8. Physical map of the A2(X) viruses.
The various duplications and deletions are

represented, as previously described in Chapter 2.

99

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Figure 8. Physical map of the A2(X) viruses.

100

A2(A9) replicates to levels comparable to A2, with an
enhancer region containing duplications nearly equivalent
to those found in A2(A8), argues against that possibility.

3. Thus, by elimination, the 123 bp deletion present
in both the A2(A8) and A2(NG-23) viruses appears to be
responsible for the loss of replication in neonatal
tissues. This suggests that a positively acting factor is
interacting with sequences between nucleotides 5141-5265 of
the polyoma genome in order to facilitate viral
replication. This factor may be responsible for the high
levels of replication demonstrated in neonatal tissues, in
particular the kidney.

The polyoma enhancer region has been dissected into a
number of subdomains in tissue culture systems,19'2°'3°'
32'44'45 as summarized in Fig. 9. Some of these domains
are functionally redundant. The polyoma transcriptional
enhancer domains are also replicational enhancer domains,
thus blocks in replication are usually accompanied by
blocks to transcription of viral early genes.9'19'43r46
The nucleotides within 5141-5265, deleted in A2(NG-23) and
A2(A8) define one of the two functionally redundant
enhancer elements, (called B and C46 or beta core and beta
auxillary sequences31). These sequences have been shown to
not be required for replication of polyoma in mouse NIH-3T3

cells (see Chapter 2), yet in actual mouse infections this

101

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TRANSCRIPTIONAL T
ENHANCER ELEMENTS

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Figure 9. The transcriptional/replicational enhancer

elements, and the factors which bind to this region.

102

region is crucial in allowing for high levels of viral
replication.

The enhancer subdomains contained within this region
have been demonstrated to bind to a number of cellular
factors,21'33"35 see Fig.9. The most intriguing of these
factors in the context of our findings is EBP-20, a protein
isolated from rat liver which was purified based upon its
ability to bind to sequences 5211-5233 of the polyoma
genome.15'21 Possibly, a factor similar to EBP-20 is also
present in neonatal mouse tissues, and controls the high
level of viral replication shown by wild type viruses.

Interestingly, it has been demonstrated previously
that a polyoma virus mutant with the B and C domains
replaced by the LTR of the Moloney murine leukemia virus
had lost the ability to replicate at normal sites in
infections of neonate mice, and had simultaneously acquired
a pancreatic tissue tropism.36 Our results suggest that
the pancreatic tropism was found because of the loss of the
B and C domains, decreasing the levels of overall
replication enough to allow visualization of the new tissue
tropism induced by the LTR sequences.

The ability of the A2(X) viruses to persist in kidney
tissue up to 4 w.p.i. was also investigated, and while the
results are a bit more difficult to explain, they are
intriguing nevertheless. The A2(SD-15), and A2(II-5)

viruses persist in kidney tissue at a level equivalent to

103

that observed for wild type at 4 w.p.i., suggesting the
sequence alterations in A2(SD15) and A2(II-5) are not
deleterious to persistence. As expected, no persistence is
observed with A2(A8) and A2(NG-23), most likely due to
their inability to replicate to high levels early in the
course of infection. Interestingly, the A2(A9) virus does
not persist at 4 w.p.i. in kidney tissue, despite a normal
early replication pattern. This suggests that persistence
is not solely dependant on high level viral replication
early in infections of mice, but that fulfillment of other
criteria is also required.

Comparing the sequences of the viruses (see Figure 8)
as we did earlier suggests two possible reasons for the
persistence defect of A2(A9). One is that the additional
duplications of sequences within nucleotides 5088-5141 (in
relation to the single duplications found in the A2(SD-15)
and A2(II-5) viruses) may allow for increased interactions
with negatively acting factors in kidney tissue. This
phenomenon could also contribute to the lack of persistence
_ seen with the A2(A8) and A2(NG-23) viruses. The second
possibility is that the single bp substitution at nt 5351
found in A2(A9) and not in A2, A2(SD-15), or A2(NG-23) may
interfere with a positively acting factor required for
viral persistance in kidney tissue. These two possibilites

are being presently explored.

104

In conclusion, we have determined that the sequences
of the polyoma genome between nucleotides 5141-5265 are
required for high levels of viral replication, most likely
by interactions with a positively acting factor present in
neonatal mouse tissues. Secondly, persistence of polyoma
DNA in kidney tissue at 4 w.p.i. is not only dependent on
high levels of viral replication early in infection, but
also on interactions with other positive or negatively

acting factors.

10.

105

W

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110

Summary and Conclusions

The role of the polyoma enhancer region in directing
replication of the viral genome was investigated. The
experiments described in Chapter 2 utilized tissue culture
systems and recombinant viruses to define a subdomain of
the polyoma enhancer as either essential for allowing viral
replication in PCC4 embryonal carcinoma cells, or
expendable in mouse fibroblasts. The presence of this
domain had been thought to be required of all viable
viruses since none had ever been isolated which lacked this
domain. The results presented here suggest that further
studies should be done to describe the exact nature of the
functional redundancy exhibited by the viruses A2(A8) and
A2(NG-23). Possibly interactions with new host proteins,
and/or modified interactions with previously described
factors is the cause of the effect. Thus, a firmer
understanding of the processes of gene expression and DNA
replication may be gained by such studies.

The studies in Chapter 3 demonstrated that viruses
which could replicate to wild type levels in tissue culture
systems did not always behave accordingly when actual mice
were infected. This demonstrates one of the few
shortcomings of tissue culture systems, that the results

obtained in tissue culture may not accurately reflect the

111

true interactions at the host level. Nevertheless, the
results demonstrated that modifications of the enhancer
region alone, (without changes in viral capsid structure,
or structural proteins) could have a profound effect on the
ability of viruses to grow in normally permissive host
tissues. The regions of the enhancer defined as crucial
(in Chapter 3) to allow for high-level viral replication
indicates that there may be specific factors in the mouse
tissues interacting with this particular enhancer domain.
Protein extracts from mouse tissues may contain specific
enhancer binding proteins specific for this region. These
factors may be central to gene expression in general, since
loss of this enhancer domain abolished viral replication in
a number of mouse tissues.

Finally, the results presented here suggest that the
pathogenicity of other viruses may be altered simply by
altering their respective enhancer regions, preventing
viral gene expression in host tissues. This sort of
approach, along with the more traditional ways of
attenuating viruses, may allow for better vaccine

production.

