UNITED STATES OF AMERICA
DEPARTMENT OF HEALTH AND HUMAN SERVICES
CBER-NCI-NICHD-NIP-NVPO
SIMIAN VIRUS 40 (SV40):
A POSSIBLE HUMAN POLYOMAVIRUS WORKSHOP
MONDAY, 27 JANUARY, 1997
Morning Session
The Workshop took place in the Natcher
Auditorium, National Institutes of Health, Bethesda,
Maryland, at 8:30 a.m., Kathryn C. Zoon, Director,
CBER, presiding.
PRESENT:
KATHRYN C. ZOON, M.D. DIRECTOR, CBER
ROB BRIEMAN CO-CHAIR
MIKE FRIED CO-CHAIR
RUTH KIRSCHSTEIN CO-CHAIR
DIXIE SNIDER CO-CHAIR
BONNIE D. BROCK, V.M.D. SPEAKER
JANET BUTEL, Ph.D. SPEAKER
MICHELE CARBONE, M.D., Ph.D. SPEAKER
KRISTINA DOERRIES SPEAKER
ELLEN FANNING SPEAKER
RICHARD FRISQUE, Ph.D. SPEAKER
ROBERT L. GARCEA, M.D. SPEAKER
ALLEN GIBBS SPEAKER
MAURICE R. HILLEMAN, Ph.D. SPEAKER
MICHAEL J. IMPERIALE SPEAKER
KAMEL KHALILI SPEAKER
ANDREW LEWIS, M.D. SPEAKER
MARIA C. MONACO SPEAKER
LUCIANO MUTTI SPEAKER
FRANK O'NEILL, Ph.D. SPEAKER
PATRICK OLIN SPEAKER
DAVID SANGAR SPEAKER
KEERTI V. SHAH SPEAKER
HOWARD STRICKLER SPEAKER
MAURO TOGNON, Ph.D. SPEAKER
JIM C. WILLIAMS, Ph.D. SPEAKER
JOHN LEDNICKY, Ph.D. PANELIST
ALSO PRESENT:
DR. GALATEAU-SALLE
HARVEY PASS
ETHEL de VILLERS
ROBIN WEISS
CONTENTS
Introduction and Welcome by Dr. Zoon
SESSION 1 Presentations:
Dr. Fanning
Dr. Shah
Dr. Garcea
Dr. Butel
Dr. Carbone
Dr. Gibbs
Dr. Mutti
Dr. Giordano
Dr. Tognon
Dr. Shah
SESSION 2 Presentations
Dr. Dorries
Dr. Imperiale
Dr. Khalili
Dr. Frisque
Dr. Monaco
LUNCHEON RECESS
Afternoon Session
Audience Participation
Presentation by Dr. Lednicky
Panel Discussion
SESSION 3 Presentations
Dr. Hilleman
Dr. O'Neill
Dr. Lewis
Dr. Brock
Dr. Williams
Dr. Sangar
Dr. Olin
Dr. Strickler
PROCEEDINGS
8:35 a.m.
DIRECTOR ZOON: On behalf of sponsor's
agencies today, which include the National Institute
of Child Health and Human Development at NIH, the
Division of Cancer Epidemiology and Genetics at
NCINIH, the National Center for Infectious Diseases
and National Immunization Programs at CDC, the
National Vaccine Program Office, and the Center for
Biologics, Evaluation, and Research of FDA, I'd like
to welcome you to this workshop on SV40.
We, the sponsors of this workshop are
pleased that so many of the national and international
scientific community have come to discuss this very
important topic. The workshop was prompted by recent
reports demonstrating the presence of SV40 viral
sequences in tissue, including certain rare, human
tumors, and the fact that SV40 was an unsuspected
contaminant in early polio and adenovirus vaccines.
The potential connection between SV40 and
these various tumors is confounded by the finding of
SV40 sequences in individuals who are too young to
have received the SV40-containing vaccines. As a
result, we must ask whether SV40 was present in the
human population and if so, whether SV40 was present
in the human population prior to polio vaccines.
Moreover, the role of the human polyomaviruses such as BK and JC, which are closely related
to SV40, need to be explored. Therefore, the purpose
of this workshop is twofold.
