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RETROVIRAL INDUCED LYMPHOMA: PROMOTOR-INSERTIONAL
ACTIVATION OF THE CELLULAR MYC GENE BY

RETICULOENDOTHELIOSIS VIRUS

BY

Robert Alan Swift

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1985

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ABSTRACT

RETROVIRAL INDUCED LYMPHOMA: PROMOTOR-INSERTIONAL
ACTIVATION OF THE CELLULAR NYC GENE BY

RETICULOENDOTHELIOSIS VIRUS

BY

Robert Alan Swift

The avian non-acute Reticuloendotheliosis viruses(REVs)
induce B-cell lymphoma in the bursa of birds 4-10 months
post-infection. Recent evidence suggests that neoplasia
could be caused by the activation of a cellular gene by the
infecting virus. We show, by restriction enzyme analysis of
tumor DNA, that REV can activate the cellular oncogene,
c-myc, by insertion of the provirus proximal to the oncogene
and oriented in the same transcriptional direction. We have
supported our conclusion by cloning a c-myc gene activated by
proviral insertion and analyzing its structure by restriction
enzyme fine mapping. The structural analysis confirms our
conclusion and reveals that the provirus carries a major
deletion of the coding region, but has maintained both LTRs,
which are identical based on DNA sequence analysis. RNA
blots of poly(A) selected tumor RNA reveal novel c—myc

transcripts which are consistent with initiation

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sequent

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from within the LTR proximate to c—myc. However, no
transcripts are seen that initiate from the other LTR
indicating transcriptional surppresion by an unknown
mechanism. Comparison of the DNA sequence of the LTR to a
related REV strain shows major sequence divergence in the
region where retroviral enhancers are commonly located. This
sequence variation may account for the differences in

oncogenic potential observed between strains of REVS.

TO MY MOTHER AND FATHER WHO ALWAYS BELIEVED
IN ME, EVEN WHEN THEY DIDN'T

11

 

 

A
guide:
Commi1

Lee Ve

moral
Taddy

Philo

ACKNOWLEDGEMENTS

I thank Dr. Hsing-Jien Kung for financial support and
guidance. I also wish to thank members of my guidance
committee, Drs. John L. Wang, Jerry Dodgson, Alan Morris and
Lee Velcier.

A special thanks to Carol J. Fiol-Lay for all of her
moral support during my trying times. I also must thank
Teddy Fung for all that he taught me about science and

philosophy.

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LIST C

ABBREV

 

CHAPTE

I.

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II.

III

LIST OF

TABLE OF CONTENTS

FIGURES. . . . . . . . . . . . .

ABBREVIATIONS. . . . . . . . . . . . . . .

CHAPTER

I.

II.

III.

IV.

LITERATURE REVIEW. . . . . . . . . . . . .
Taxonomy . . . . . . . . . . . . . . . . . . . .
The Viral Genome . . . . . . . . . . . . . . . .
Enhancers. . . . . . . . . . . . . . . . . . .
Virus Replication. . . . . . . . . . . . . . .
Viral Integration. . . . . . . . . . . . . . .

Acute—Transforming Viruses . . . . . . . . . . .
Acute-Transforming Virus Formation: Transduction
Reticuloendotheliosis Viruses. . . . . . . . .

Cellular Oncogenes . . . . . . . . . . . . .
The c—myc Gene . . . . . . . . . . . . . . .
Proviral Insertional Activation. . . . .

Translocation. . . . . . . . . . . . . . . . . .
Amplification. . . . . . . . . . . . . . . . . .

LINKAGE OF CHICKEN C-MYC GENE TO CSV . . . . . .

Materials and Methods. . . . . . . . . . . . . .
Results. . . . . . . . . . . . . . . . . . . .
Discu881°n O O O O O O O O O I O O O O I O O

DETERMINATION OF TRANSCRIPTIONAL ORIENTATION
AND SITE OF INTEGRATION OF CSV RELATIVE TO C-MYC

Materials and Methods. . . . . . . . . . . . . .
Results. . . . . . . . . . . . . . . . . . . . .
Discussion . . . . . . . . . . . . . . .

TRANSCRIPTIONAL AND STRUCTURAL ANALYSIS OF
AN ACTIVATED C-MYC GENE. . . . . . . . . . . . .

Materials and Methods. . . . . . . . . . . . .

Results 0 O O O O O O O O O O O O O O I O O O 0
Discussion . . . . . . . . . . . . . . . . . . .
LITERATURE CITED . . . . . . . . . . . . . .

iv

Page

vii

55
57
60
75
79
81
82
90
94
95
98
114

118

 

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11

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15

16

LIST OF FIGURES

Characteristics of some commonly
studied retroviruses . . . . .

Structure of a viral RNA genome from
a replication competent retrovirus . . .

The general structure of a C-type retrovirus

A comparison between the viral RNA genome
and the viral DNA intermediate . . .

General structure of a retroviral LTR.
Synthesis of the minus-strand of viral DNA
Transduction of a cellular oncogene. . . .

Characteristics of some oncogenes not associated
with acute transforming viruses. . . . . . .

Two configurations of proviral DNA that result
in enhanced expression of a cellular oncogene.

Restriction enzyme map of CSV. .

Restriction enzyme map of the
chicken c-myc locus. . . . . . . . . . . . . . .

Hybridization of Eco RI digested tumor DNA .

Quantification of the CSV sequences in
tumor no. 15 . . . . . . . . . . . . . . . . .

Quantification of c-myc RNA from tumors.

Position and orientation of CSV proviruses
inserted in the c-myc locus. . . . . . . .

The nonrandom distribution of proviral insertion
sites in the c-myc locus of avian bursal tumors.

Page

16

19
21
23

29

36

49

63

65

68

71

74

84

89

 

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Structure of the chicken c-myc gene in B-cell
lymphoma as analyzed by Eco RI digestion . .

Restriction endonuclease cleavage map
of phage clone 713 . . . . . . . . .

Comparison of the nucleotide sequences in the
LTRs of CSV and SNV. . . . . . . . . .

Northern blot of poly(A) selected RNA
from tumor 713 . . . . . . . . . . . .

vi

Page

100

103

107

111

 

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DIAV

DNA

FSV

GTP

kb

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MuLV

MSV

PDGF

REV

RNA

mRNA

RSV

SNV

TS

ABBREVIATIONS

avian leukosis virus

chicken syncytial virus

duck infectious anemia virus
deoxyribonucleic acid

feline sarcoma virus
guanosine triphosphate
kilobase

kilodalton

long terminal repeat

murine leukemia virus

murine sarcoma virus
platlet—derived growth factor
reticuloendotheliosis virus
ribonucleic acid

messenger RNA

rous sarcoma virus

spleen necrosis virus

tumor specific

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LITERATURE REVIEW

The RNA tumor virus and oncogene story dates back to
1910 (Rous 1910, 1911) when Francis Peyton Rous discovered a
filterable agent(virus) that could cause sarcomas, a tumor
of the connective tissue, in domestic chickens. There were
other filterable agents isolated that had the capacity to
induce a variety of neoplasia; leukosis in chickens
(Ellerman and Bang 1908), mammary carcinomas in laboratory
mice (Bitter 1936), and leukemia in newborn mice (Gross
1951, Gross 1970). In the decades that followed Rous's
discovery the physical and biochemical nature of the sarcoma
inducing agent was elucidated. With the invention of
electron microscopy (Knoll and Ruska 1932) and sophisticated
biochemical techniques the structure (Gaylord 1955) and
chemical content (Crawford and Crawford 1961) of the sarcoma
virus was shown to be single-stranded RNA surrounded by a
protein coat.

M

The cancer inducing viruses discovered by Rous and
others are now taxonomically grouped as part of the family

Retroviridae (Fenner 1975). The primary criterion for

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admission to the retroviral family is the replication of an
RNA genome that procedes through a double-stranded DNA
intermediate. Some other taxonomic characteristics
associated with this family are: a diploid genome of
positive-sense single-stranded RNA(the subunits are believed
to be joined together by hydrogen bonds involving sequences
located close to the 5' end of each subunit, Kung et a1.
1975), and a DNA polymerase enzyme activity capable of using
RNA or DNA templates(reverse transcriptase) (Fenner 1975).
Retroviruses have been discovered in a wide variety of
vertebrates, including snakes, fish, birds and many
mammalian orders(Figure 1). However, the most
experimentally useful and well studied retroviruses have
been isolated from chickens, mice, cats and lower primates.

The Viral Genome

The viral RNA genome exhibits the structural features
common to eucaryotic mRNA; a 5'-m7Gppme cap structure, a
poly adenylic acid(poly A) tail and a low level of internal
methylation (Teich 1982). The general structure of a
replication competent retrovirus is divided into the three
coding regions shown in Figure 2. The gag region encodes a
polyprotein which is cleaved to form the four or five
proteins which make up the major structural components of
the virus partic1e(for review Eisenman and Vogt 1978;
Dickson et al., 1982). The pol region encodes a reverse
transcriptase, a RNA:DNA specific RNase H exonuclease
activity (for review see, Verma 1977) and an enzyme activity

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the host cellular DNA (Schwartzberg et al., 1984;Donehower
et al., 1984). The env region encodes a polyprotein that is
glycosylated, cleaved and transported to the plasma membrane
(Hayman et al. 1978) to form a component of the viral
membrane that surrounds the core of the virus. The envelope
glycoproteins are targets for neutralizing antibodies and
are a major determinant of the host range of the virus
(Payne and Biggs 1964,1966;Weiss 1982). Treatment of intact
virions with protease results in the removal of envelope
proteins and the concommitant loss of infectivity (Rifkin
and Compans 1971;Dickson 1982).

In addition to the viral replication genes other
portions of the genome serve important functions in the life
cycle of the virus. The R region, repeated at both ends of
the viral RNA genome, is presumed to be used during
replication of the viral RNA genome to provide a means for
transferring the nascent DNA chain from the 5' end to the 3'
end of the viral genome(see section on viral replication).
The primer binding site(PBS), located at the 3' boundary of
05, is a stretch of 18 nucleotides that binds a specific
tRNA molecule that is used to prime the synthesis of the
first(minus) strand of viral DNA during reverse
transcription. The leader region(L), an untranslated
sequence preceeding the coding region of the viral proteins,
has recently been shown to contain recognition sequences
necessary for the proper encapsidation of the RNA genome
into virons (Shanks and Linial 1980;Watanabe and Temin 1982)

and a splice donor site used for the generation of

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feat.

of b.

9

subgenomic viral mRNAs. The U3 region contains eucaryotic
regulatory sequences important for transcriptional
initiation, capping and polyadenylation of the viral RNA
transcript (Varmus 1983). In addition, the 03 region has
been shown to contain "enhancer" sequences that can elevate
gene transcription irrespective of the position or the
orientation of this sequence to the affected gene.

Enhancer Sgggences

Enhancers are short cis-acting regulatory sequences
that appear to increase transcription from eukaryotic
promotors irrespective of their orientation and relatively
independent of the location of the affected promotor.
Enhancer sequences were first discovered and have been most
throughly characterized in Simian Virus 40(SV 40), a small
DNA tumor virus, (Benoist and Chambon 1981;Gruss et al.,
1981). The transcriptional regulatory sequences of the
early region of SV 40 contains several important structural
features, including two 72 base—pair repeats. The deletion
of both 72 bp repeats results in a drastic reduction in the
transcriptional activity of the SV 40 early region genes.
Interestingly, the transcriptional activity can be restored
by reinserting the 72 hp repeats at a variety of positions
and in either transcriptional orientation relative to the
SV 40 early region RNA cap site (Moreau et al., 1981;Fromm
and Berg 1982). These experiments with SV 40 laid the
foundation for the operational definition of enhancers as a
sequence that can increase the transcription of a gene

independent of orientation and that their influence is not

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10
strongly affected by the distance from the promoter.

The molecular mechanism of enhancer activity is not
known, although several models have been proposed. Perhaps
the most popular model, suggested by Moreau et al.(1981), is
that enhancer sequences provide a bi-directional entry site
for either RNA polymerase II or one of its subunits. This
hypothesis is consistent with the position and orientation
independence of enhancer activity and is supported by the
observation that enhancement decreases when functional
promotors are inserted between the enhancer and the
initation site of the gene being assayed (Moreau et al.,
1981;Wasylyk et al., 1983). Another hypothesis suggests
that enhancers may alter chromatin structure and/or
superhelicity to create an 'open window' within the
nucleosome structure, thereby increasing the accessiblity of
the DNA within the enhancer for binding of macromolecules
involved in transcription. This might be accomplished
indirectly if the enhancer sequence contains a topoisomerase
binding site or directly if the enhancer sequence contains,
for example, alternating purine and pyrimidine tracts which
favor a left-handed form of the double-stranded
helix(Z-DNA). It has been proposed that Z-DNA assumes a
more "open" conformation then the normal right-handed B-form
of the helix and that it can not form around nucleosomes
(Groudine 1983). Perhaps in support of all these hypotheses
is the observation that the enhancer sequences are generally

hypersensitive to DNase digestion, a result that is

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interpreted to mean that the DNA sequences of enhancers are
in regions of the chromatin that are "open". For example,
hypersensitive regions reside close to the SV 40, polyma,
MMTV and immunoglobulin enhancers within cell types in which
promotors under their control are active, but not when
promotors are inactive. Consistent with the Z—DNA
hypothesis is the recent finding that anti-Z-DNA antibodies
(Lafer et al., 1981) bind to some viral enhancer regions
(Nordheim et al., 1982;Nordheim and Rich 1983). These
hypotheses are not necessarily mutually exclusive. The
generation of an open chromatin structure and
sequence-specific interaction between enhancers and the
transcriptional machinery could act in concert to facilitate
the activation of promoter elements.