Appendix

Cover Sheet

The following paper, in which I appear as an author, is included

here because I was responsible for a large portion of the data. This

includes all data referred to in Figures 3-6, and Experiments 2b-Sc
listed on Table I.,

112

Prepared for Hol. Cell. Biol.

Appendix
LOH PROBABILITY 0? DOUBLE INTEGRATION IN TRANSFORMATION
OF NONPERHISSIVE CELLS BY POLYONA VIRUS

Saw Yin Oh’, Andrea Amalfitano, Karen Friderici”, Hing Chu Chen,

and Michele H. Fluck'

Department of Microbiology, Interdepartmental Program
in Molecular Biology
Michigan State University
East Lansing, MI 48824-1101
(517) 353-5014

Running title: Integration of unselected polyoma genomes

This is Journal article no. from the Michigan Agricultural Experiment

Station.

Present Address: *Department of Pharmacology, Fluiders University,
Bedford Park, South Australia 5042
’*Department of Pathology, Michigan State University,

East Lansing, Michigan_ 48824

113

Abstract

F-111 rat and BHK hamster cells were infected with mixtures of wild
type polyoma virus and transformation-defective deletion mutants. The viral
integration patterns of‘ resulting transformants were determined. In the
majority of the transformants the unselected deletion mutant was not
present, even when the unselected parent was used in large excess over the
wild type in the infection mix. These results were obtained for transfor-
mants derived with a variety of parental viral strains. Overall, the
unselected parent was recovered in only 111 of the transformants analyzed
(i.e. in 14 of 142). When two transformation competent strains were used
to infect rat cells, a low frequency of double parental integration was
also observed. It was shown that both parents in the infection mix are
infecting the same cells and that both replicate and persist in the
infected cells up to the time when transformants arise. These results
suggest that the probability for two independent integration events per
transformant is low, in contradiction to previous results on the analysis
of the number of integration sites determined by hybridization of restric-
tion endonuclease fragments separated by electrophoresis. We present a

reinterpretation of these data in agreement with the present data.

114

Introduction

The pattern of viral integration concomitant with neoplastic transfor-
mation by papovaviruses polyoma and Sit-40 has been extensively studied.
Analysis of cellular DNA from transformants by electrophoretic separation
of restriction endonuclease fragments has shown that the viral genome is
apparently inserted at multiple sites in the host chromosome, where it is
arranged in a head-to-tail manner (4,5). The variation in patterns from
transformant to transformant suggests a great diversity in chromosomal
integration sites. A review of the published literature concerning the
number of integration sites of the polyoma viral genome in rat transfor-
mants suggests multiple sites in 801 of the cases reported--the number of
sites per transformant averaging over three (1,4,8,15,20,29). In experi-
ments using gels with more resolving power, this number appears to be even
higher (i.e., eight to ten) (16 and K. Friderici and H. Pluck,
unpublished). Thus, it has generally been assumed that the number of
chromosomal sites available for integration is very high, if not unlimited.
The simplest model would assume 1) that each of the multiple integration
events in any transformant represents a random interaction between avail-
able sites on the host chromosome and available members from the pool of
integratable viral genomes and 2) that each event happens independently
from the events occurring at other sites.

Assuming that all genomes present in the parental infection mix have
equal probability to become integrated, we would expect the integrated
genomes to be a faithful representation of all the viral genomes present in
the input for the infection. To test this prediction, we have carried out

mixed infections of nonpermissive rat and hamster cells with wild type and

115

transformation defective deletion mutants, using various multiplicities and
ratios of the two parents. We isolated transformants from such infections
and studied the viral integration patterns of the two parental genomes. It
was expected that, in addition to the selected transforming wild type
parent, the transformation-defective mutant would be recovered in the
transformants at a frequency and a dosage which reflect the ratio of the
two genomes in the input of infection. The results of our test are
described in the present report. They failed to support the prediction
from the model, and consequently, generated hypotheses for an alternate

model.

 

116

Materials and Methods

Cells and Viruses. Fischer rat F-111 (10), and hamster BHK cells (28)
were grown in Dulbecco's modified Eagle's medium supplement with 10: new-
born calf serum.

wild type strains A2 and A3 are widely used strains which have been
sequenced (16,24). The pseudo-wild-type strain NGS9RA (RA) was obtained by
marker rescue (11) of the middle T-antigen defect of hr-t mutant N059 (3).
An unexpected large T-antigen defect of this strain has been described
elsewhere (18). Differences between RA and A2 in the enhancer region have
been noted previously (24). RA and A2 also differ in plaque morphology:
RA is a small plaque virus with a heat stable hemagglutination pattern (6),
while A2 is a large plaque virus. Transformation-defective hr-t mutants
82, 30'8, and 3A3 were derived from the Pasadena small plaque strain by
mutagenesis with ICR-191 (27). All hr-t mutants used contain rearranged
enhancer regions (11 and K. Higgins, K. Friderici and M. M. Fluck,
unpublished) as well as a deletion in the middle T-antigen gene located in
HpaII/MspI fragment 4 (3,11,16). This deletion is also easily detectable in
PvuII digestions. Transformation defective mutant 18-5 was constructed from
wild type A2 and was a gift of M. Fried (20). It too contains a deletion
in MspI/HpaII fragment 4. Hild type RA-BZOri was constructed by ligating
the small BglI-BamHl fragment encompassing the enhancer-origin region of
hr-t mutant B2 to the large genomic BglI-Bamfll fragment of pseudO'wild type
RA. Mutant d145, derived from wild type A3 (2), has a deletion of 66
nucleotides in the overlapping large T-antigen/middle T-antigen reading
frames. The deletion can be visualized in digestion with Sst I. This

mutant is reported to have no transformation nor growth defects (2).

117

Infections. F-111 or BHK cells were seeded at a density between 105
and 1.5 x 106 cells per 60 mm plate (as described in the text and Table 1)
and infected at the multiplicities and ratios of mutant to wild type virus
indicated in the text and Table 1. In most instances, the relative ratio
of the two parental genomes in the infection mix, established from the
titer of the two parental viral stocks, was confirmed by hybridization
analysis of the viral sequences in the input. Cells were fed with DMEM
supplemented with Si newborn calf serum and became confluent within one to
eight generations postinfection (see Table 1). In experiment 4b and 5b,
each infected 60 mm dish was passed to ten 100 mm dishes (leading to an
approximate cell confluency of 1.5 and 31, respectively) and fed with DMEM
supplemented with 10: serum until confluent. In most cases, transformants
from infections carried out under the various conditions described were
isolated as foci overgrowing a cell monolayer. In some cases, transformants
were isolated directly (or were recloned) by growth in agar.