The first is to consider the possibility
that SV40 is an infectious agent that is endemic in
the human population; and second, is to stimulate the
effort required to determine if SV40 is a causative
agent in human disease.
We look forward to discussions, both formal
and informal, over the next two days, that will better
define these scientific issues. Furthermore, we hope
that these discussions will lead to important new
research collaborations.
Finally, at the outset I would like to
acknowledge the enormous effort by those individuals
who organized this conference, including Drs. Strickler, Levine, Egan, and particularly, Dr. Lewis.
Before I turn the meeting over to the Chair
of the first session, Dr. Ruth Kirschstein, there's
several housekeeping issues I'd like to go through.
Lunch will be available upstairs at the cafeteria.
There will be coffee breaks served in the foyer down
here. Buses will return participants to the Marriott
Hotel.
Transcripts and videotapes of the workshop
will be available and how to obtain them is presented
in your registration package. In the context of that
I would like to ask those of you who come to the
microphone to please identify yourselves so we will
have a record of it.
The registration package also contains a
comprehensive bibliography of papers relevant to the
topics being discussed at this meeting. If participants know of additional materials, please send them
to Dr. Lewis and we will see to it that they will be
disseminated to the registrants.
Finally, we welcome the media coverage of
this meeting but we ask that in the spirit of facilitating the scientific discussions that are so vital to
the success of this meeting, that the media refrain
from questioning the participants during the meeting.
There will be a press-availability session
at the end of the meeting in conference room B, and
representatives from each of the sponsoring agencies
will be available. Other workshop participants are
also welcome.
With that, I'd like to now turn the meeting
over to Dr. Ruth Kirschstein.
CHAIRMAN KIRSCHSTEIN: Good morning. I want
to add my welcome on behalf of NIH as a whole. We're
pleased to be helping to sponsor this conference.
This conference is a little bit of a
nostalgia trip for me since I started working in this
field many, many, many years ago. And indeed, I was
scheduled to Chair a different session than this one.
For reasons of scheduling, I had to change that, and
when I looked at the program, it is perhaps even more
appropriate that I chair this session since some of
the talks relate to work that I did over 30 years ago.
With that, I'd like to call on our first
speaker, Ellen Fanning, who is in the Department of
Molecular Biology at Vanderbilt University, who will
give us a brief review of SV40 biology, and an
overview of the organization and expression of the
SV40 genome.
Dr. Fanning.
Return to Table of Contents
DR. FANNING: Thank you very much. When I
got a call from Andy Lewis asking me to review the
biology at the beginning of this meeting -- of SV40 --
in 20 minutes, I felt a little bit like Sarge in the
Beetle Bailey cartoon. He said, you've been working
on SV40 for 20 years; you know a lot about it. I says
yes, but I don't know all about it, and 20 minutes?
Andy persisted, however, so it's of course a little
bit daunting.
SV40 has been studied from the beginning of
its first reports in 1960 by Sweet and Hilleman, by
some of the brightest minds in science. This virus
provided a model system to study cell transformation
and growth control, eukaryotic gene expression and DNA
replication, chromatin structure.
It's led to the discovery of enhancers and
promoters -- many of the factors that bind to them --
to the discovery of RNA processing and eukaryotes to
signals for nuclear protein transport and to the first
reports of the tumor suppressor, p53. SV40 has taught
us a lot about eukaryotic cell biology.
So what I'm going to try to do in the next
20 minutes is to start at the beginning, to mention a
few of the milestones along the path in learning about
this virus, and then over the course of the next two
days, my colleagues will provide you with some data on
the questions that are at hand and to be discussed in
this meeting.
As was already mentioned, SV40 was a
byproduct of the early polio vaccines. The virus was
discovered in polio vaccines that had been produced in
monkey cells in culture. The virus was named after
the cytopathic effects that it produced in infected
monkey cells which became highly vacuolated, hence the
name. The SV40 standing for simian virus, or vacuolating virus number 40.
The virus particle is relatively simple.
It's small, it's made up of 72 capsomeres that contain
three different viral proteins. It's just slightly
larger than ribosomal subunits. Inside the virus
there's a mini-chromosome of 5,243 base pairs, which
is complexed with cellular nucleosones made up of
cellular histones.