More recently, enhancers have been found within the 03
region of a number of retroviral LTRs. The biological
implications of enhancer function in this family of virus is
consistent with the observation that LTRs have been shown to
activate adjacent cellular oncogenes following the
integration of viral DNA upstream or downstream of the
affected gene (Hayward et al.;1981;Fung et al., 1981;Payne
et al., 1982;Nusse et al., 1984;5wift et al., 1985). This
activation, referred to as enhancer insertion, has been
implicated in the formation of tumors in hosts infected with
certain retroviruses.

Viral enhancers may determine the host range and/or the

oncogenic potential of the virus. Studies show that the

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SV 40 enhancer is 5 times more effective in elevating gene
transcription in monkey cells(CV—1) then in mouse L cells.
Conversely, the enhancer of Mo-MSV is more effective in
mouse cells then in monkey cells (Laimins et al., 1982).
The RAV-O and RAV-i strains of avian leukosis virus are very
similar in DNA sequence, except for the U3 region of the LTR
which is highly divergent (Robinson et al., 1982). However,
RAV-O is non-oncogenic in an avian host. In contrast, RAV-l
can induce B-cell lymphoma and erythroblastosis (Fung et
al., 1982,1984). Experiments with a recombinant of two
strains of murine leukemia virus(MuLV), Fr-Mulv and
Mo-Mulv, that induce erythroleukemia and T—cell lymphoma,
respectively, were examined for their oncogenic properties
(Chatis et al., 1983). It was found that a recombinant
whose genome is derived primarily from Fr-MuLV, but has the
enhancer region of Mo-MuLV induced T-cell lymphomas rather
then erythroleukemias.

Another example of the enhancer region affecting the
oncogenicity of the retrovirus was shown by Lenz et
al.(1984), who observed that a recombinant of the
non-oncogenic endogenous retrovirus Akv and the highly
leukemogenic murine virus SL3-3 that was primarly derived
from the Akv genome, was leukemogenic. Nucleotide
sequencing showed that the recombinant differed from Akv
only in the region thought to contain the enhancer sequence
of SL3-3. It appears that the observed difference in

oncogenicity is due to sequences present in the LTR of SL3-3

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13
that exhibit significantly enhanced transcriptional activity
in T-cells compared with the corresponding region of the Akv
LTR (Celander and Haseltine 1984).

Experiments with recombinants of two strains of MuLV, a
strain that replicates efficiently in the thymus of
mouse(T+) and a strain unable to replicate in this
organ(T-), show that the region associated with the

+ strain is sufficient to allow the

enhancer of the T
replication of the T- strain in the thymus (DesGroseillers
et al., 1983).

Several groups have recently reported experiments which
demonstrate that sequences which lie adjacent to the
immunoglobulin heavy chain locus are functionally analogous
and structurally homologous to viral enhancers. As well as
conforming to the operational definition of an enhancer
Gilles et al.(1983) and Banerji et al.(1983) have shown that
the immunoglobulin heavy chain enhancer has a somewhat more
restricted tissue specificity and functions most efficiently

in cells of the B lineage. There is evidence based on

in vivo dimethyl sulfate protection experiments and genomic

 

sequencing (Ephrussz et al., 1984) that alterations in the
octamer CAGGTGGC found in the mouse immunoglobulin heavy
chain enhancer correlates with the B verus non—B lineage
specificity observed by Gilles et al.(1983) and Banerji et
al.(1983). These results are consistent with the binding of

a tissue-specific molecule to the enhancer region.

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Virus replication

The first step in the life cycle of the virus is the
adsorption and penetration of the host cell by the virus
particle. The architecture of a type C retrovirus is shown
in Figure 3 with protruding spikes covering the surface of
the viron that represent the envelope glycoproteins. The
adsorption of the virus to the cell surface involves some
form of env glycoprotein-cellular receptor interaction.
This interaction, between the viron envelope glycoproteins
and specific receptor sites on the host cell surface is the
major determinant of host susceptibility to infection (Weiss
1982). Susceptible cells can be rendered resistant to a
specific retrovirus infection by pre-infection with a virus
bearing the same viral glycoprotein specificity, apparently
by blocking the cell receptor (Rubin 1960). This
interference based on subgroup specificity for cell
receptors can be used to divide a group of related viruses
into different classes.

It has been suggested that virons enter the cell by
first becoming attached to cell surface receptors located
primarily in regions of coated pits (Marsh and Helenius
1980). In some cases, the internalized virons appear first
in intracellular coated vesicles, then in large uncoated
collection vacuoles and finally in secondary lysosomes. In
the lysosomes(pH<5), the glycoproteins of the viral envelope
can become strongly fusogenic. They may fuse rapidly with
the lysosomal membrane(<1 min) thereby permitting transfer

of the viral genome to the cytoplasm in such a way that the

 

 

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viral genome is constantly protected from the hydrolases
present in the lysosomal environment (Lenard 1982). After
the viron is uncoated, the single stranded RNA genome is
available in the cytoplasmic compartment in a form suitable
for conversion to double-stranded DNA.

The synthesized linear double-stranded viral DNA
intermediate is co-linear with the viral RNA with respect to
the coding region(Figure 4). However, the ends of the viral
RNA are different from the ends of the viral DNA. The viral
RNA has a short terminal repeat(R), while the viral DNA has
a long terminal repeat(LTR). The general structure of a LTR
with some important regulatory features is depicted in
Figure 5.

The current model for the conversion of the
single-stranded viral RNA to a linear DNA duplex is
complex. A model for the synthesis of the minus strand of
viral DNA is presented in Figure 6. The final product of
this complex process is a blunt—end linear duplex with two
copies of the 03-3-05 region which constitutes the long
terminal repeat(LTR), as was seen previously(Figure 4).

This molecule is transported to the nucleus and converted to
one of two monomeric closed circular forms; containing
either one copy of the LTR or two copies of the LTR linked
in tandem.

Viral Intgggation

The process of retroviral integration into the host

cellular DNA has two direct consequences for the structure

 

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of the retroviral DNA and the host cellular DNA. By
comparing unintegrated viral DNA to the integrated viral DNA
it has been shown that the terminal 2 bp from each inverted
repeat are lost from the viral DNA. In addition, a short
direct duplication of the cellular DNA results. Every
proviral-host junction examined has lost exactly 2 bp,
however the size of the direct duplication varies between
different retroviruses (Varmus 1983). It was shown
elegantly by Panganiban et al. (1984) that a closed circular
molecule with the two LTRs in tandem is the precursor to the
integrated retroviral DNA. The fact that the precursor to
the retroviral DNA is a closed circular molecule and the
integrated viral DNA is always colinear with the linear
duplex synthesized in the cytoplasm(Figure 4) implies that
viral integration involves some enzymatic activity that
recognizes specific sequences, presumable at the LTR circle
junction. These observations are consistent with an earlier
proposition that the 3' portion of the retroviral pol gene
encodes an integrative function, since the protein product
of this region from the RSV and the MuLV genome display
endonuclease and DNA binding activities (Grandgenett et al.,
1978;Misra et al., 1982;Nissen—Meyer et al., 1980;Kopchick
et al., 1981). More recently, Schwartzberg et al.(1984) and
Donehower et al.(1984) have examined deletion mutants of
MuLV that are defective for integration. Both have shown
that the deletions map in the 3' pol region of the

retroviral genome. Neither the late stages of the viral

11f:

 

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25
life cycle(transcription, translation and virion assembly)
nor early events such as adsorption and reverse trancription
of the RNA viral genome are affected by the deletions.
Therefore, it is believed that one or more of the steps
involved in proviral integration has been pertrubed.

There are structural similarities between the termini
of the integrated retroviral DNA and transposable elements
found in bacteria, yeast and insects (Ju and Skalka 1980;
Varmus 1983). Both have a short inverted repeat at each
terminus and both integration and transposition result in
direct duplications of the adjacent cellular DNA. In
addition, with the exception of the mouse mammary tumor
virus(MMTV) LTR, all proviral inverted repeats examined are
bounded by TG....CA (Temin 1980;Majors and Varmus
1981;Hughes 1981;8cott 1981;5himothono and Temin 1980) the
same 2 bp found at the end of all eukaryotic transposable
elements. This has lead to the proposal that the provirus
could "jump" in and out of the cellular genome in a fashion
similar to transposable elements. However, it is known that
the terminal 10—12 nucleotides of the LTR, located in the
inverted repeat region(Figure 5), are required for
integration of the viral DNA (Panganiban et al., 1984) and
integration of the provirus results in the loss of the two
terminal nucleotides from the inverted repeats in the LTR.
Taken together, these results would generally preclude the
direct DNA-DNA "transposition" of the provirus by the same

mechanism used for the original integration event.

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26

Aggte-Transformigg Viruses

The family of RNA tumor viruses can be divided into two
classes based on their biological properties; the rare and
highly oncogenic class of acute-transforming viruses that
harbor oncogenes and the almost ubiquitous class of
non-acute transforming virus that do not harbor oncogenes.
Acute transforming viruses, with exception of some Rous
strains, are replication defective, induce neoplastic
disease in host animals rapidly(2-4 weeks) and transform the
appropriate target cells in tissue culture (Hanafusa et al.,
1977;Graf and Bueg 1978;Shih and Scolnick 1980). In
contrast, the group of non-acute transforming virus are
replication competent, induce neoplastic disease in the host
after a prolonged latent period(4-1O months) and they do not
transform cells in tissue culture.

All of these differences in biological activity between
the two classes of retrovirus can be accounted for by the
presence of specific oncogene(s) within the genome of the
acute transforming virus (Hanafusa et al., 1977;Vogt
1977;Bishop and Varmus 1982). It was demonstrated by
Stehelin et al.(1976) that the viral oncogene of RSV(v—src)
has a homologous counter-part(c-src) in normal uninfected
avian cells. Hanafusa et al.(1977) showed that
transformation defective mutants of the acute transforming
virus RSV that retained a portion of v-src could recombine
with c-src to regenerate the acute transforming virus.

These were the first clues that the origin of the viral

onco
fact
infei
been
large

al.,

 

 

27
oncogenes found in the acute transforming viruses were, in
fact, derived from cellular genes already present in an
infected host. Since that time other viral oncogenes have
been shown to have homologous cellular counterparts in a
large range of vertebrates (Spector et al., 1978, Rosseul et
al., 1979;Bishop and Varmus 1982;Figure 1).

It was suggested by Graf and Bueg (1978,1980) that the
susceptibility of target cells to transformation by a
particular retroviral oncogene might correlated with the
expression of the homologous cellular oncogene in the
uninfected target cell. This was predicated on the
assumption that the cellular oncogenes were involved with
and expressed at specific stages of cellular
differentiation. Therefore, the expression of the
homologous viral oncogene interfered with further
differentiation, resulting in continued cellular
proliferation. However, this appears not to be the case for
a number of acute transforming viruses. For example, avian
myeloblastosis virus(AMV) transforms cells in the
myelomonocytic lineage, but the homologous cellular
oncogene(c-myb) is expressed at high levels in cells from
the erythroid lineage (Gonda and Bishop 1983).

Aggtefgragsforligg V1133 Formation: Transggctiog_

Since all oncogenes harbored by acute-transforming
viruses have homologous cellular counter-parts, it is
believed that acute-transforming viruses arose by capturing

or transducing the cellular gene. It is apparent from the

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A)

B)

C)

D)

E)

28

Transduction of a cellular oncogene.

The process may begin with the continued passage
inevivo of a non-transforming virus. During the
viral replication and infection cycle the virus
has the opportunity to integrate many times in
the host cellular DNA. The open box represents
the U3 region and the closed box represents the
R-Us region of the viral LTR. A cellular
oncogene is shown with 2 exons separated by an
intron. The An represents the polyadenylated
tail of eucaryotic mRNA.

On occasion the virus may integrate upstream
and in the same transcriptional orientation of a
cellular oncogene.

A deletion that includes the 3' LTR could fuse
the proviral and cellular oncogene DNA, such
that a hybrid RNA transcript is formed.

The hybrid viral-oncogene transcript could be
post-transcriptionally medified, i.e., removal
of introns, capping, and poly adenylation, and
transported to the cytoplasm as mRNA.