DNA Analysis. Total cellular DNA was isolated from infected or trans-
formed cells as described (12,21). For gel electrophoresis, each sample
contained 10-20 ug DNA digested with the restriction enzymes indicated in
the text and figure legends. Endonucleases were chosen for their ability
to generate fragments with which wild type and deletion mutant sequences
can be distinguished. MspI/HpaII or PvuII were chosen for all infections
with hr-t mutants (exp. 1-8 and 10, Table 1), and Set! to distinguish
between A3 and dl45 (experiment 9, Table 1). After transfer to nitro-
cellulose (21,26), the blots were hybridized to polyoma nick translated
probes (1-2 x 108 cpm/pg). '“Ibridization was at 65°C for 3 days in

2XSSC/1X Denhardt's (0.1 ml/cm2 using 1 x106 cpm of the labeled probe per

118

ml of hybridization solution). In many of the hybridizations a probe
consisting of HpaII fragment 4 and designated pPyH4 was utilized. Since the
hr-t deletions are contained within the fragment 4 generated by restriction
endonuclease HpaII or isoschizomer MspI, this probe allows the identifica-
tion of wild type or mutant sequences spanning this area of the genome

(12).

119

RESULTS

An assay for the integration of unselected viral genomes. To analyze
the integration of viral genomes unselected by the transformation process
we established transformants from infected nonpermissive cells, with
mixtures of transforming and nontransforming viral strains containing dele-
tions in their genomes (see Materials and Methods). Such transformants are
expected to contain an integrated :ild type genome selected in the trans-
formation process. The integration of the unselected transformation-
defective deletion genome present in the infection mix was detected by
means of restriction endonucleases which distinguish between the wild type
and the deleted parental genomes (see Materials and Methods). A total of
120 transformants were analyzed; these were derived from ten different
infections involving four different wild type viruses and four deletion
mutant viruses. These results are summarized in Table 1.

Normal overall integration patterns in transformants derived from
mixed infections. The viral integration pattern of transformants resulting
from the mixed infections were analyzed by restriction endonuclease diges-
tion, electrophoresis, Southern blotting and hybridization to viral probes.
The patterns obtained for transformants derived from co-infections of
Fischer rat F-111 cells with wild-type strain RA and transformation defec-
tive hr-t deletion mutant 82 are shown Fig. 1. In the experiment shown,
the two parental genomes were used at equal multiplicity of infection. In 8
out of 10 transformants presented in Fig. 1 (experiment 1, Table 1), a
typical integration pattern is observed: 1) first and most important,
multiple apparent sites of integration are present, as evidenced by

multiple bands in digestions with restriction endonuclease BglII which does

120

a
Table 1. Integration of an unselected viral genome.

3 Summary of the transformants analyzed in this report. Rat or hamster
cells were co-ihfected as described in Materials and Methods by the
deletion mutant and transforming parent indicated. The multiplicities of
infection for each parent are indicated.

5 The deletion mutants described in Materials and methods which can be

distinguished from the wild type by the presence of deletions in their

genomes. 82. 30'b, and 3A3 are transformation defective “hf-t" mutotta.
which are not selected in the transformation process; these are easily
distinguished from wild type sequences in digestion with MspI/Mpali or
PvuII. Mutant d145, used in experiment 9, is a transformation competent
strain. which can also be distinguished from wild type A] DdcawSO of a
66 bp deletion in its genome. The deletion is easily visualized in
digestions with Sstl.

c HT refers to the source of the transforming parent. For a description of
these strains, see Materials and Methods. In experiment 9, either parent
(A3 or d145, or both) can contribute to the transformation phenotype.

9 The ratio of the multiplicities of nontransforming to transforming mutant
which were used to Obtain the transformants. In most cases, the ratio of
the two parental genomes in the infection mix, established by relying on
the titles of the parental viral stocks, was confirmed by hybridization
analysis of the infection mix. '

‘ The number of cell divisions allowed postinfection until infected cells
formed a confluent monolayer. This number depends on the cell density at
the time of infection (see Materials and Methods). Cells were set in
60 mm dishes at: 1) 2 x 105; 11) 5 I 105; 111) 1 x 106 cells per dish. In
'I. infected cells were passed to 10 plates within 24 hrs postinfection.
In a, infected cells were passed to agar immediately postinfection.

f Summary of the number of transformants containing the unselected viral
genome over the number of transformants analyzed as determined by

Southern blotting analysis and illustrated in Fig. 3.

8 The ratio in percent of transformation frequency in the mixed infection
over transformation frequency in the infection with the transforming

parent alone. NA no applicable. ND not done.

 

 

 

121

' Table 1. Integration of an Unselected Viral Genomea

 

l of cell Mutant 1 HT

Exp. 525325“ flflllc Egg? Egg; divisionse Integrations Transformstionb
1 32 RA 5:5 5-111 2-31 0/10 no
2a 32 RA 1:1 3-111 2.31 0/4 A00
2b . 5:1 013 1700
3 32 RA 20:0.1 2-111 8‘:' 1/1u .200
ua 32 RA 20:1 3-111 1H 2/6 ND
ub u-s‘o' 1/6 ND
5a 32 RA 10:10 3-111 01“ 2/12 ND
5b 6-7‘1‘v' 0/12 no
So 2:2 6-7"‘ 1/6

6 32 RA-BZOrl 20:3 r-111 2-31 0/12 150
7 301a A2 5:1 2-111 2.31 1/5 t 30
8 3A3 A2 20:1 9-111 2-3' 0/5 13
9 dius A3 10:10 5-111 3, 0A both 2112, u/22 NA

4135 2112; 10/22
‘ A3 8/12. 8122

10 32 RA 5:5 aux 2-3‘ 1/13 ND

 

Total ' 16/1u2

I22

not cut the viral genome (Fig. 1A) (for example, the transformant analyzed
in lane 2 shows seven bands apparently representative of integration sites.
including an unresolved band at limit entry which appears to represent more
than a single band); 2) free viral genomes are evidenced by a band of
identical size in all samples co-migrating with supercoiled viral DNA in
the same Bglll digests (Fig. 1A), and 3) the viral genome is integrated as
a head-to-tail tandem, as revealed by a genome size 5.3 kbp band in diges-
tions with EcoRI which cuts the polyoma genome at a single site (Fig. 18).
Only two transformants in 10 show a single site of integration containing
an incomplete viral genome and no free viral DNA in this particular set
(lanes 1 and B Fig. 1A and 18). Another 63 rat transformants derived from
other mixed infections with RA and 82 were analyzed and found to have
”normal" integration patterns (experiments 2. 3, ll, 5, Table 1) as well as
12 transformants derived from infections with 82 and wild type RA-BZOri
(exp. 6). This is also the case in transformants derived from co-infec-
tions of hr-t mutants 30'b or 353 with wild type A2 (10 cases analyzed)
(experiments 7, 8, Table 1). Apparently, most of the Fischer rat transfor-
mants analyzed (95$) contain evidence for multiple integration sites as
Judged by analysis with a restriction endonuclease which does not cut the
viral genome. Note that the number of flanking bands in digests with EcoRI
is less than expected (compare lane 2 in Fig. 1A and 18). He will return
to this apparent paradox in the discussion.