The mini-chromosome as shown up here, it's
a very compact structure. When it's prepared for
electronmicroscopy sometimes it opens up like this so
that you can see the typical beads on a string. These
over here are intermediates in viral packaging.
SV40 normally infects permissive cells --
that was how it was discovered -- such as this little
-- according to this little scheme which is typical of
the way that it infects CV1 monkey cells in culture.
The virus particle enters the cell, progresses into
the nucleus. The viral mini-chromosome is set free in
the nucleus, it's transcribed by cellular transcription factors and enzymes to produce the so-called
early messenger RNAs.
One of the products, protein products,
that's generated from this early messenger RNA is
called T-antigen. This is a short abbreviation for
tumor antigen. This protein was discovered by virtue
of the fact that very early on, it was established
that SV40 could cause tumors in rodents and that these
rodents produced antibodies against a new antigen that
was not found in the virus particle. It was called
tumor antigen.
This protein, T-antigen, as it's now known,
is found in infected cells as well as in these tumor
cells. This protein migrates back into the nucleus
where it carries out the next stage of the productive
infection, initiating viral DNA replication and
stimulating late transcription.
The late messenger RNAs encode the capsid
proteins which again migrate back into the nucleus,
assemble around the mini-chromosomes to produce the
new virus particles.
Not all cells are permissive for SV40. Most
cells in fact, are semi-permissive like this species
here, or non-permissive like the rodent shown down
here in the grass. The infection in non-permissive
cells is depicted here in a slide borrowed from Arnie
Levine's book, Viruses. It stops after the transcription of the early viral genes.
The virus particle comes into the cell, the
early messenger RNAs are made, the T-antigen is
synthesized, it migrates into the nucleus, and
normally the infection stops there. However, infrequently, the viral genome can become integrated in the
host DNA in such a way that the T-antigen continues to
be expressed on the other early genes as well. This
can lead to cell transformation.
All right. So we have seen that somehow the
virus infection does not progress beyond the very
early phase. Viral DNA replication does not take
place and late gene products are not synthesized.
The year 1978 was a milestone year in SV40
research. One of the important advances that was made
in this year was that the first reports of the
sequence of the SV40 genome appeared by Walter Feur's
lab, and independently from Sherman Weisman's lab.
You can notice several things about the SV40
genome here. There are two sets of genes. The early
genes are here; the late genes are over here; one
transcribed on the Watson strand; the other transcribed on the Crick strand.
You can also note that the genes are
overlapping. That is, the early genes contain several
protein products, there's a large T-antigen, the small
T-antigen -- and not depicted on this slide is another
early viral gene product called the 17KT antigen which
was recently identified.
The late genes are also overlapping. The
three capsid proteins: VP1, VP2, and VP3. In between
these two sets of genes is a control region which is
depicted in more detail on this slide down at the
bottom, here.
Here we have the early messenger RNA being
transcribed this way, the late messenger being
transcribed this way, the alternative splice products
of these two sets of transcripts lead to the different
viral gene products.
If we look at these control sequences in
more detail we see two prominent binding sites here
for SV40 T-antigen. Now, as the concentration of T-antigen increases in the cell, T-antigen binds to
these sites specifically. It down-regulates the early
transcription through this binding event, and initiates viral DNA replication through this second binding
event.
The sequences that control transcription are
located here. There's a so-called tatabox promoter,
and you'll hear more about these copies of the 72 base
repeats which are called enhancers. All of these are
required for transcription.
Now, as viral DNA replication takes place
and T-antigen changes the activity of the cellular
transcription factors, the late genes are turned by
indirect mechanisms.
So if you're starting to get the idea that
T-antigen is a rather complex protein, you've got the
right idea. T-antigen is perhaps the most multi-functional protein that's ever been discovered.
Notice all of the arms here. T-antigen is something
that we still don't know everything about. Notice the
mysterious spatial expression here.
We do know quite a bit about it, however.
The strategies that the T-antigen follows in directing
the viral infection in permissive cells is depicted
here.
First of all, the T-antigen must prepare the
cell to support the viral infection. It does this by
kicking a quiescent, differentiated, resting cell back
into the cell cycle and forcing it into the S-phase.