The 5' viral portion of the hybrid RNA permits
the formation of heterodimers with the genome of
a superinfecting or co—resident retrovirus and
packaging of the hybrid RNA into virions.

The high frequency of retroviral recombination
during replication may allow the joining of the
sequences in the hybrid RNA on the 3' side of
the oncogene to a 03-3 sequence from a normal
viral subunit, thereby creating an
acute-transforming virus structure.

29

 

 

 

provirus c-onc

 

 

 

30
structure of the acute and the non—acute transforming virus
that at least two recombination events must occur in order
to capture an oncogene and form an acute-transforming virus
genome structure. Retroviruses are adept at recombination:
their genomes frequently exchange domains of unpredictable
size during mixed infections (Coffin 1979). Their proviral
forms can enter the host genome with great facility (Bishop
1978;Varmus and Swanstrom 1982) and furthermore, they are
diploid, the linkage between viral genomes could assist
cross-over between heterozygous combinations of different
RNAs within the virions (Weiss et al., 1973;Hunter 1978).

Support for this model comes from two fronts. First,
the comparison of viral and the homologous cellular oncogene
(Wyke 1983;Wang et al.,1984) have shown that the viral gene
probably derives from the cellular gene via normal RNA
processing events. Second, structures similar to those
shown in Figure 7 have been observed in some tumors (Payne
et al., 1982;Pachl et al., 1983).

Reticuloendotheliosis Viruses.

Reticuloendotheliosis viruses(REVs) are a group of
avian retroviruses related by their similar morphology (Kang
et al., 1975), common group-reactive antigen (Maldonado and
Bose 1976) and extensive RNA sequence homology (Kang and
Temin 1974). The histopathological lesions consist of
marked proliferation of reticuloendothelial cells(RE) around
blood vessels and lymph follicles, especially in the liver

and spleen (Sevoian 1964). The proliferating cells are

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31
usually fusiform (Theilen 1966), with basophilic cytoplasm
(Robinson and Twiehaus 1974). The nuclei are large and have
prominent nucleoli (Olson 1967). In more extensive lesions,
large areas of central necrosis are observed (Theilen 1966),
usually in the gastrointestinal tract, bursa of Fabricius,
thymus, heart and bone marrow (Olson 1967).

REVs can be propagated in many different avian hosts
and cell types and although REVs natural hosts are birds,
there is much data to indicate that they evolved from a
mammalian C-type retrovirus. The major core protein(p29) of
REV is antigeniclly distinct from the Avian Leukosis Virus
core protein p27 (Maldonado and Bose 1971,1976;Moelling et
al., 1975), but exhibits 40% sequence homology with MLV p30
core protein (Hunter 1978;Charman et al., 1979) and over 50%
sequence homology with the major core protein of the
endogenous retroviruses of macaques (Oroszlan and Gilden
1980).

The REV reverse transcriptase is antigeniclly unrelated
to other avian retroviral reverse transcriptase enzymes
(Misutani and Temin 1973,1974,1975;Kieras and Faras
1975;Moelling 1975;Waite and Allen 1975), but it is
antigeniclly cross—reactive with reverse transcriptase from
mammalian retroviruses (Bauer and Temin 1980). In addition,
REV reverse transcriptase has a divalent-cation requirement
for manganese, as do most of the reverse transcriptases
associated with C-type mammalian retroviruses (Kang et al.,

1975;Moelling et al., 1975;Waite and Allen 1975).

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32

Furthermore, the tRNA primer binding site for the reverse
transcriptase of REV is tRNApro, the same as the primer
binding site used by many mammalian retroviruses (Waters
1975). Recent studies show that REV—A, the helper virus of
REV-T, can replicate in dog and rat cell lines and can act
as a helper virus for the replication-defective
Moloney-Murine Sarcoma Virus (Varmus and Swanstrom 1982).

The prototype virus is considered to be REV strain T,
originally isolated by Twiehaus in 1958 from turkeys with
lymphoproliferative lesions(1958). REV-T is highly lethal
for young chicks, ducklings, goslings, pheasants, turkeys,
quail and guinea keets (Sevoian 1964;Olson 1967;Taylor and
Olson 1972;Theilen 1966,1977;Rodriguez and Field 1969;
Rodriguez 1971;Mcdougall 1980,1981;Bagust 1981;Dren 1983)
inducing an acute systemic proliferative disease which is
characterized by RE lesions in the liver and spleen.

Initial attempts at transforming cultured cells with
REV-T failed (Witter et al., 1970;Temin and Kassner 1974).
However, this was due to the loss of the transforming virus
during serial passage. It has since been recognized that
REV-T is a replication defective acute transforming virus
(Chen et al., 1981) and therefore requires the presence of a
helper virus for continued replication. REV-T can transform
avian fibroblasts (Franklin 1977) and hematopoietic cells
in vitro (Franklin 1974;Hoelzer et al., 1980;Lewis et al.,
1981;Shibuya et al., 1982). Until recently, REV-T was the

only member of the REV group that has been characterized as

an

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but

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and
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33
an acute transforming virus. However, an Australian strain
of REV can also induce neoplasia in inoculated chickens
within 8-20 days post-infection (Ratnamohan et al., 1982).

The REV-T genome appears to be derived from REV-A (Rice
et al., 1982), the helper virus found associated with REV-T,
but with two major differences: an insertion in the env
region of a 1.4 kb DNA segment derived from normal turkey
DNA (designated the c—rel locus) and a deletion of about 3.0
kb of helper virus related sequence. By making deletions
and substitutions in an infectious clone of REV—T,
recovering the mutated virus by transfection and assaying
the transforming capacity in chicken spleen cells, Chen et
al.(1982) showed that only the v-rel gene and a small amount
of helper virus is necessary to transform cells igeyitgg,

An interesting result from these studies has been the
observation that sequences present in the helper virus of
REV-A can suppress the transforming capacity of v-rel and
therefore the deletion of REV-A sequence is necessary for
the transforming capacity of REV-T.

Recently, attention has been focused on the
replication-competent non-acute transforming members of the
REV group. The four best characterized strains are: spleen
necrosis virus(SNV) isolated by Trager in 1959, chicken
syncytial virus (CSV) isolated by Cook in 1969, duck
infectious anemia virus (DIAV) isolated by Ludford in 1969
and reticuloendotheliosis associated virus (REV-A) isolated

by Hoelzer in 1979. CSV, isolated from a domestic chicken

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34
with nerve lesions, can induce syncytia in cultured chicken
embyro fibroblasts(CEF). Both SNV and DIAV were isolated

from a duck infected with Plasmodium lophurae. SNV induces

 

a rapid enlargment and necrosis of the spleen resulting in
death, and DIAV induces an acute anemia. It is interesting
that a new REV isolate (Li et al., 1983), RU—i, was also

isolated from a duck infected with Plasmodium lophurae. The

 

significance of this observation is unclear. All of these
REV strains have the capacity to suppress the immune system
response, but the suppression appears to be non-specific.

In contrast, REV-A induces an immunodepressive runting
disease (Hoeltzer et al., 1979), by activation of a
suppressor cell population in the spleen, within 3 days
post-infection (Carpenter et al., 1981;Rubin et al.,
1978;Carpenter;Kempf et al., 1982). The nucleotide sequence
of these four REVs show greater than 90% homology (Kang and
Temin 1973). Natural occurrence of REVs have been reported
in chickens (Cook 1969;Ratnamohan et al., 1980;Witter et
al., 1981,1982), ducks (Grimes and Purchase 1973;Paul 1978),
turkeys (Robinson and Twiehaus 1974;Paul et al., 1976;Sarma
et al., 1975;McDougall et al., 1978) and Japanese quail
(Carlson et al., 1974;8hat et al., 1976). In addition, new
natural isolates of REV have been described (Ratnamohn et
al., 1980;Mcdougall et al., 1978;Li et al., 1984;6rimes et
al., 1979;Witter and Crittenden 1979). The non—acute REVs
can induce B-cell lymphomas or lymphosarcomas, depending on

the genetic background of the chicken, characterized by a

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long latent period of 4 to 12 months post-infection (Fadly
et al., 1982, Nazerian et al., 1982). The B-cell lymphomas
resemble those induced by avian leukosis virus(ALV) on the
basis of organ distribution, pathology, latency, surface
immunoglobin(IgM) production and activation of the same
cellular oncogene, c-myc (Grimes et al., 1979;Witter and
Crittenden 1979;Fadley et al., 1982;Hayward et al.,
1981;Norri et al., 1981;Fung et al., 1981;Payne et al.,
1982). However, the lymphosarcomas have a different organ
distribution and pathology. Consistent with these
biological observations is the recent finding that the
activated oncogene is not c-myc (Swift unpublished data).

gellglar Oncogenes

Oncogenes were first identified as the genetic
component of acute transforming viruses responsible for the
capacity of these viruses to induce neoplasia rapidly in the
infected host and to transform cells in_vitro. Additional
oncogenes have been identified by experimental designs
involving the transfection of tumor DNA into NIH 3T3 cells
or by the examination of common proviral integration sites
in neoplasms induced by non-acute transforming viruses
(Figure 8). The prototype viral oncogene is v-src, the
viral oncogene of an avian acute transforming virus(RSV).
Stehelin et al.(1976) and Spector et al.(1978) showed that
both avian and other vertebrate DNAs contain nucleotide
sequences homologous to the v-src gene. This has become a

common theme as additional retroviral oncogenes have been

36

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mascaaouooonoum omsoz A>ezzvnlunn

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masonaaefl Haooua omaox Amazes can

Amoun>oun unaosuna.
acauo>uuoo Hosanupoone

o>alo Ou poumnom ooeouoo~nouuoc amen: o>EIz

eawaou ocom own no whom mmeoonmo.monaoxson.meaouomnnonao: noes: omen:
ecumooon mom 0» mononom unfloumoanouvo: vooapcnllouuooouvacaenum so:
moaonuonmo endgame poonpnnt>ezx lo-

oocou onexmo ou oouonom mononnlhn HHOOIe onset Beale
mnaouonm nwnuoumnonu cu poumnom mmaonnaem nHoolm noospnnl>q< Henna

42a «0 acuuoouenmue
>ua>nuo< nuunuo ocomoono

momflun> uanHOMonmne oufio4 nu“: noHMnooomd won monomooco oaom uo oofiumauOuoonono

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37
identified. In fact, the homologous sequence to a number of
oncogenes have been observed in many vertebrate DNAs (Bishop
and Varmus 1982), Drosophila DNA (Shilo and Weinberg
1981;Shilo et al. 1984), Dictyostelium DNA (Reymond et al.,
1984) and yeast DNA (Papageorge et al., 1984;Defeo-Jones et
al., 1983).

The conservation of the cellular oncogenes in such
evolutionary diverse species coupled with the observation
that viral oncogenes and several of their "activated"
cellular counterparts can induce uncontrolled cellular
proliferation and interfere with or arrest cell
differentiation processes (Graf et al., 1978) has led to the
proposal that these cellular homologs of viral oncogenes
function in normal pathways of cellular proliferation and
differentiation. This proposal is very attractive in light
of recent findings that link the oncogene products of c-fos,
c-myc, c-ras, and c-sis with cellular growth control and
differentiation (Waterfield et al., 1983;Doolittle et al.,
1983;Downward et al., 1983;Kelly et al. 1983;Campsi et al.
1983;Weston et al. 1984;Muller and Muller 1984).

An early suggestion for the mechanism of oncogenesis by
viral oncogene products proposed that oncogenes might be
analogs of the small polypeptide growth factors that
stimulate cell division after binding to receptors on the
the cell surface (Todaro et al. 1976;for review see Heldin
and Westermark 1984). Specificially, it was proposed that

v-onc products from MuSV and Fesv, acting as agonists,

.-."
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38
blocked the EGF receptors resulting in a relentless
stimulation of cell division. Though this does not appear
to be the mechanism in the case of the MuSV and FeSV gene
products, it may very well explain the mechanism of
transformation by the simian sarcoma virus gene product,
v-sis.

Recent experiments by Waterfield et al.(1983) and
Doolittle et al.(1983) show that there is a striking near
identity of the amino acid sequence of v-sis(the oncogene of
SSV) and the B polypeptide chain of platelet-derived growth
factor(PDGF). In addition, the v-sis gene product may be
functionally equivalent to PDGF in many respects since
conditioned media from SSV—transformed cells contain a PDGF
like growth factor that will interact with specific PDGF
receptors on responsive cells (Huang et al. 1984) and whose
activity is neutralized by PDGF specific antibodies (Deuel
et al., 1983).

The structural homology between v—sis and PDGF, taken
together with the functional similarity, lends support to
the proposal that v-sis protein functions as an agonist of
the PDGF receptor resulting in the continued stimulation of
cell division. The observation that addition of antibodies
to the culture media of SSV-transformed cells does not slow
down their proliferation may imply that v-sis activates the
PDGF receptor from the cytoplasmic side of the plasma
membrane or that continued expression of v—sis is not

necessary for maintenance of a transformed phenotype.