Low frequency of integration of the unselected parent. The transfor-
mants described in Fig. 1 were further analysed for the presence of the
two parental genomes. Each parent can be easily distinguished by the

presence of a normal or a deleted restriction nuclease fragment. Fig. 2

Fig.

123

1. Integration patterns in F-111 cells.

Rat F-111 cells (2 x 105 cells per 60 mm dish) were infected at a
multiplicity of five plaque-forming units each of transforming strain
RA and transformation defective deletion mutant B2 (experiment 1,
Table 1). Independent transformed foci were selected and cloned by
anchorage independence in soft agar. 10 to 20 pg of high molecular
weight DNA isolated from these clones was digested with BglII (A) or
EcoRI (B) and fractionated on 0.6: agarose gels. Hybridizations were
as described in Materials and Methods using 106 cpm/ml of a probe
(pPy-1) representing the whole genome. M shows the position of marker
viral DNA digested with EcoRI and the arrow shows the position of
supercoiled form I unlntegrated viral DNA. Lane 1-10 contain DNA from

individual foci.

124

M1234567891O

 

 

ii
A 23.... 9 ---e11 190
9.5—. ' C V "' .
- w
. O
O
5.3-.
7 I,“ ‘
> 3‘.
i: i
S! . ’2.
1:
a;
M12345678910“
. i"
- ‘3
.

          

Figure 1. Integration patterns in F-lll cells.

125

shows the results for 10 Fischer rat transformants from Table 1, experiment
1, digested with restriction enzyme MspI. The probe used in these experi-
ments is the MspI/HpaII fragment '1 in which the B2 deletion (nucleotides
1191-730) is located (6). All transformants contain a normal size fragment
4 corresponding to the presence of the wild type parent. This is expected
since fragment If is in the coding region for middle T-antigen which is
required for transformation. In contrast, none of 10 F-111 transformants
contain (the 82 parent. This is surprising for 8 of 10 of these trans-
formants which apparently have evidence for multiple integration events.
Similar underrepresentation of the B2 mutants was observed in other experi-
ments in which the RA and 82 parental genomes were used in equal ratios at
varying total multiplicity of infection. The B2 parent was recovered in
none of four transformants analyzed in experiment 2a. in two of 12, four of
12 and one of six in experiment 5 a.b.c, respectively.

To increase the probability of mutant recovery. the ratio of the
unselected to the selected parent was varied in the infection mix in favor
of the unselected parent. In the most extreme case. the-experiments were
repeated using a ZOO-fold excess of the unselected parent (i.e., multipli-
cities of 20 of the unselected parent and 0.1 of the selected transforming
parent [experiment 3]). In such conditions, one should be able to achieve
an effective ratio of mutant to wild type of 20 to 1 per transformable
cell, i.e., in each cell which receives a wild type (experiment 3, Table
1). The high ratio of unselected to selected- parent was confirmed by
hybridization of the HpaII digested viral DNA extracted either from the
infecting mix or from infected cells at 2 hours post infection. In these,

the mutant shows a dense band while the wild type sequences are below the

126

Fig. 2. Low frequencyiof integration of the unselected parent among the
integrated viral sequences in rat transformants.

The rat F-111 transformants described in Fig. 1 were further analyzed

for the presence of the unselected parental genome. 10 to 20 pg of

DNA was digested with MspI and fractionated on 2: agarose gels.

Blotting and hybridizations were as described in Materials and

Methods. The probe used is pPyHu, representing the MspI fragment A in

which the hr-t deletion is located.

127

M1234567891O

H4mm .

 

Figure 2. Low frequency of integration of the unselected parent
among the integrated viral sequences in rat transformants.

128

detection level. As summarized in Table 1, the high level of mutant to wild
type did not alter the results: only one in 1H transformants analyzed from
this infection contained the unselected parent.

Similar results were obtained in further experiments: for example 82
was recovered in no transformant of three analyzed in experiment 2b
performed at a 5:1 ratio of mutant to wild type, and only in three of 12
cases from experiment A, carried out at a ratio of 20 to 1. As can be seen
in Table 1 the recovery of the unselectes parent seems independent of the
total multiplicity of infection which ranged from 1:1 (experiment 2a) to
10:10 (experiment 5a,b) to 20:1 (experiment 3, A). Fig. 3 illustrates that
the ratio of mutant to wild type genome in the infected cells in experiment
A is indeed as high as expected from the ratio of the two parents in the
infection mix, as established by using plaque-forming units. The DNA shown
in the figure was extracted 3 days postinfection [a time at which there has
been little or no replication of the input genomes (19)] and was digested
with PvuII and hybridized with the: pPyMu probe which spans the region
deleted in mutant 82. The 0.911 kb deleted PvuII fragment is clearly in
vast excess over the wild type 1.2 kb fragment.

Low probability of recovery of two parental genomes was also
demonstrated in transformants derived from mixed infections with
transformation defective M09103] and transformation competent tsB. In
these, only 9 of 69 transformants analyzed contained both parental genomes
(17).

Underrepresentation of the mutant sequences in mixed transformants.
In those few instances in which the unselected parent was recovered in the

transformed cells, the level of sequence from the deletion parent was not

129

Fig. 3. Ratio of wild tyge and mutant genomes in the infection mix.

Rat F-111 cells (5 x 105 cells per 60 mm dish) were infected with a

 

mixture of wild type RA and hr-t mutant 82 at (multiplicities of 1 and

20 respectively) (experiment. A, Table 1). Total DNA was extracted

3 days postinfection, DNA was digested with PvuII and hybridized with
pPyHN as in Fig. 2. The 1.2 kb wild type fragment and 9.98 kb mutant
fragment are shown. A: confluent cells (experiment l1a). 8: one-tenth
of cells shown in A were passed 10 hours postinfection and allowed to

grow to confluency (experiment Nb, Table 1).