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39
The ras oncogenes are a multigene family which encode
highly related proteins of 21-24 kd that have the capacity

to transform cells in vitro and i vivo. They have been

 

localized at the cytoplasmic side of the plasma membrane
(Willingham et al. 1980) and ras-related proteins can be
detected in normal mammalian cells (Furth et al. 1982),
Drosophila and Yeast (DeFeo-Jones et al. 1983;Papageorge et
al. 1984). Furthermore, the level of conservation of the
gene between species is such that the human ras gene is
capable of substituting for some functions of the
inactivated endogenous yeast ras genes (Kataoka et al.,
1985). Studies of temperature sensitive mutants of the
viral ras protein from the murine acute—transforming virus
Ha-MSV have shown that ras proteins have the biochemical
property of binding GTP and autophosphorylation of threonine
(Scolnick et al. 1979). Epidermal growth factor or insulin
stimulates the GTP-dependent phosphorylation of the viral
ras protein and the guanine nucleotide binding activity in
ras-transformed cells (Kamata and Feramisco 1984). While
the function of the ras proteins is not yet clear, several
observations concerning their role in cell proliferation
have been described.

The transformation of some mammalian cells can occur if
the ras gene is expressed at an abnormally high level (DeFeo
et al. 1981:0hang et al., 1982;8pandidos et al., 1984) or if
mutations alter the protein primary structure (Furth et al.,

1982;Tabin et al., 1982;Yuasa et al., 1982;Santos et al.,

p»

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40

1983). A particularly potent mutation is one that changes a
glycine at residue 12 to a valine. This mutated or
activated ras gene was first isolated from an urinary
bladder carcinoma cell line, T24 (Tabin et al., 1982).

Consistent with these observations from human tumors
and cell lines are the results of Feramisco et al.(1984)
which show that microinjection of purified T24 mutant ras
protein into a variety of quiescent somatic cells induces
dramatic morphological changes followed by transient
proliferation of the cells. In contrast, the microinjection
of the normal ras protein has little effect on the cells.
In BPA31 cells, a chemically transformed Balb/c 3T3 cell
line, the c-rasK1 is expressed in a cell cycle-dependent

K1 mRNA begins to

manner. The relative abundance of c-ras
increase in mid- to late GO/Gl'

Recently, a number of laboratories have reported that
the purified ras protein possess GTPase activity that is
decreased by the same mutations that activate the ras
protein, a valine at residue 12 or threonine at residue 59
(Gibbs et al., 1984;3weet et al., 1984;McGrath et al.,
1984). This is intriguing since there is some structural
homology between ras proteins and the G protein, which is
known to regulate the hormone sensitive adenylate cyclase
activity (Gilman et al. 1984).

Activation of the G protein requires GTP binding and

appears to utilize the hydrolysis of GTP to end a cycle of

activation of the adenylate cyclase activity. If the ras

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41
protein has an analogous regulatory function in some
proliferation control pathway, then the deficiency in GTPase
activity of the T24 mutant could result in prolonged
stimulation of a normally regulatable activity.

These observations suggest that the ras proteins may be
involved in growth factor receptor activity or in the signal
transduction that may follow receptor activation with a
mechanism similar to that employed by the G protein (Kamata
and Feramisco 1984).

FBJ murine osteosarcoma virus(FBJ-MuSV) is an acute
retrovirus that transforms fibroblasts cells in vitro and
causes osteosarcomas in new born mice (Finkel and Biskis
1966). FBJ—Musv harbors the oncogene v-fos, that is derived
from the cellular gene c-fos by an out of frame deletion of
104 bp (Curran and Miller 1984). The c-fos gene product is
a nuclear phosphoprotein that may be involved in cell
differentiation and cell proliferation. The level of the
c-fos transcripts is loo-fold greater in human term fetal
membranes than in other normal human tissues and cells.
These levels of c-fos expression in human amniotic and
chorionic cells are close to the level of v—fos expression
that results in the induction of osteosarcomas in mice and
transformation of fibroblasts inevitro. Transcription of
c-fos is detected at very high levels in murine
extra-embryonic tissues, especially amnion, viseral yolk sac
and mid-gestation fetal liver. The level of c-fos

expression in fetal membranes increases markedly at late

 

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42

stages of gestation (Muller et al., 1983) and in cells of
monomyelocytic lineage, high levels of c-fos are detected
only at late stages of differentiation (Muller et al.,
1984b). Also, a rapid induction of the expression of the
c-fos gene is observed when the promyelocytic cell line
HL-60 (Collins et al., 1977,1978) or monocytic cell line
0-937 (Sundstrom and Nilsson, 1976) is induced with phorbol
esters to differentiate into nondividing, adherent
macrophages (Mitchell et al., 1985).

Recent experiments show that stimulation of a murine
cell line with PDGF or whole serum results in a transient 15
fold increase of c-fos mRNA within minutes of stimulation
(Greenberg and Ziff 1984). Perhaps most intriguing are the
recent experiments of Muller and Wagner(1984) that strongly
suggest that c-fos plays a pivotal role in cell
differentiation. Using F9 cells, a teratocarcinoma cell
line, they showed that transfection of the c-fos gene into
F9 cells results in two phenotypically distinct cell types.
One consists of proliferating cells with normal F9
morphology and the other comprises enlarged, flat cells that
assume an epithelial morphology and cease to proliferate.
The morphologically altered F9 cells develop an ordered
array of intermediate filaments that carry several antigens
characteristic of endoderm tissue. Therefore, high levels
of c-fos expression in F9 cells can result in phenotypic

properties characteristic of differentiated cells.

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43

The c- c Gene

The structure and function of the c—myc gene has been
the subject of intense investigation and is implicated in a
number of vertebrate neoplasms through a plethora of
mechanisms. C-myc is a normal cellular gene that has been
conserved during evolution and is expressed in all tissue
types examined (Bishop and Varmus 1982;Persson et al.,
1984). Alterations at the c-myc locus have been observed in
many B-cell malignancies of chicken, human and mouse
origin. These alterations include the proximate integration
of a retrovirus (Hayward et al., 1981;Fung et al.,
1982,1984;Noori-Daloii et al., 1981;Payne et al.,
1982;Corcoran et al., 1984:Li et al., 1984;8teffen 1984),
point mutations (Rabbits et al., 1983;Westway et al., 1984),
chromosomal translocations (Shen—Ong et al., 1982,Adams et
al., 1982;Taub et al., 1982, for review see Klein 1983;Perry
1983;Leder et al., 1983) and amplification (Noori-Daloii et
al., 1981;Collins and Groudine 1982;Little et al., 1983;for
review see Klein 1981;8chimke 1984).

The c-myc gene is the cellular homolog of the oncogene
v-myc, carried by the acute transforming virus, MC-29. This
virus has a very wide oncogenic spectrum, inducing solid
tumors composed of malignant myelocytes (Beard 1980),
hepatomas, endotheliomas, carcinomas (Mladenov et al, 1967),
and sarcomas (Graf and Bueg 1978). This is in contrast to
the observation that the activated c-myc gene is almost
always associated with the induction of tumors of B-lymphoid

cells. The cause of this variance in target cell

 

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44
susceptability is unknown, however, a M029 mutant that
induces mainly B and T cell lymphoid tumors and some
myelocytomas has recently been isolated (Enrietto et al.,
1983) that may address this question.

The c-myc protein has been localized to the nuclear
matrix of normal and transformed cells (Hann et al., 1983;
Donner et al., 1982;Alitio et al., 1982;Persson et al.,
1984;Eisenman et al., 1985) and appears to display DNA
binding properties (Donner et al., 1982;Persson et al.,
1984). This is consistent with earlier reports that the
v-myc gene product is predominatly located in the nucleus
(Donner et al., 1982;Abrams et al., 1982). The data to
suggest that the c—myc gene product is a DNA binding protein
is supported by affinity chromatography assays that show
c-myc protein binds to single stranded and double stranded
DNA (Persson et al., 1984) and the structural similarity of
the carboxy terminal region of the c-myc protein to other
DNA binding proteins (Pabo and Sauer 1984).

A comparison of the DNA sequence of v-myc to the
chicken c-myc gene reveal seven amino acid changes and 10
nucleotides at the 5' end of v-myc not shared with c-myc
(Watson et al., 1983). The 10 extra nucleotides are
apparently derived from the first intron by differential
splicing during transduction of the c-myc gene (Shih et al.,
1984). In addition, the v-myc gene lacks the first exon of
the c-myc gene (Shih et al., 1984). Though the first exon

is non-coding, it could serve an important function in c—myc

TE

 

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45

regulation at the transcriptional, post-transcriptional or
translational level. It has been suggested that a stem—loop
structure of the c-myc mRNA, between the first and second
exon, hinders translation of the gene, thereby causing
translational suppression that could be important in the
regulation of the levels of c-myc protein (Saito et al.,
1983). However, Nilsen and Maroney (1984) showed that the
presence or absence of the first exon did not affect
initation or elongation of the c-myc gene product. Also,
Hann and Eisenman (1984) could detect equal levels of c-myc
protein in cells having an intact and unmutated first exon
compared with cells lacking exon 1, suggesting that the
predicted stem-loop structure does not affect translation.

The expression of c—myc mRNA has been examined in many
different cell types. The level of c-myc mRNA in
non-neoplastically transformed cells is low during
quienscence and increases 10-30 fold prior to cell division
(Kelly et al., 1983;0ampisi et al., 1984). An interesting
experiment by Kelly et al. (1983) indicated that the c-myc
gene may be involved in the normal response of quiescent
lymphocytes and fibroblasts to a growth signal appropriate
to those cells. They have been able to show that
platelet—derived growth factor(PDGF), a fibroblast mitogen,
and concanavalin A and lipopolysaccharide, two lymphocyte
mitogens, stimulate the expression of c-myc in those cells
that normally respond to them. All three mitogens are

believed to act early in the cell cycle to induce

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competence; and all stimulate transient expression of c—myc
within 1-2 hours, with a return to baseline well before the
onset of DNA synthesis. The activation of c-myc by PDGF is
particularly interesting because the sequence of the B-chain
of P06? and the gene product of the c—sis oncogene show a
high degree of homology (Doolittle et al., 1983;Waterfield
et al., 1983).

In a related experiment, Makino et al.(1984) examined
the expression of c-myc in liver tissue induced to
regenerate by partial hepatectomy. This system allows the

observation of how cells 1 vivo, that are reasonably

 

synchronized, respond when they move from a differentiated
resting state(Go) to a proliferative state. The results
show that the expression of c-myc is increased 10-15 fold
within 1-3 hours after partial hepatectomy. This expression
begins to decrease rapidly after 4 hours, to a level of less
than two fold the normal level after 8 hours, at which time
DNA replication has still not begun. A still larger
increase in c—myc transcription( 600 fold) is observed when
protein synthesis is inhibited by an injection of
cycloheximide. These results are consistent with previous
findings ig_vitro with fibroblast or lymphocyte cells
pre-treated with cycloheximde and stimulated by PDGF. The
large increase in c-myc expression after pre-treatment with
cycloheximide is believed to imply that c-myc transcription
is under negative regulatory control by a protein with a

high turnover rate.

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47

During differentiation of F9 teratocarcinoma cells to
nonproliferating endoderm cells, expression of c-myc mRNA
decreases upon terminal differentiation (Muller et al.,
1983). The c—myc gene product has also been implicated in
the terminal differentiation of mouse erythroleukemia(MEL)
cells (Lachman and Skoultchi 1984). It has been observed
that c-myc mRNA expression shows a rapid bi-phasic change in
MEL cells induced to differentiate by dimethyl sulphoxide or
hypoxanthine that may be important in the irreversible
commitment of MEL cells to terminally differentiate. In
addition, Reitsma et al.(1983) have shown that treatment of
HL-60 cells with a vitamin D metabolite results in the rapid
differentiation to monocyte-like cells that is preceded by a
decrease in the expression of c-myc. These observations
suggest that c-myc may be involved in some aspect of cell
differentiation.

Proviral Insertional Activation.

It has been demonstrated that avian leukosis
viruses(ALVs), a group of non-acute transforming viruses,
frequently induce B-cell lymphomas after a latency of 4-10
months post-infection (for review see Purchase and
Burmeister 1978;0rittenden 1980). Since ALVs do not harbor
transforming genes, the mechanism by which this retrovirus
induced neoplasia was unclear.

It was shown by Hayward et al.(1981) that ALV, RAV-l
strain, induce B-cell lymphomas by activation of the c—myc

gene. Their results showed that the majority of bursal

 

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50
tumors contained proviral sequences inserted near the c-myc
gene. Subsequently, they showed that most proviral
insertions occur upstream of c—myc and oriented in the same
transcriptional direction. Consistent with this
configuration, in most tumors, an elevated expression of a
novel c-myc mRNA is detected that contains the R and 05
regions of the viral LTR. Therefore, the insertion of
proviral regulatory sequences upstream of c—myc results in
initiation of transcription from within the viral LTR and
continues into the adjacent c-myc gene. This model of
cellular oncogene activation is referred to as
promotor-insertion activation(Figure 9A). Similar results
have been reported by Fung et al.(1982). In addition, Swift
et al.(1985) have reported that promotor—insertion is the
predominant mode of c-myc activation by chicken syncytial
virus, an avian non-acute transforming virus unrelated to
ALVs(see section on Reticuloendotheliosis virus).

In contrast to these results, Payne et al.(1982) have
reported, using a RAV-z strain of ALV, two configurations of
proviral insertion adjacent to c-myc that are inconsistent
with the promotor-insertion model. In one configuration the
provirus is inserted upstream of c—myc, but the viral LTR is
in the opposite transcriptional orientation relative to the
c-myc gene. In this orientation, transcription of c-myc can
not initiate from the viral promotor. In the second
configuration, the provirus is found downstream of the c—myc
gene and in the same transcriptional orientation(Figure

QB). In both cases, the elevated expression of c—myc mRNA

51
is observed. These exceptions prompted the hypothesis, later
found to be correct, that the LTR contains 'enhancer'
sequences that can elevate the level of gene expression
independent of the position or orientation of the 'enhancer'
relative to the affected gene(see section on Enhancers).
This mechanism of gene activation is referred to as
enhancer-insertion.

Though the promotor-insertion configuration predominates
in B-cell lymphomas induced by avian retroviruses, it appears
that this system my not represent the most common
configuration of proviral oncogene activation. In the small
percentage of T—cell lymphomas, induced by murine non-acute
transforming viruses, that show c-myc alterations,
enhancer—insertion is the predominant mode of activation.

The murine provirus is located upstream of the c-myc gene and
in the opposite transcriptional orientation (Corcoran et al.,
1984;Li et al., 1984;8teffen 1984). In addition the
majority of reported activations of other putative oncogenes
by proviral insertion(Figure 8) show a preference for the
enhancer-insertion mode of transcriptional elevation.

Translocation.

A substantial body of evidence indicates that
chromosomal translocations involving the c-myc locus and one
of the immunoglobulin loci are common in certain types of
B-cell lymphomas found in mice and men (Klein 1983;Perry
1983). Most of the translocations that are observed in the

human disease, Burkitt's lymphoma, involve the movement of

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52

part of chromosome 8, containing the c-myc gene, to the
heavy-chain locus of chromosome 14 and in rare cases to the
light-chain locus of chromosome 22 and 2 (Hollis et al.,
1984). As a result of the translocation, alterations in
c-myc expression have been observed in either the amount of
c-myc mRNA or the size of the c-myc transcript. An early
hypothesis for the elevated levels of c-myc mRNA was the
influence of the cellular enhancer located in the
heavy-chainswitching region. However, subsequent reports
(Battey et al., 1983;Rabbitts et al., 1983;6elmann et al.,
1983) indicate that the translocation of c-myc often results
in the movement of the cellular enhancer to chromosome 8
where it can no longer influence the transcription of c-myc
on chromosome 14. Another hypothesis is that the c-myc
regulatory sequences are altered due to the translocation.
There is strong, but circumstantial, evidence for the
suggestion that the first exon serves a regulatory function,
since the c-myc gene has 3 exons, but only the second and
third exons code for the c-myc protein.

The first exon of c-myc is highly conserved between mice
and man, but is absent from v-myc (Shih et al., 1984).
Therefore, exon 1 may serve a vital function in the normal
cellular context that is either unnecessary or even
deleterious to the transforming activity of v—myc. The
translocations that are typical of mouse plasmacytomas result
in the separation of some or all of exon 1 from exon 2 and

exon 3. In contrast to the mouse plasmacytomas, in some

.3
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53
Burkitt‘s lymphoma, all three exons are translocated intact.
However, DNA sequencing and S1 mapping reveal mutations in
the intact exon 1 in five Burkitt's lymphoma cell lines.

A Burkitt's lymphoma cell line carring an 8:14
translocation has been identified that has all three c-myc
exons and the nucleotide sequence of the first exon is
identical to the normal c-myc sequence with the exception of
two single-base substitutions (Wiman et al., 1984). As
suggested by the authors, it is possible that one or both of
these subsitutions are in the region involved with c-myc
regulation. It is also possible that sequences proximal to
the first exon are also important in regulating c-myc
expression. Interestingly, a similar situation has been
reported in a mouse plasmacytoma, but in this case only one
base substitution is observed (Stanton et al., 1984).
Perhaps, only by coincidence, the next four nucleotides at
this site are identical to those found in the Burkitt's
lymphoma just mentioned.

ggplification.

The concept of gene amplification as a model for the
generation and/or progression of cancer has been considered
(Pall 1981:Klein 1981) and is supported by a number of
results. Perhaps the first report of amplification of the
c-myc oncogene was observed in CSV-induced bursal lymphomas
(Noori—Dalloii et al., 1981). There are now a number of
examples of oncogene amplification in both fresh tumors and
established cell lines (Noori-Daloii et al., 1981;Collins and
Groudine 1982;Little et al., 1983;Lee et al., 1984;Kohl et

al., 1983;Schwab et al., 1983a;Schwab et al.,

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54
1983b;Alitalo et al., 1983). In the vast majority of the
amplifications the oncogene c—myc or a closely related gene,
N-myc, is involved.

The c—myc gene has been shown to be amplified in the
human promyelocytic leukemia cell line, HL—60 (Collins and
Groudine 1982). This is interesting in view of the
complementation experiments of Land et al.(1983) and
Ruley(1983), since coexisiting with the amplified c-myc gene
is an activated N—ras gene (Murray et al., 1984). Other
examples of c-myc amplification have been reported in two
neuroendocrine tumor cell lines derived from a colon
carcinoma (Alitalo et al., 1983) and in 8 out of 18 human
lung cancer cell lines (Little et al., 1983). However, most
intriguing is the consistent amplification of the N-myc gene
in tumors of neural origin. The N-myc gene is amplified in
14 out of 18 neuroblastoma cell lines and 5 out of 8 fresh
neuroblastomas (Yunis et al., 1984;5chwab et al., 1985). In
contrast, only 4 out of 36 nonneuroblastoma human tumors and
tumor cell lines tested showed N-myc amplification, of which
three were of neuroectodermal origin (Kohl et al., 1984).

In addition, Brodeur et al.(1984) find N-myc amplified in 24
of 48 tumors from late stage retinoblastoma tumors, but find
no amplification in 15 tumors from early stages of
retinoblastoma tumors. The authors conclude that N-myc
amplification may be involved in the progression of these

tumors to later, and more deadly, stages of retinoblastoma.

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CHAPTER II

LINKAGE OF CHICKEN C-MYC GENE T0 CSV

The family of RNA tumor viruses is generally separated
into two classes that appear to cause neoplasia by different
molecular mechanisms. The class of acute transforming
virus, with the exception of some Rous strains, are
replication defective, harbor oncogene(s) for cell
transformation and induce neoplasia in the host with a very
short latency (2 weeks) between infection and neoplasia. In
contrast, the class of non-acute transforming virus are
replication competent, do not harbor an oncogene and can
induce neoplasia, albeit, with a long latency of 4-10 months
post-infection.

Chicken syncytial virus(CSV) is an avian non-acute
transforming virus genetically unrelated to avian leukosis
virus(ALV). However, similar to ALV, if affects the primary
lymphoid organ for B-cell maturation, the bursa of Fabricus,
inducing a malignant B-cell lymphoma in chickens 4-6 months

post-infection.

55

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56

It has been demonstrated by Hayward et al.(1981) that
in the majority of tumors the ALV(RAV-1 strain) provirus
integrates near c-myc, the cellular homolog of the oncogene
of the acute-transforming virus MC-29. Analysis of the RNA
from these tumors shows that c-myc transcription was
elevated (Neel et al., 1981). These observations have led
Hayward and co-workers to propose that increased production
of c-myc protein triggers the transformation process.

In the present study we have characterized the
structure of the CSV provirus in the induced tumors and
report here that the proviral DNA is integrated adjacent to
c-myc in over 90% of the tumors examined. In addition,
similar to the results seen in the ALV system deletions of

proviral DNA occurs frequently in these tumors.

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MATERIALS AND METHODS

Induction 0 old Leukosis.

Day-01d chicks from line 1515x71(Regional Poultry
Research Laboratory) were inoculated intra-abdominally with
105 infectious units of end-point purified CSV. The
chickens were free of common avian pathogens and reared in
plastic canopy isolators. Both the virus stock and the
tumor samples were shown serologically and biochemically to
be free of common avian pathogens and reared in plastic
canopy isolators. Both the virus stock and the tumor
samples were shown serologically and by hybridization
analysis to be free of avian leukosis virus. Approximately
20 weeks post-infection birds that developed lymphoma were
sacrificed and both tumor and non—tumor tissue were
collected for analysis. All tissue samples were immediately
transferred to liquid nitrogen and then stored at - 70° C
until use.

DNA Extracting and Eggygg Digestigp.

Approximately 0.59 of frozen tissue was pulverized in a

stainless steel tissue smasher in the presence of liquid

57

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58
nitrogen. The powdered tissue was transferred to a sterile
tube containing 20ml SET buffer/0.5x SDS,10mM EDTA,20mM Tris
pH 7.5 and 200ug per m1 proteinase K/ preheated to 60°C.
After incubation for 1hr at 60°C, 500ug/ml of pronase was
added and the mixture incubated overnight at 37°C with
constant shaking. The mixture was extracted with
phenol/chloroform and the DNA concentrated by precipitation
with 2.5 volumes of ethanol. DNA was digested with a two
fold excess of restriction endonuclease for 3 hours. The
digested DNAs were fractionated on 0.7% agarose gels,
immobilized on nitrocellulose and hybridized with the
appr0priate nick-translated probe.

RNA Isolation.

Tissue was pulverized similar to DNA, but the powdered
tissue was transferred to 10 mls of a 4 M guanidinium
thiocyanate mixture (Chirgwin et al., 1979) pre-heated to
60°C. The mixture was disrupted by homogenization in a
polytron mixer. An equal volume of phenol preheated to
60°C was added and the mixture passed through a syringe
fitted with a 25-gauge needle until the viscosity of the
suspension was reduced. The mixture was extracted twice
with chloroform and the aqueous phase transferred to a
sterile tube. One gram of CsCl/2.5 mls of mixture was added
and then the contents was transferred to a sterile
polyallomer centrifuge tube with 1.5 mls of 5.7 M CsCl in
0.1 M EDTA(pH 7.5). The tube was spun at 35k rpm in a
SW50.1 rotor for 12 hrs. The supernatant was removed and

the pelleted RNA was suspended in a small volume of 4 M

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59

guanidinum thiocynate. A 1/40th volume of glacial acetic was
added and the contents transferred to a 1500u1 eppendorf.
The mixture was precipitated in 0.5 volumes of ethanol over
night at —20°C. The tube was spun 5 minutes in a
microfuge. The precipitation step was repeated and RNA was
suspended in, DEPC treated, sterile water.

gybridization and lick-Translation.

Filter hybridizations were carried out in 50%
formamide, 5xSSPE/20x=3.6M NaCl, 0.2M NaH2P04 pH 7.4,
0.02M EDTA/, 1xDenhardts/100x-10g polyvinyl pyrolidone, 10g
Picoll and log BSA in 500ml DDHZO/ and 100ug per m1
single-strand salmon sperm DNA at 42°C. The filters were
washed at room temperature for 10 minutes in 2xSSPE and then
washed twice in 0.1xSSPE, 0.1x 30$ for 45' at 50°C with
shaking. The SNVt probe, a gift from Dr. H. Temin,
consisted of permuted copy of the SNV genome cloned into the
Sal I site of pBR322. The c-myc probe, gift from Dr. T.
Robbins, consisted of a subclone of the second exon of
c-myc. The probe was cloned into the Sac I and Sal I sites
of pDH24. All probes contained in recombinant plasmids were
digested with the appropriate restriction endonuclease(s)
and gel purified. The gel purified fragments were generally

8

nick-translated to a specific activity >10 cpm/ug of DNA

(Rigby et al., 1977).

Re-hygridization of filters.
To remove the hybridized radioactive probe from
nitrocellulose filters for subsequent re-hybridizations, the

filters were placed in a baking dish with 20 mM Tris and 20

fi
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mM EDTA, pH 7.5 and heated in a Tappan(Model 120) microwave
oven, set on high, for 25 minutes. After cooling the
filters were placed on 3MM paper to air dry.

RNA Dot Blot.
The RNA samples were denatured for 25' at 60°C in 15xSSPE
and 2.2M formaldehyde. Dilutions of RNA were made in wells
of a microtiter plate. Nitrocellulose was equilibrated with
15xSSPE and placed on a Minifold apparatus(S&S). A 100 ul
of each dilution was placed in the Minifold wells. After
suction was removed from the Minifold, the nitrocellulose
was air dried and baked for 2 hours in a vacuum oven at
80°C. Hybridization and washing conditions were similar

to DNA, except the hybridization temperature was 45°C.