130

 

Figure 3. Ratio of wild type and mutant genomes in the

infection mix.

131

represented proportionally to its level in the input. This is illustrated
in Figs. 3 and u for the three mixed transformants derived from
experiment A, Table 1. Fig. 3 described above shows that the ratio of non-
transforming to transforming parent in the infected cells at early times
postinfection is very high: i.e. in the 20:1 ratio range established for
the infection mix using stock titers. In contrast, those transformants
which do carry the nontransforming mutant (Fig. A) show ratios of wild type
to deletion sequence which varies from, at most, equal (lane 1), to much
less than equal (lane 3). Similar results were obtained in the other
experiments involving high ratios of mutant to wild type (experiment 3, 7,
Table 1). The presence of free viral DNA in those transformants does not

alter these conclusions since the unlntegrated DNA is produced by i situ

 

replication from the integrated sequence (15) and the composition of
unlntegrated copies is a faithful copy of the integrated sequences (15,17).
The loss of the deletion mitant is not due to underreplication.
Persistence of the mutant and wild type sequences in the infected cells was
compared from early times after infection until the appearance of trans-
formed foci over the monolayer. For this purpose, F-111 cells were
infected at equal multiplicities of the two parents RA and 82 to allow the
detection of both parents throughout the course of infection. Furthermore,
this experiment was also designed to test that the lack of recovery of the
hr-t mutant was not due to segregation to different cells by cell division.
Thus, one-half of the plates were maintained at confluency postinfection
while the other half were diluted 1 to 30 to allow cells to divide. Total
DNA was isolated from infected cells at various times post infection and

analyzed by Southern blotting. Results from experiment 5 are shown in

132

Fig. H. Underrepresentation of the unselected parent among mixed trans-
formants.

Rat transformants were obtained as described in Fig. 1 except that the

ratio of mutant to wild type was 20:1. The three mixed transformants

among 12 analyzed from experiment 8 in Table 1 which contained

integrated wild type and mutant genomes are shown. High molecular

weight DNA was extracted, digested with PvuII 'and hybridized with

pPyHN as described in Fig. 2. Markers shown are as in Fig. 3.

133

1.2 )1

0.941>

 

Figure 4. Underrepresentation of the unselected parent

among mixed transformants.

a--- —— o

134

Fig. 5. It is clear that the signal from the deletion mutant is retained
in the population of infected cells. As is most evident in the infection
of confluent cells, the ratio of wild type and mutant is not altered during
the course of infection and the mutant persists up to the time of
appearance of transformed foci. In the population of dividing cells, the
signals of ‘both wild type and mutant DNA decrease, relative to cellular
DNA. Ne have recently demonstrated that a low level of viral DNA synthesis
occurs in F-111 cells maintained at 37°C (19). Clearly, the amount of
synthesis does not ensure efficient synthesis and segregation of the viral
genomes in daughter cells, so that under conditions of low cell density,
the signal is lost after a few generations. However, there is no preferen-
tial loss of the deletion parent in these conditions. If anything the 82
genome is preferentially retained. This may be due to differences in the
enhancer region between the two parents (M. C. Chen and M. M. Fluck, in
preparation).

The loss of the nontransfbrming deletion mutant is not due to a segre-
gation problem. Most of the experiments described above were carried out
in conditions designed to optimize transformation. These usually allow for
a few cell divisions post infection before the monolayer becomes confluent.
Since the time at which the integration events are fixed is not known, and
may not be during the first generation, we considered the possibility that
the transformation-defective hr-t mutant and wild type might segregate to
different cells during cell division, resulting in loss of the hr-t mutant
from cells which contain wild type and can potentially become transformed.
Thus, infections were carried out with monolayers derived from

exponentially growing cells reseeded at confluency prior to. infection

135

Fig. 5. Fersistence of the viral genome in the infected cell populggigg.
Confluent F-111 rat cells were infected as described in Materials and
Methods (experiment 5, Table 1), with transforming parent RA and non-
transforming parent 82 at multiplicities of'ten for each parent. One
half of the plates were maintained at confluency (lanes 1 to 3) while
the other half (lanes 8 to 7) were diluted 1 to 30 to allow cells to
divide. Total cellular DNA was isolated at the times indicated
(dpi = days postinfection), digested with PvuII, electrophoresed and
analyzed by Southern blotting and hybridization as. described in
Materials and Methods and in Figs. 1 to 3. Hybridization was carried

out with pPyHN as described in legend to Fig. 2.

 

 

 

136

123 4567

WT

32

 

dpi 0 5’ 15 2.5 5 10 15

Figure 5. Persistence of the viral genome in the infected cell

population.

 

137

(experiment 11a and 5a, Table 1). As summarized in Table 1 and illustrated
in Fig. 6, for nine transformants from experiment 5, this procedure did not
increase the recovery of the hr-t mutant. Of 112 transformants analyzed in
experiments A and 5, four in 18 contained the hr-t mutant in the infections
with nondividing cells, versus six in 211 from the dividing cells. As
already noted above, in mixed transformants the ratios of the intensity of
mutant to wild-type fragment were considerably in favor of the wild-type
fragment even for those transformants which arose in the nondividing
condition. These experiments suggest that integration (or the events which
fix it) occur early after infection.

The underrepresentation of molested genomes is a general phenomenon.
He considered the possibility that the underrepresentation of the hr-t
mutant 82 in transformants derived from mixed infection with strain RA is
specific to this particular pair of genomes. For example, differences in
adsorption, decapsidation, histone acetylation (25), might either retard
one of the parents or segregate them to separate .cell compartments.
Different affinities for transcription factors or large T-antigen could
also differentiate their integration potential. This possibility needs to
be entertained since RA and 82 differ in multiple sites: the origin-
enhancer (K. Higgins, K. Friderici, M. C. Chen, and M. M. Fluck,
unpublished), in the large T-antigen coding region (between EcoRI and Nsil)
(18), and in the capsid region (6 and K. Friderici and M. M. Fluck,
unpublished). To analyze the role of differences in the origin-enhancer
region (encompassing binding sites for the large T-antigen and transcrip-
tional factors), a strain was reconstructed in which the small 8glI-8cli

fragment comprising the origin-enhancer region in strain RA was replaced by

138

the equivalent region from mutant 82. As can be seen (experiment 6,
Table 1). this exchange did not relieve the underrepresentation of 82, as
82 was not recovered in any of 12 transformants analyzed. To further test
the generality of the underrepresentation, other pairs of parental strains
were used including wild type A2 and other hr-t mutants such as 30'b and
3A3 (experiments 7 and 8, Table 1) or wild type A3 and mutant dlAS (see
below). As can be seen, the unselected parent was also underrepresented in
these infections.