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RESULTS

Li e of c- to CSV.

A map of the CSV genome is presented in Figure 10 and a
map of the chicken c-myc locus is presented in Figure 11.
As indicated by the restriction enzyme maps of CSV and
c—myc, neither have Eco RI sites in their coding regions.
To examine the linkage of CSV proviral sequence to nearby
cellular sequence the restriction endonuclease Eco RI was
used. Since this endonuclease does not cleave within the
CSV genome the whole proviral sequence and adjacent cellular
sequences are obtained on a single Eco RI fragment.

The tumor DNA was digested with Eco RI, fractionated on
a 0.7x agarose gels and transferred to nitrocellulose.

Hybridization of these filters to the SNV probe is shown

t
in Figure 11A. With most tumor samples, a single band of
variable size is observed. Each band represents a
tumor-specific(TS) fragment that corresponds to CSV proviral
sequence. Only one proviral band is seen in most tumors,

indicating that the CSV induced bursal tumors are derived

from the outgrowth of a single transformed cell.

61

 

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.H Homnnmm "H 0mmI0m “HHH ocamnmm "Hmammum

“H m>¢u€ .>mo nu“: >m0~080£ mucmdvmm mom cmnu umumoum mommnm umcu >mm

mo cfimuum m .moua> mwmonooc condom aonm mocoHonzn spas c0«umN«0nun>c vOHn
anocunom >0 0030~H0u .ocum Hamo unnosoonm >m0 m ..Nmmfi ..am we :mfiumwmz.
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To demonstrate the linkage between the CSV provirus and
the c-myc gene, the Eco RI cleaved tumor DNAs were
re-hybridized with a myc specific probe. As shown in Figure
123, each tumor contains both the unaltered c-myc allele of
13.5 kb and an altered c-myc allele that comigrates with at

least one of the TS bands detected with the SNV probe.

t
This data strongly implies that a CSV provirus is located
adjacent to one of the c—myc alleles. These results
implicate the c-myc gene in lymphomagenesis and CSV
integration as the activator of this gene. The variation in
relative intensity of the bands representing the unaltered
and altered c-myc alleles implies that the unaltered c-myc
allele is less abundant than the altered allele in some
tumors. Presumably, this is due to aneuploidy for the
chromosome bearing the unaltered c-myc gene. Similar
results have been observed in bursal tumors induced by ALV
(Payne et al., 1982).

The size of the Eco RI linkage fragments vary from 8-22
kb. We assumed that the size variations are due to
deletions or structural alterations of the viral or
surrounding cellular sequence. In order to examine the
possibility that the alteration are in the viral sequences
the tumor DNA was digested with Bam HI and hybridized to the
SNVt probe(data not shown). In an unaltered provirus
there are 5 internal CSV fragments produced ranging in size

from 2.2 - 0.75 kb. The Bam HI digestion pattern of the

tumor DNA shown in figure 12A indicated the loss of some of

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69
the viral specific fragments, thereby confirming that the
size variations seen in the bands in Figure 12A are due to
deletion or structural alteration of the integrated viral
genome. The TS bands that are smaller than the unaltered
c-myc allele(lane 9 and 10) are probably due to the presence
of a new Eco RI site in the structurally altered viral
genome.

Amplification.

An interesting result, presented in Figure 12A, is the
unusual intensity of the hybridization signal of the TS band
in lane 9. We believe this to be caused by the
amplification of the provirus and c-myc sequences. In order
to obtain a better estimate of the amount of CSV sequences
present in a tumor with an intense TS band a titration
experiment using SNVt as quantization standards, was
carried out( Figure 13). Thirty micrograms of DNA from
tumor number 15 that showed amplification of CSV sequences
(data not shown) and 30 ug of DNA from a tumor with a
proviral band of similar size, but with a "normal" band
intensity ( tumor number 18), were digested with Eco RI and
loaded on an agarose gel together with varying amounts of
the purified insert of SNVt(Figure 13, lanes c-f). The
quantities of the cloned DNA in lanes c-f were such that
they represent, respectively, 2, 4, 10, 20 copies per cell
equivalent of CSV sequence in 30 ug of chicken DNA. When
the TS bands in lanes a and b were traced densitometrically
and their intensity compared with that of the standards in

lanes c-f, it was found that while tumor no. 18 in lane b

 

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71

 

72
acquired only one copy of a CSV provirus, tumor number 15
appears to carry approximately eight copies of the proviral
sequences.

Enhanced ggpression og c-myc.

The structural analysis of the tumor DNA showing the
CSV-myc linkage implies that c-myc gene expression is
elevated based on the results of Hayward et al.(1981) and
Payne et al.(1982). To confirm the transcriptional
activation of the c-myc gene by the CSV provirus, the RNA
from tumors 1, 2, and 3 were examined by RNA dot blot. The
results in Figure 14 show that c-myc expression is elevated
in all 3 tumors when compared with controls. Densitometric
tracing indicated that the amount of elevation is 100, 80,

and 50 fold, respectively.

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DISCUSSION

Recent studies by Hayward et al.(1981) strongly
implicate the activation of the c-myc gene in the bursal
lymphomas induced by ALV infection. In this study we wished
to determine whether CSV, which induces a lymphoid leukosis
similar to ALV, was also integrated near the c-myc gene.

Our data shows that in over 90% of the tumors examined, 28
of 30 tumors, c-myc is linked to CSV proviral sequence.
These results are consistent with results from Hayward et
al.(1981) and support the authors suggestion that the
insertion of a provirus near c-myc is instrumental in
altering the expression of the cellular gene, thereby
triggering the oncogenic transformation.

The fact that CSV and RAV-i share very little sequence
homology, including their LTRs, and yet they are both found
integrated near the c-myc gene in lymphoid leukosis strongly
implicates this gene in the transformation of lymphocytes.
In addition, our RNA dot hybridizations indicate that CSV is
able to increase the transcriptional activity of c-myc as

much as 100 fold, supporting the proposal that elevated

75

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76
levels of c-myc gene product may trigger the progression to
neoplasia.

It is very interesting that the deletions of proviral
sequence observed in the CSV induced lymphomas is strikingly
similar to those observed in RAV-i induced lymphoid
leukosis. These results imply that deletion is important
for some aspect of the transformation process. It is
possible that deletions of the viral genome that disrupt the
transcriptional program of viral RNA facilitate the
transcription of the downstream cellular sequences. Perhaps
the transcription of viral RNA from the left LTR extending
into the right LTR may affect the initiation at the right
LTR. A disruption of the transcriptional program caused by
a deletion in the proviral DNA may expose the right LTR and
allow efficient transcription of the downstream cellular
oncogene.

Alternatively, the deletion of viral sequences may play
a role in the selective growth of the tumor clones. Those
cells in which the expression of viral antigens is
eliminated by deletion may therefore be rendered less
immunogenic and able to escape the host immune response.
Histopathological examination shows that at onset of the
disease, there are many microscopically observed enlarged
bursal follicles(considered to be transformed cells) (Cooper
et al., 1968;Neiman et al., 1980). Immune selection may
account for the finding that only a limited number develop

into tumors.

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The possiblity that gene amplification may relate to
the generation and/or progression of neoplasia is supported
by a variety of findings (Pall et al., 1982;Varshavsky et
al., 1982). Chromosomal aberrations, including double
minute chromosomes, 'homogeneously staining regions' and
trisomy have been found in a number of tumors and tumor cell
lines (Klein 1981). In some cases the cell lines have been
shown to contain amplified oncogenes, including c—myc, c-abl
and c-Ki—ras (Schwab et al., 1983). In most virally and
non-virally induced murine T-cell leukemias trisomy of
chromosome 15 is often the only chromosomal abnormality
detected (Dofuku et al., 1975).

Our results indicating that c-myc is amplified in tumor
no. 15 may point to another mechanism whereby a non-acute
transforming virus can induce neoplasia. However, we have
examined 30 tumors for CSV-myc linkage, in only two tumors
have we observed amplification of the c-myc gene. These
results indicate that if amplification is involved in a
mechanism for c-myc activation for certain bursal lyphomas,
it is relatively rare.

We have shown that c-myc is linked to proviral CSV
sequence in over 90% of the bursal tumors examined and the
majority of CSV provirus have undergone some structural
alteration. In addition, the RNA dot blots indicate the
enhanced expression of c-myc in these CSV induced tumors.
Presently, we do not know the exact insertion site nor the

orientation of the provirus. Further analysis by

4.

 

78
restriction enzyme digestion and northern blot analysis
should tell us whether the elevated expression of c-myc is
consistent with promotor-insertion, enhancer insertion or
perhaps some other molecular mechanism of oncogene
activation. Also, it is not clear how the structural
alterations in the provirus contribute to the transformation
process nor what the exact nature of the alterations are,
but detailed structural analysis of the molecularly cloned
TS bands should provide insight into the molecular mechanism
of the alteration and its possible function in the

transformation process.

 

 

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CHAPTER III

DETERMINATION OF TRANSCRIPTIONAL ORIENTATION
AND

SITE 0? INTEGRATION OF CSV RELATIVE TO C-MYC

The mechanisms by which ALV activates the c-myc gene in
B-lymphomas have been extensively characterized (Hayward et
al., 1981;Payne et al., 1982;Fung et al., 1982;Westway et
al., 1984). Although important exceptions have been noted
(Payne et al., 1982;Pachl et al., 1983), ALV proviruses
usually integrate upstream of the c-myc coding exons and are
arranged in the same transcriptional direction, enabling the
provirus to utilize its 3' LTR promoter to transcribe and
deregulate the downstream c-myc gene (Hayward et al.,
1981;Payne et al., 1982;Fung et al., 1982). We have
previously shown that in CSV induced B-lymphomas, CSV
proviruses and the c-myc gene are physically linked
(Noori—Daloii et al., 1981). The arrangement and insertion

sites of CSV proviruses however were not analyzed in detail.

79

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Here we describe our analyses of the provirus—c-myc
linkage structure in twenty—two CSV induced lymphomas. Our
results indicate that the integration pattern of CSV
proviruses show a striking similarity to those observed for

ALV proviruses involved in c-myc activations.

MATERIALS AND METHODS

Inductign o; Lygphoid Leukosis.
Day-old chicks from line 15I5x71(Regional Poultry
Research Laboratory) were inoculated and housed as described
in Chapter II.
DNA gxtragtign andggnzyme Digestipn.
The isolation of DNA from tissue and digestion with
restriction endonuleases was as described in Chapter II.
gzgridiggtion and lick-Translation.
Conditions for hybridization and nick-translation were as
described in Chapter II. The SNV-LTR probe, derived from
the SNV proviral clone, was subcloned into the Sac I-Bam HI
sites of pDH24. The mycs and myc3 probes were derived from
gel purified Sma I-Sac I and Cla I-Eco RI fragments from a
chicken c-myc clone, gift from Dr. T. Robbins.
Re-ggbridization of Filters.
The conditions for washing off hybridized probe were the

same as described in Chapter II.

81

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RESULTS

The chicken c-myc gene, like its mammalian counterpart,
has three exons; only the second and the third exons contain
protein-coding sequences. As indicated before, the majority
of the ALV proviruses in B—lymphomas are situated upstream
from the second c-myc exon and are in the same
transcriptional direction as the c-myc gene. Figure 15A
shows what would be predicted if the CSV provirus also
inserts in a similar manner. To facilitate the mapping of
the proviral integration sites in a large number of samples
we adopted the following strategy, relying principally on
Sac I and 8am HI digestions. We took advantage of the fact
that a Sac I site cleaves near the 5' end the second c-myc
exon, thereby dividing the c-myc locus into a 5' and a 3'
region (Watson et al., 1983), and we prepared probes (myc5
and myc3, Figure 14) specific for each region. The CSV LTR
contains a Sac I and a Bam HI site near its 5' and 3'

boundaries, respectively. If the diagram in Figure 15A

82

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represents the CSV/c-myc junction structure, then Sac I
digestion should yield the following results. One, the 5'
Sac fragment of c-myc should be shortened when compared to
the normal control, due to the presence of the Sac I site in
the viral LTR, but the 3' Sec fragment of c-myc should not
be affected. Two, the LTR probe and the myc5 probe should
cohybridize to the shortened 5' Sac I fragment. As shown in
Figure 15C to 150. the predicted results are exactly what we
observed. In these experiments, Sac I digested tumor and
control DNA were fractionated by electrophoresis in 0.85%
agarose gels. immobilized on nitrocellulose paper and
hybridized with the myc5 probe (lanes a of Figure 150).
The normal control (C) DNA displays a 6.8 kb band,
corresponding to the normal c-myc locus. Each tumor sample
carries an additional shortened c-myc band (indicated by
). In contrast, none of the tumors showed any evidence of
alteration when hybridized to the myca probe (Figure 15).
This indicates that the proviral insertions occur 5' to the
Sac I site in the second c-myc exon.