Evidence that the selected and the unselected parent are in the same
cells. One can imagine that the underrepresentation of the unselected
parent in the transformed cells reflects a low frequency of co-infection
which may be linked to an exclusion phenomenon. This possibility is not
ruled out by the persistence of viral genomes of both types in the infected
cells, since these might reside in different cells. However, a physio-
logical aspect of the co-infection suggests that this is not the case.
Nhen Fischer rat F-111 cells are co-infected with RA and hr-t mutants such
as 82, the yield of transformants in the mixed infection. is higher than
those observed in infection with wild type alone used at the same multipli-
city. This effect is dependent on the dose of 82 mutant present in the
infection. As is illustrated in experiment 2, Table 1, the yield of trans-
formants was increased H-fold at a 1 to 1 ratio or 17-fold at a 5:1 ratio.
Re are assuming that this effect is due to complementation by the 82 parent
of a large T-antigen mutation which was very recently discovered in strain
RA (18). Similar examples of complementation have been documented
previously between large T-antigen and middle T-antigen mutants (91. It is

worth emphasizing that even when a high level of complementation is

 

139

Fig. 6. Recovery of an unselected viral genome from transformants

obtained from nondividing or dividing infected F-111 rat cells.

Infections of F-111 rat cells were carried out as described in the

 

legend to Fig. 5 and Materials and Methods. Transformants were iso-
lated and high molecular weight DNA extracted and analyzed as
described in legends Fig. 1 to 3. DNA digestion with PvuII and hybri-
dization were as described in legend Fig. 2. Lanes 1 to N were from
cells maintained at confluency postinfection. Lanes 5 to 9 were

diluted 1 to 3D postinfection.

 

140

123456789

m8

*
m»..- -~$. WT

- seem B2

 

Figure 6. Recovery of an unselected viral genome from transformants

obtained from nondividing or dividing infected F-lll cells.

141

observed, the complementing parent is underrepresented in the transformant.
In experiment 2b of Table 1, a 17-fold complementation factor was observed
in co-infections with 82, at a 5:1 ratio; yet the mutant was not recovered
in three transformants analyzed.

In contrast, when co-infections are carried out with wild type A2, the
yield of transformants is depressed proportionally to the dose of hr-t
mutant. Depression of transformation was recorded in experiments 7 and 8,
Table 1. For example, in experiment 6, the yield of transformants Obtained
in co-infection of wild type A2 with hr-t mutant 3A3 was reduced five times
compared to the yield obtained with the same dose of wild type in single
infection. This effect has also been noted previously and referred to as a
dominant lethal effect (9). He believe that its potential origin lies in
competition for a limited factor (M. C. Chen and M. M. Fluck, in
preparation). At very high levels of hr-t mutant 82 (beyond the comple-
menting range), a depression of transformation is also observed in mixed
infections between RA and 82 (M. C. Chen and M. M. Fluck, in preparation).

The depressive effect (competition) of hr-t mutants on wild type
transformation and the positive effect (complementation) of these same hr-t
mutants on strain RA transforming potential reflect a complex biological
system with multiple levels of interaction. It is worthwhile pointing out
that, in mixed infections between hr-t middle T-antigen- and ts-a large T-
antigen-deficient mutants, similar complex interactions are observed (9).
Nhen the mixed infections are carried out at 33°C, a situation in which the
ts-a mutant can transform, a depression of transformation is observed in
the mixed (ts-a e hr-t) infection, compared to infection with ts-a alone,

indicative of what we believe to be competition for a limited factor. In

142

contrast, when the mixed infections are carried out at 39°C, a situation in
which the ts-a mutant cannot transform, an enhancement (complementation) is
observed in the mixed infection (ts-a o hr-t) compared to infection with
ts-a alone (9). In this case, complementation of the large T-antigen
defect of the ts-a mutant by the fUnctional large T of the hr-t mutant, is
stronger than the competition between the two strains for limiting
factor(s) and net complementation is observed. Experiments in progress
suggest that the differences in competition between A2 e 82 and RA v 82 co-
infections may be due to differences in the enhancer region between these
two strains (M. C. Chen, and M. M. Fluck, unpublished), in addition to the
complementable RA defect in large T-antigen noted above.

In summary, we believe that both :33 cooperation and the depressive
effect in the co-infection reflect the fact both parental genomes are
co-infecting the same cells.

The reductive effect of mutant 82 on wild type transformation opens
the possibility that in the population of infected cells only those with
relatively high ratios of wild type to mutant can become transformed, thus
providing an explanation for the rare recovery of the transformation-
defective mutant genome in transformed cells. However, this explanation is
not applicable to the infections performed with strain RA.

Underrepresentation of an unselectable genome is also observed in
hamster transformants. To analyze whether the low frequency of co-integra-
tion of two parental genomes in transformed cell can be generalized to
different hosts, we repeated the experiment using the other nonpermissive
host for polyoma transformation, i.e., the hamster. Baby hamster kidney

(811)!) cells were infected with mixtures of strains RA and 82

 

 

143

(experiment 10, Table 1). Thirteen transformants were analyzed in detail.
Only one of those contained the 82 parent in addition to the wild type
(Fig. 7).

In contrast to the results described above for F-111 transformants,
the integration patterns of the 8111! transformants were unusual. Ten out of
13 transformants analyzed contained a single viral insertion site with an
incomplete copy of the viral genome including a deletion of the carboxy
terminus of large T-antigen. Only two transformants in 13 contained
multiple sites of integration with tandem repeats of the viral genome. The
8111i transformant in which the unselected parent was recovered is one of the
two transformants for which “typical“ integration patterns were observed.
The basis for the differences between the 811K and “Fischer rat integration
pattern in mixed infections with strain RA is not fully understood.
Further experiments demonstrated usual integration patterns for strain RA
and A2 in single infections of 811! cells (K. McNilliams and M. M. Fluck,
unpublished). The tendency to inactivate large T-antigen in transformants
has been noted before (1). He believe that an unusual selection pressure
was applied during the selection of the 811K transformants (selection of
very well-developed foci, followed by recloning in agar), which may have
selected for a very early inactivation of large T-antlgen.