Hybridization to the LTR probe(lanes a in Figure 15D)
reveals the linkage between LTR and c-myc. In each tumor
sample, at least one of the bands (marked by ) detectable
with the LTR probe comigrates with the altered c-myc band
shown in lanes a of Figure 150. The other LTR-containing
bands presumbly represent the junction fragments from the
other end of the individual proviruses or from other
proviruses present in the same tumor DNA. This analysis

also defines the transcriptional orientation of the

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proviruses as the same as that of c—myc. If the provirus
and c-myc were oriented in opposite transcriptional
directions. then regions to which the LTR and myc5 probes
hybridize would be unlinked by Sac I digestion and no
cohybridization would have been detected. To further
substantiate the conclusion that the LTR-c-myc linkage
structure shown in Figure 15A is correct, tumor DNA was
doubled-digested with Sac I and Dam HI, and hybridized to
the myc5 probe (lanes b in Figure 150), the resulting
altered c~myo band(indicated by ) was found to be
approximately 500 bp shorter than the band in Figure 150
(lanes a) in all cases. In addition, the fragments in lanes
b of Figure 150, in general, did not comigrate with those in
lanes b of Figure 15D, when the LTR probe was used as the
hybridization probe. The LTR probe uniformly detects a ca.
500 bp fragment(indicated by ). We anticipated this
result since Sac I and Ben HI double digestion of the DNA
should liberate the 500 bp fragment from which the LTR probe
was originally derived. Based on all the criteria discussed
above, we conclude that the proviruses have the structure
depicted in Figure 15A. This type of analysis was extended
to a total of 22 tumor DNA samples. With no exception, all
tumor samples displayed digestion patterns similar to the
eight representative samples illustrated, suggesting that
the diagram in Figure 15A indeed represents the predominant
configuration of the CSV proviruses involved in c-myc

activation. This conclusion has received additional support

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from detailed mapping and sequencing of a genomic clone
(from tumor 4) carrying the entire activated c-myc gene and
the inserted provirus ( Swift et al., in preparation).

Knowing that all the proviruses are oriented in the
same way as the c—myc gene, we could then use the sizes of
the Junction fragments to accurately locate the individual
proviral insertion sites. The Sac I-Bam HI Junction
fragments (lanes b Figure 150) are especially useful for
this purpose, since they are usually smaller than 700 bp and
readily resolved in the gel. The data summarized in Figure
16 shows that most of the insertion sites are located within
500 bp immediately 5' to the second exon of c-myc. five of
the proviruses reside within the first exon and two are
located further upstream. However, none are located in the

5' one third of the first intron.

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DISCUSSION

It is Interesting that the pattern of integration
observed for the CSV proviruses is identical to that
observed for the ALV proviruses found in the B-lymphomas
induced by ALV (Shih et al., 1984). Specifically, most of
the ALV proviruses are concentrated in a region 500 bp
preceding the second c-myc exon and a few are scattered
within the first exon. The 5' one third region of the first
intron of c—myc which is devoid of CSV proviruses is also
silent for ALV proviral integration. Intuitively, the
insertion of a viral promoter between the natural promoter
and the coding sequence of the resident gene should render
the resident gene under viral control.

One would anticipate. in the c-myc case, that the viral
insertion sites would be distributed along the entire region
between the start of the first and second exon. This is
apparently not the case in both CSV and ALV induced

B-lymphomas. One possibility is that the nonrandom

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91

distribution of the proviruses may be a consequence of
integration specificity of the viruses. However, given the
dissimilarity of ALV and CSV, it is unlikely that such a
specifity is based on the primary sequences of the target
sites. (The terminal nucleotides, implicated in integration
of CSV and ALV are very different and the viral enzymes
involved in the cleavage of Junction also have different
recognition specificities (Shimotohno et al., 1980;8wanstrom
et al., 1981)). Furthermore, the fact that CSV and ALV both
have multiple insertion sites in the clustered region also
argues against a strict sequence requirement for
integration. Though, the local DNA structure and chromatin
conformation may have a profound effect on the proviral
insertion frequency. As pointed out by Shih et al.(1984),
the intron region contains many complementry sequences that
may form dyad symmetries. However, computer sequence
analysis (Staden 1984;8wift et al., 1985) of the intron does
not reveal a correlation between dyad symmetries (Staden
1984;Tinoco et al., 1973) and proviral integration sites.

There are two possiblites for the lack of integration
in the 5' portion of the first exon. First, it may be that
the chromatin in this region is inaccessable to the provirus
during integration. Second. perhaps integration in this
region may not result in cellular transformation and the
formation of a tumor. The first possiblity could be
mediated by a lack of specific viral integration recognition
sequences in this region of the DNA. However, as mentioned

above, sequence specificity during integration is not likely

 

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to be the answer. It is also possible that the chromatin in
this region adopts a structural organization, such that the
proteins necessary for viral integration are unable to gain
access to the host DNA.

It may be that there is some DNA sequence at the 3' end
of the silent region that affects post-transcriptional
processing, mRNA stablity. translation or perhaps all of the
above. An example of what might cause translational
inhibition is translation initiation codons in the 5'
untranslated region of the mRNA. Fewer then 10* of over 200
eukaryotic mRNAs examined have ATGs in the 5' untranslated
region (Kozak 1983). Recent reports demonstrate that the
presence of ATGs 5'-proximal to the authentic initiation
codon results in a dramatic decrease in the amount of
protein synthesized (Kozak 1984;Lui et al., 1984). We find
four ATGs positioned from 575 to 515 bp 5'-proximal to the
second exon of c-myc, that may interfere with the
translation of the c-myc gene. This location demarcates the
the approximate 3' boundary of the silent region. In this
regard, it is interesting that the 5' end of the silent
region is bounded by a consensus splice donor site (Mount
1982), presumably used to join the first and second exon of
c-myc during post-transciptional processing (Shih et al.,
1984). Proviral integrations 5' to this splice donor site
would allow the removal of the initiation codons during

post-transcriptional processing.

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93

The several possibilities discussed above are not
mutually exclusive and more than one factor is likely to be
involved in defining the activation scheme. In this regard,
it is interesting to note that the situation for c-myc
activation in mouse or rat lymphomas induced by murine
non-acute retroviruses are rather different. In these
instances, the proviruses are exclusively found to be
upstream from the first c-myc exon and are arranged in the
opposite orientation (Tsichlis et al., 1983;Nusse et al.,
1984). Thus, enhancement, rather than promoter insertion
appears to be involved in c-myc activation non-acute murine
retroviruses.

In summary, the data presented in this communication
show that despite its completely different origin, CSV uses
a mechanism very similar to that of ALV to activate c-myc
gene causing B-lymphomagenesis. The majority of CSV
proviruses are arranged in a configuration that allows the
use of the viral promoter for c-myc transcription, resulting
in enhanced expression of a truncated c—myc message with

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CHAPTER IV
TRANSCRIPTIONAL AND STRUCTURAL ANALYSIS

OF AN ACTIVATED C-MYC GENE

We reported previously that the CSV provirus integrates
near the c-myc locus in CSV-induced chicken B—cell lymphomas
(Noori-Daloii et al., 1981). Based on Southern analysis,
the majority of the proviruses have large deletions, are
located in the first intron of the c-myc locus and oriented
in the same transcriptional direction as the c-myc gene
(Swift et al., 1985). In order to confirm and extend these
findings, we wished to isolate from tumor DNA clones
containing the CSV provirus and the perturbed c-myc allele
to analyze its structural features by restriction enzyme

mapping and DNA sequencing.

94

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MATERIALS AND METHODS

induction of Lygphoid Leukosis.
Day-old chicks from line 15I5x71(Regional Poultry
Research Laboratory) were inoculated and housed as described
in Chapter II.
DNA Extraction and gggyge Digesiion.
The isolation of DNA from tissue and digestion with
restriction endonuleases was as described in Chapter II.
gypridization and lick-Translation.
Conditions for hybridization and nick-translation were as
described in Chapter II. The SNV-LTR probe, derived from
the SNV proviral clone, was subcloned into the Sac I-Bam HI
sites of ponzc. The myc5 and myc3 probes were derived from
gel purified Sma I-Sac I and Ole I-Eco RI fragments from a
chicken c-myc clone, gift from Dr. T. Robbins.
Rs-hzgridiggtion of Filters.
The conditions for washing off hybridized probe were the

same as described in Chapter II.

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Construction of a Genomiciiibragy og_1gmgg Tia.

A library of tumor 713 genomic DNA was contructed
according to methods described by Maniatis et al.(1982).
High molecular weight DNA from tumor 713 was prepared (see
Chapter II) and partially digested with Mbo I. The DNA was
fractionated on a 10-40% sucrose gradient (Maniatis et al.,
1982), fragments of 16-22 kb were pooled and ligated into
the Dam HI site of phage vector BFlOl. Recombinant molecules
were packaged igrvitro and screened for inserts homologous
to both myc3 and LTR probes by inrgigg_hybridization (Benton
and Davis 1977).

DNA Sggggncigg.

DNA sequencing was carried out according to the method
of Maxam and Gilbert (1980). A subclone of the 5' LTR and
the INT region were derived from the 2.0 kb viral Sac-Sac
fragment of clone 713 after digestion with Sam BI and
ligation to Bam—Sac digested pDH24. A subclone of the 3' LTR
and the 3' viral Junction region were derived from the 2.2
kb Sac-Sac viral—myc Junction fragment of clone 713 after
digestion with Ham HI and ligation to Ban-Sac digested
pDHZt. The subcloned DNAs were digested with either Eco RI,
this site is in pDH24 adJacent to the Sac I site, or Bam HI,
treated with calf intestinal phosphatase, end labeled by T4
polynucleotide kinase and digested with either Bam HI or
Sac I. The fragments were gel isolated and sequenced.

Southern blag.
As described in Chapter II.

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Northern biot.

RNA blot analysis was performed on poly(A) selected RNA
(Aviv and Leder 1972). Samples and DNA markers were
denatured for 25' at 60°C in 25mM mops pH 7.0/lmm
BDTA/2.2M formaldehyde/50$ formamide. The RNA samples and
markers were fractionated by electrophoresis in a 1.5%
agarose gel with 2.2 M formaldehyde (Maniatis et al., 1982).
Conditions for transfer and hybridization were similar to
those used for DNA, except hybridization was carried out at

45°C and washing was at 55°C.

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RESULTS

We have previously used Eco RI, a restriction enzyme
which cleaves neither the CSV provirus nor the c—myc exons,
to screen for CSV DNA insertion near the c-myc locus
(Noori-Daloii et al., 1981). Figure 17 shows the Eco RI
digestion analysis of three representive tumor DNA samples
and one non-neoplastic control(C) hybridized with v-myc
probe. In all lanes, including the control lane, a 13.5 kb
fragment corresponding to the normal allele is seen.
However, in the tumor lanes is an additional band, of
varying size, but larger than the normal c-myc band. Since
Eco RI does not cleave in the CSV provirus it is unlikely
that the altered c-myc bands of varying mobilities can be
attributed to differences in proviral insertion sites;
rather, they are likely consequences of proviral or cellular
DNA deletions. The c-myc band in tumor 713(Figure 17, lane
1) is only 16 kb, implying there is a deletion of ca. 6 kb

of viral or cellular sequences.

98

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nonuo ca conunomup handcu>mmm cocoaoconn m .maaoo scams cu oHoHHM chain
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any a" menu .>Hnmadmumm .onnu ma umnu>ou any .Au mommy .u.o. momma

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may .>adm5ma .co«uommca >mm no menu cmxoano m sown ma onmcmfi Howucoo
one .onoum o>el> cmxoano may Ou canumwfipwnn>m .co«umm0au HM oou

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101

Cloning and mapping of the activated c-myc gene in

tumor 713.

To ensure that the c-myc alteration is due to CSV
proviral insertion, Eco RI digested 713 DNA was hybridized
to SNV probe (data not shown). As expected, the SNV probe
cohybridized to the altered 16 kb c-myc band indicating that
the CSV proviral sequences are contained in the same 16 kb
fragment. The fact that no other CSV-related sequences are
detected indicated that the c-myc linked CSV DNA is the only
provirus present in this tumor.

We have isolated the perturbed c-myc allele from a
genomic library of tumor 713 DNA. The genomic library was
constructed from 713 tumor DNA partially digested with Mbo I
and ligated to phage vector BFIOI (Mantiatis et al., 1982).
The CSV linked c—myc clone was selected from the library by
its ability to simultaneously hybridize to myc and REV
probes. The map of this clone, lambda 713 (Figure 18), is
consistent with the presence of a complete c-myc gene with
all three exons intact, but between the first and second
exon, a deleted CSV provirus has been inserted. Southern
hybridizations with LTR probe and probes representing the
first(myci) and second(myc2) exons confirm the linkage of
CSV to c-myc(data not shown). For example, Bam HI digestion
yields three fragments with respective sizes of 2, 6.3, and
ca. 10 kb. The 2 and 10 kb fragments are detectable by the
LTR probe and the 6.3 kb fragment is detectable by myc2

probe. The 10 kb fragment is also detectable by mycl probe,

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104

thereby defining the 5' Junction fragment. The 2 kb band
represents the internal portion of the provirus and is only
detected by LTR probe. The 6.3 kb fragment is only detected
by myc2 probe, defining the 3' Junction fragment. Sac I
digestion yields two fragments detectable by LTR; the 2 kb
internal fragment and the 1.65 kb 3' Junction fragment which
can be detected by both the myc2 and the LTR probe. This
and other data are consistent with the physical map shown in
Figure 18.