Low probability of double integration events in transformants derived
from mixed infections with two selectable genomes. The experiments
described above all make use of hr-t deletion mutants whose integration is
not selected in the transformation process. He considered the possibility
that the underrepresentation of hr-t genomes represents a problem specific

to all hr-t mutants. This hypothesis is not highly likely since mixed

144

Fig. 7. Underrepresentation of an unselected viral genome in hamster

transformants.
Baby hamster kidney (BHK) cells were infected as described in legend
to Fig. 1 and Materials and Methods. Transformants were isolated and

analysis is identical to legend to Fig. 2. Digestion is with MspI and

hybridization with pPyHH.

 

 

 

 

 

 

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146

infections with two non-overlapping hr-t mutants generate transformants by
recombination at relatively high frequency (compared to wild type infec-
tion) (S. Kalvonjian, C. Priehs, H. H. Chen, and M. M. Fluck, manuscript in
preparation). However, differences in capsid phosphorylation (14) and in
chromatin acetylation (25) have been documented between the hr-t mutant and
wild type, and it is conceivable that these might lead to differences in
processing by the host. To determine whether the low recovery of a second
parental genome is specific for transformation-deficient hr-t mutants, we
set up a nuxed infection with an isogenic pair of strains in which both
parents can transform and which can also be distinguished from each other
by restriction endonuclease analysis. He chose wild type A3 and a deletion
mutant, d145, which was isolated from A3. Thus, these two strains have
identical sequences, except for the leS deletion of 66 bp in the
overlapping middle T-antigen-large T-antigen reading frames (2). This
deletion can be detected by digestion with Sst! and observation of fragment
3. The dlflS mutation does not result in a noticeable transformation nor
growth deficit (2). Thus. this pair of strains offers a further advantage
in our analysis since both genomes are selectable. The result of two
experiments ('9) are presented in Table 1 and one is presented in Fig. 8.
In one of these experiments (9b shown in Fig. 8). infected cells were
passed to agar after infection, to prevent cell division and segregation of
the two parental genomes to different cells. in the latter experiment,
cells were infected at a multiplicity of 10 each of the two parental
viruses. Analysis of the viral genomes in the imput is in agreement (not
shown). Transformation frequencies in the mixed infection and in the

controls were very close (1.31 for the infection with A3, 0.61 for aius and

 

 

147

Fig. 8. Low probability of double integration events in transformants
derived from mixed infections with two transforming strains.

F-lii rat cells were infected with wild type A3 and transformation

competent deletion mutant all-65 at a multiplicity of infection of 10

p.fJJ. of each parent (experiment 9, Table 1). Transformants were

isolated and high molecular weight cellular DNA was analyzed as

described in legend of Fig. i and 2. Digestion is with Ssti and

hybridization to a probe for the whole viral genome (on-i).

148

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Lou probability of double integration events in transformants derived

from mixed infections with two transforming strains.

Figure 8.

149

1.81 for the inixed infection). supporting the notion that A3 and divs
transform equally efficiently. Twenty-two transformants were analyzed in
detail; all showed normal patterns of integration. in eight transformants.
the A3 wild type genome was present alone, in 10, the dlAS genome alone was
found. The equal recovery of A3 and dIAS genomes among the transformants
suggest that these two strains have equal abilities to integrate and
transform. Both parental genomes were recovered in only four transformants
of the 22 twelve analyzed. Results are shown in Fig. 8 for 14
transformants derived from the mixed infections. including the four
transformants in which both parental genomes are integrated. Note that in
all four cases. one of the level of one of the parental genomes is higher
than the other. One clone each derived from single infection with either
A3 or dIMS alone is also shown (lanes 1 and 2). Finally. note that all the
fragments expected after digestion with Sstl are present (except for one
fragment missing in lane 12). This pattern supports the finding that the
viral genome is integrated in head to tail tandem in the transformants. in
agreement with these results, double parental integration was detected in
only one of 23 transformants derived from mixed infections with two

transforming strains Py3-33 and ts3 (D. L. Hacker and M. M. Fluck, in

preparation).

150 _

Discussion

The experiments presented above were designed to analyze how two
independent viral genomes integrate into the host chromosome during the
normal process of neoplastic transformation of rat and hamster cells by
polyoma virus, i.e.. a context in which integration of a at least one viral
genome is known to occur. For this purpose, rat transformants were derived
from mixed infections with wild type virus (parental genome whose
integration is selected) and a nontransforming deletion mutant (hr-t
mutant) (whose integration is not selected). The recovery of the non-
selected parent in the transformants was analyzed by restriction
endonuclease digestion of cellular DNA. followed by. electrophoresis (and '
hybridization, using appropriate endonucleases. which resolve the two
parental genomes. Our results are as follows:

The overall integration pattern of transformants derived from such
infections are typical: i.e. each transformant displays head-to-tail tandem
structures of the viral genome, apparently integrated at multiple sites in
the host genome. This integration pattern is. compatible with the
occurrence of normal integration events in these mixed infections. As
expected, all transformants contain the wild type transforming parent,
since selection for the retention of that parent was applied by selecting
for transformation. Surprisingly, the unselected parent is vastly under-
represented among the transformants. at least that fraction of the viral
genome encompassing the deletion marker mutation. Among 108 transformants
analyzed from mixed infections using an unselectable second parental
genome, the unselected genome was recovered in only 12 cases. Forty

percent of the transformants were derived from infections using equal

 

151

amounts of the two parents while the remainder 60 percent of the transfor-
mants analyzed were) obtained from infections in which the unselectable
parent was used in considerable to high excess over the transforming
selectable parent (a condition used in "0 percent of the infections). This
increase of the ratio of unselectable to selectable parent in the infection
did not increase the probability of recovering the unselected parent in the
transformants. Furthermore, in those transformants in which the unselected
parent was recovered, the ratio of mutant to wild type sequences was at
most equal, even though some were derived from infections performed at an
extreme ratio of mutant to wild type (200:1 or 20:1).

Control experiments demonstrated that the hr-t mutant genomes persist
as well as the wild type in the infected cells, suggesting that the mutants
have no major adsorption--decapsidation--replicatlon problem. Previous
experiments have demonstrated that an hr-t like mutant integrates normally
(20). as do the recombinants generated in crosses between 2 nonoverlapping
hr-t mutants (S. Kalvonjian, C. Priehs, and M. M. Fluck, manuscript in
preparation). The presence of both mutant and wild type genomes in the same
cells early in infection is a prerequisite to any conclusion regarding the
integration of the two genomes in the same cell. This was insured by infec-
ting confluent cells which were not allowed to divide between the time of
infection and transformation or by passing the infected cells into a semi-
solid agar suspension. Furthermore, biological features of the mixed
infection indicated that co-infection was taking place since mixed
infections with transforming and hr-t mutants lead to an increase in trans-
fermation frequency when the transforming parent was RA or, conversely, a

decrease in transformation when the transforming parent was wild type A2.

 

152

This strongly suggests that both parental genomes were in the same cell and
in one case with strain RA, complementing each other. The apparently
opposite effect of the hr-t parent in mixed infection with wild type A2
compared with pseudo wild type RA has been addressed in details in the
results section. The possibility that cells cannot become transformed when
both the wild type and the transformation defective parents have integrated
appears highly unlikely. Multiple examples of complementation in the
process of transformation have been demonstrated previously in mixed infec-
tions with large T-antigen mutants and hr-t mutants (9). Furthermore, we
have previously described a highly transformed cell line which contains the
hr-t mutant in addition to a single copy of the wild type middle T-antigen
gene (23). as well as cases of transformants from nuxed infections which
express a wild type and a deleted middle T-antigen protein (9). Thus, the
presence of both wild type and hr-t mutant alleles in the same cell does
not prevent transformation. In summary, it appears that both the trans-
forming and the non-transforming parent infect the same cells. and persist
equally in those cells during the course of infection: Yet, the frequency
of recovery of the two parents in cells selected for the presence of the
transforming parent (i.e. transformed cells) is low. The nontransforming
parent is highly underrepresented in those cells. This underrepresentation
is not specific to any pair of strains used. In fact, it is not peculiar
to the recovery of an unselectable genome, since a second selectable genome
was only recovered in six cases of 36 transformants selected from a nuxed
infection carried out with two transforming parent strains. The

underrepresentation of a second parental genome in transformants derived

 

153

from mixed infection was observed in the two cell lines tested i.e., in the
F-111 Fischer rat and in the BHK hamster cell lines.

Interestingly, the frequency at, which, a second parental genome is
recovered is not affected by the number of times the infected cells are
allowed to divide after infection before the selection for transformation
is applied. This suggests [as do other unpublished results from our lab and
previous results (13)] that the integration events are fixed early
postinfection.

The present results do not fit the expectations from the simplest
model of integration reviewed in the Introduction. The analysis of integra-
tion pattern of viral genomes in a large collection of transformants,
derived both from polyoma virus and SVLHO infections of nonpermissive
cells, has suggested that the viral genome can integrate at a large number
of sites in the host genome (11,5). Judged by restriction endonuclease
analysis, multiple integration events have usually occurred within a single
transformant (8-10 as reviewed in the introduction). ‘The simplest model
would be that a large number of sites are available" for the integrating
viral genomes and that each integration event within a single cell in the
transformation pathway occurs independently from the other integration
events within the same cell. Thus, in transformants derived from mixed
infections, we expected to find both parental genomes integrated at
independent sites and we expected them to be represented within a single
transformant proportionally to their ratio in the infection mix. As Judged
by the usual criteria of multiple bands in digests with enzyme which do not
cut the viral genome, multiple integration sites are apparently present-an

example with seven apparent sites is shown-; however, the nonselected non-

 

154

transforming parent is very strongly underrepresented in the population of
integrated viral genomes in the transformants. These resolts can be
interpreted to mean that viral integration is infrequent as suggested in
the past (28) based on the low frequency of stable transformants. Such an
interpretation contradicts the picture based on Southern blot analysis.
Low frequency of integration could be due to limited amounts of host
factor(s) involved in integration or to limited numbers of sites in the
host chromosome available for integration. Some indirect support for the
former hypothesis is available. The progressive depression of
transformation in response to increasing doses of transformation deficient
mutant in co-infection with wild type is compatible with competition for a
limited factor, or a limited site, or both. The factor could be an
enhancer binding factor required for the transcription-replication of the
viral genome and linked to its integration (22). Re are presenting data
elsewhere which support a role for the enhancer in integration (M. C. Chen
and M. M. Fluck, manuscript in preparation). However, in co-infections with
pseudo-wild type RA, the sufficient levels of factors are present to allow
replication of the nonselected (82) parent, present in excess and yet
integration of B2 is rare, points towards a limit for integration, separate
from a limit on replication. Possibly integration sites (or a structure
linked to them) are limited. Or the probability of integration is low. The
latter is compatible with the low frequency of stable transformation by
polyoma virus, and suggests by comparison with the frequency of abortive

transformation, that integration may be a rate limiting step in trans-

formation (28).

 

155

If the above explanation is correct, we must reinterpret the
apparently elevated number of integration sites revealed in pfeVlOuS
analyses of integration patterns and reviewed above. Preliminary results
from our lab suggest that at least part of the multiple bands obtained in
digests with enzymes which do not cleave the viral genome may consist of
multiple forms of the same integration site (K. Friderici, L. J. Syu, and
M. M. Fluck, unpublished) and we are testing the hypothesis that these are
produced by in situ replication of the integrated viral genomes.

Recent experiments from our lab (6) have demonstrated a high level of
interviral recombination in the viral genomes which become integrated in
the transformed cells. The present experiments are not in contradiction
with these results since in most cases analyzed, the integrated recombinant
viral genomes have undergone recombination in two adjacent to intervals,
generate a genome such that for any given interval the sequences of only
one of the parent are present. Thus, in the case of the present
experiments, the wild type sequence must be selected over those of the hr-t
mutant. Interestingly in a few cases analyzed so far in which both wild
type and hr-t mutant sequences were recovered in the same transformants,
these were present at the same site demonstrating double parental
integration by an interviral recombination event which involves a single
integration into the host chromosome (8,12).

. in conclusion, the experiments presented above suggest that in the
neoplastic transformation of rat cells by polyoma virus a highly limited
number of viral genomes become integrated. This number appears more
restricted than either the number of viral genomes which can be transcribed

since complementation or competition can be observed, or that which can be

 

 

 

156

replicated (since both parents persist). It is possible that the viral
genomes which serve as templates for transcription and replication are not
in the same compartment as those which become integrated. Thus, it may be
that there are integration centers, at which multiple genomes interact
since high recombination has been observed among those integrated viral

sequences, and the number of integration centers is highly limited.

 

 

157

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