Sequencing analysis of 713 clone.

The mapping data described above suggests that the
provirus has a large internal deletion, but retains two
intact LTR's. The latter finding is in contrast to the
structures of several ALV provirus clones isolated from
chicken B-lymphomas, where the deletion usually extends to
or beyond the 5' LTR (Payne et al., 1982;Pachl et al.,
1963). To ascertain the intactness of both LTR's and the
nature of the large internal deletion, we have determined
the DNA sequence of the relevant portions of the proviral
clone.

We have divided our sequencing results into four
regions: 5' LTR, 5V (5' viral sequence), 3V (3' viral
sequence), and 3' LTR. Using the appropriate subclones
derived from the 5' and 3' LTR's. we have determined the
entire sequence of the 3' LTR and all but the first seven
nucleotides of the 5' LTR. The data shows that the two

LTR's are identical to each other. The 3' LTR is also

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105
identical to a CSV provirus LTR isolated from the DNA of a
producer cell line(data not shown). This strongly suggests
that the 713 provirus indeed contains two intact and
potentially transcriptionally active LTRs.

The CSV LTR sequence (Figure 19) is 510 bp long. The
03 region is ca. 334 bp long, the terminal repeat region(R)
is 76 bp and the U5 region is ca. 100 bp, based on a
comparison of the 03, R and U5 regions to the SNV LTR
(Shimotohno et al., 1980). The CSV LTR has the same
regulatory features that are characteristic of most
retrovirus LTR's (Varmus 1983). A CCAT box is present at a
position 75 bp upstream of the RNA cap site and 23 bp
upstream of the cap site is a TATA box. The signal sequence
usually associated with eukaryotic polyadenylation, AATAAA,
is located 24 bp upstream of the R-U5 Junction.

A comparison of the published sequence of the SNV LTR
(Shimotohno et al., 1980) to the sequence of the CSV LTR
reveals several interesting features. A variability in the
size of the U3 region of the viral LTRs of different
recombinant clones of SNV provirus has been reported
(Shimotohno and Temin 1962). In each case a deletion of one
of two duplication elements consisting of a 46 bp and 26 bp
unit, each is bounded by smaller penta or hexa-nucleotide
repeats is observed to account for the variation. In the
CSV clones analyzed here, only one copy of each repeat is
present. While the 46 bp repeat in CSV is almost identical

to that of SNV, the sequences of the 26 bp repeat diverge

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108

significantly. In fact, it accounts for 40% of the
divergent sequences between the CSV and SNV LTRs, not
counting deletions. Although the functions of the 46 and 26
bp repeats are not clear, they map in to the general area
where retroviral enhancer sequences usually reside (Dhar et
al., 1980;8chwartz et al.. 1983;Hampe et al., 1983). The 72
bp repeat of Mo-MSV is in a similar location to the SNV and
CSV repeats. Since the two repeats, 46 and 26 bp, add up to
72 and they are in a region where viral enhancers have been
localized, a possible analogy might be made to the Moloney
murine sarcoma virus (Dhar et al., 1980) which contains two
72 bp repeats that can act as a transcriptional enhancer.

The restriction map of the 713 provirus already
suggests that the deletion break point probably lies very
close to the 5' end, since the 5' Sac I and Sal I sites are
both missing in the clone. Sequence analysis reveals that
the breakpoint is only 18 nucleotides away from the 5'
LTR(data not shown). The 18 nucleotides correspond to the
tRNApro primer binding site(PBS), but the sequences after
that are completely different from the published leader and
gag sequences of SNV (O'Rear and Temin 1982). We presume
this stretch of sequences belongs to the pol or env genes,
however, there is no available sequencing data from either
CSV or related REV-A and SNV to confirm this. Nevertheless,
it is clear that deletion occurs rather early, since the
leader sequence, the packaging signal and the splice donor

site for env gene are all lost, making this defective

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109
geneome incapable of synthesizing properly processed and
packagable transcripts. Interestingly, a deletion that
begins in approximately the same place was observed in an
ALV provirus integrated upstream from c-myc (Westway et al.,
1984). The 3'V sequences match closely with those in the
corresponding region of the SNV DNA. It represents the
carboxyl terminus of the env gene and the region between env
and the 3' LTR. The overall homology in this region between
CSV and SNV is ca. 95%. However, only 180 bp is available
for comparison from the literature.

The 5' viral sequencing data suggests that the 113
provirus carries a large deletion and is likely to contain
only a portion of the env sequences. Hybridization to
cloned SNV DNA with a radio-labelled DNA fragment
corresponding to the internal 2 kb sequence of the 713
provirus confirms that only the env sequences are present.

Expression ofgproviral and c-myc sequences in tumor 713.

 

Having obtained the structural information about the
perturbed c-myc gene, we wished to examine the mode of its
activation. The RNA expression of c—myc and viral related
sequence were examined by northern blot hybridization using
myc3, LTR, and INT( the internal viral region of 713,

Figure 20). If the CSV provirus activates c-myc in a manner
similar to ALV provirus, then we would predict the presence
of a 2.3 kb transcript which initiates in the 3' LTR and
contains the two c-myc exons (Hayward et al.. 1981). We

observe two myc transcripts in lane 1 (2.3 and 2.0 kb).

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112
However, only the 2.3 kb transcript cohybridizes to the LTR
probe (lane 2). These results indicate that the 2.3 kb
transcript has LTR sequence covalently linked to the c-myc
mRNA and therefore initiation for this transcript very
likely begins at the viral promotor located in the 03 region
of the LTR. This result is consistent with the
promotor-insertion configuration of the 713 provirus.

Then what is the origin of the 2.0 kb myc transcript
that does not contain LTR sequences? We believe that the
2.0 kb transcipt may initiate from a cryptic promotor
located in the first intron of c-myc. There are many TATA
sequences in this region, especially within the first 80 bp
of the second exon (Shih et al., 1984). Based on the
elevated level of expression, it is likely that the CSV
enhancer sequences are influencing the transcription from a
cryptic promotor.

We observe two LTR transcripts in lane 2, a 2.3 kb
transcript and a 1.8 kb transcript. However, as mentioned
above, only the 2.3 kb transcript hybridizes to the c-myc
probe. Since the 1.8 kb transcript hybridizes only to the
LTR probe, it is possible that this transcript initiates
upstream of the first exon of c-myc and terminates in the 5'
LTR of CSV. If one assumes that inititation begins 300 bp
upstream of the first exon, there are TATA sequences in this
location, and terminates at the 03-R junction of the 5' LTR
the size of the expected transcript is ca. 1.8 kb. This

utilization of the 5' LTR is analogous to the result of

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Payne et al.(1982) for the case of ALV integration
downstream of the c-myc gene and very common in the MMTV
integration near the int-1 region (Nusse et al., 1983,1984).

Since the 5' LTR is identical to the 3' LTR and the 3'
LTR is transcriptionally active, we would expect an
abberrant viral transcript or transcripts initiated at the
5' LTR. However, when the northern blot was hybridized to
probe INT, a probe which spans the internal region of the
713 provirus, there is no detectable viral transcript
carrying the internal sequences, indicating either the
transcripts are not stable, abberrant termination, possibly
as a result of the deletion, or some mechanism of
surppression of the transcriptional potential of the 5' LTR.

Due to the scarcity of the intact RNA samples, more
detailed characterization to explore the nature of these two
messages could not be accomplished. Nevertheless, it is
evident that the viral genes are not present in the final
transcripts and there is one c-myc message generated that is
consistent with the promotor-insertion mode of oncogene

activation.

 

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DISCUSSION

It has been observed that c-myc activation, by the
insertion of avian leukosis virus, is often accompanied by
deletion of one of the two LTRs (Neel et al., 1982;Payne et
al., 1982:Fung et al., 1982,1984). One suggestion for the
observed high frequency of LTR deletion is that
transcription from the 5' LTR would interfere with the
amount of transcription from the 3' LTR. Cullen et
al.(1984) have demonstrated that early after transfection of
quail cells(QCi-a) with DNA containing a provirus-like
structure and a preproinsulin II gene linked to the 3' LTR,
i.e., 5' LTR-env-a' LTR-preproinsulin II, the
transcriptionally active 5' LTR surppressed the
transcription of the downstream preproinsulin II gene. They
also observed that deletion of the 5' LTR or insertion of a
transcriptional terminator from SV 40 Just downstream of the
5' LTR increased transcription of the preproinsulin II gene

5—fold.

114

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115

It might be expected that the presence of the 5' LTR in
the 713 provirus would suppress transcription from the 3'
LTR. One possiblity that could explain both the
unsuppressed trancriptional activity of the 3' LTR and the
unobserved viral transcript(s) is the presence of a
eukaryotic transcriptional termination signal near the 5'
LTR/5' viral Junction. In this case, the situation would be
analogous to the results observed by Cullen et al.(1984)
when the SV 40 early termination region was placed near the
5' LTR Junction. We have examined the 5' viral junction
region for sequence homologies to termination signals found
in Ad2 virus, SV 40, drosophila, yeast and phage (Hay et a1,
1982;Maderious and Chen-Xiang 1984;Henikoff et al.,
1983;Hatfield et al., 1983). Though we do not find any
sequence similarities, there could still be sequences
present in this region that attenuate or terminate
transcription.

It is possible that the promotor in the 5' LTR is
active in initiating transcription of the internal viral
region, but during post-transcriptional processing the
internal region is spliced out of the transcript using the
concensus splice donor site (Mount 1982) located 195 bp
downstream from the 5' LTR—PBS junction. However, this
would still leave 231 bp of homologous sequence in the
spliced transcript, a sufficient length to be able to detect

the viral transcript with the INT probe.

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116

An alternate possiblity is suggested by the results of
Emerman and Temin(1984). They have shown that suppression
of transcription of two proximal promotors does not depend
on the relative position of the two promotors, but on which
promotor is transcriptionally active. Using a retrovirus
vector with two dominant selectable markers, i.e.,
5' LTR-tk-promotor-neo-S' LTR, they have shown that
suppression of expression is dependent on which gene is
under selective pressure. They find suppression of the 3'
promotor when selection is for the tk gene, consistent with
promotor interference. However, selection for the neo gene
results in suppression of tk expression by the 5' promotor.
In this case. inhibition of expression cannot be attributed
to promotor interference. The authors hypothesize a model
in which transcription from one promotor causes a change in
the chromatin structure of the surrounding DNA so that
transcription from a nearby promotor will be inhibited. The
activated c-myc gene could be considered a gene under
selective pressure, since its consitutive expression may be
required to maintain a transformed phenotype. Analogous to
the results of Emerman and Temin, the transcriptional
activity of the 3' LTR and the c-myc gene would result in
the suppression of the viral transcript(s) from the 5' LTR.
It might be possible to test this proposition by
transfecting the 713 clone into an appropriate cell where

the expression of c—myc is no longer required.

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117

Several recent observations suggest that the 03 region
of viral LTRs may determine the oncogenic potential of a
retrovirus by conferring the capacitiy to replicate in the
appropriate target cell. Murine viruses capable of inducing
T-cell lymphomas replicate in the thymus, but
non-leukemogenic viruses do not (Cloyd 1983;0'Donnell et
al., 1983;Celander and Haseltine 1984). In addition, the
capacity of Friend leukemia virus and Moloney leukemia virus
to induce erythroid or lymphoid neoplasia, repectively, is
probably determined by 03 sequences in the viral LTR (Chatis
et al., 1984). It has also been demonstrated by Robinson et
al.(1982) that the RAV-O and RAV-i strains of avian leukosis
virus are almost indentical in DNA sequence, except for the
03 region which is highly divergent, however RAV-O appears
to be non-oncogenic in an avian host. In contrast, RAV-l
can induce B-cell lymphoma and erythroblastosis.(Fung et
al., 1982,1984).

The divergence observed between the 03 region of CSV
and SNV LTRs is intriguing in light of the fact that CSV and
SNV have different oncogenic capacities (R.L. Witter.
personal communication). SNV is approximately 3 times more
potent in inducing bursal lymphoma in chicken line
1515x71 than CSV and has greater than 3 times the
capacity to induce non-bural lymphomas(thymic

lymphosarcomas) in chicken line 6 Therefore, it is

3'
possible that the differences observed in the oncogenic
capacity of SNV and CSV is related to the variation observed

in the U3 region.

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LITERATURE CITED

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118

 

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119

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