It needs this in order to replicate its own viral DNA
which is dependent on cellular replication enzymes.
Having done that, T-antigen then initiates
replication of the viral DNA, recruits the cellular
proteins to replicate it, and through the indirect
mechanisms that I mentioned earlier, leads to stimulation of late transcription -- viral transcription.
T-antigen somehow then senses that it should
not initiate DNA replication anymore, but allow the
viral genomes to be packaged into new virus particles.
In other words, the T-antigen function changes with
time after infection.
All right. So now, how does T-antigen do
all of this? Generations of graduate students and
post-docs have mutagenized the T-antigen gene, and
this is the simplified version of some of what they've
found.
The protein encodes 708 amino acids; it's
sensitive to proteases at the sites marked by the
asterisks, which tells you that the protein is folded
up in different folding domains. These domains tend
to correlate with functional properties of the T-antigen molecule. For example, this domain is
responsible for specific binding to those two sequences in the viral control region.
T-antigen has a number of other intrinsic,
biochemical activities. It binds to ATP and hydrolyzes ATP in a DNA-dependant manner using sequences
located in the carboxyl terminus of the protein. It
uses both of these domains to encode a DNA helocase
that was first recognized in 1986 in Rolf Knipper's
lab.
It needs all of these activities in order to
replicate viral DNA. Now in addition to these
intrinsic biochemical activities, T-antigen also
interacts with a large variety of cellular proteins.
Some of the proteins that it interacts with are
depicted here.
The DNA preliminaries alpha primase,
interacts with T-antigen at two independent sites
diagramed here. T-antigen interacts with nuclear
location protein transport machinery located at this
region here, designated NLS. T-antigen also interacts
with tumor suppressor proteins such as Rb and the
other members of the pocket protein family.
T-antigen also interacts with p53, and
actually there are two independent regions that are
involved in binding p53: one here and one here, as
shown in Judy Tevethia's lab. There's also a sequence
down here which was not very well understood, which
helps SV40 determine what type of monkey cells it can
replicate in.
Now, T-antigen's phosphoprotein, the sites
have been mapped to serienes and threonines and two
clusters in the amino terminus and the carboxyl
terminus.
All right, so having said all that, let's
try and look at how T-antigen carries out this
strategy. I'm going to discuss first -- because we
know most about it -- how T-antigen directs the
replication of viral DNA, very briefly; and then say
a few words about how T-antigen prepares the cell to
support the viral infection.
All right. More than ten years ago Tom
Kelly's lab developed a system which would replicate
SV40 DNA in a test tube. This system was dependent
only on one viral protein of course, the SV40 T-antigen, as well as ten cellular proteins that have
been defined and studied in some detail in Bruce
Stillman's, Jerry Hurwitz's, and Tom Kelly's lab.
T-antigen's functions in this system are
threefold, basically. First of all, T-antigen binds
to the viral origin of replication and assembles there
as a multimer. Having done that, it proceeds to
unwind the two strands of the parental DNA so that
they're available to be replicated by the cellular
proteins.
You can see here that a mutant T-antigen --
this is the wild type up here -- this mutant T-antigen
is stuck at the origin and cannot proceed further to
these bidirectional unwinding of the parental DNA.
The third function that T-antigen carries
out in the viral replication phase of the infection is
that it interacts with key cellular proteins involved
in replication -- in getting replication started --
such as DNA preliminaries alpha primase.
Its DNA preliminaries alpha primase is the
molecule which is responsible for determining the host
specificity of viral replication at least, in a cell-free system, as first shown by Jerry Hurwitz's lab.
And in fact, as it's turned out in studies that were
carried out in my lab, it's only one of these subunits
of DNA preliminaries alpha primase which is sufficient
to determine whether or not SV40 DNA can be replicated
by this preliminaries alpha primase.
What we did was to generate recombinant
enzymes, human or mouse enzymes, or rehybrid enzymes
which contain only one subunit from mouse or one
subunit from human. And using this system what we
were able to find was that only a single subunit, the
large subunit of DNA preliminaries alpha primase must
be from humans in order to allow SV40 replication. If
that subunit's from mouse cells, SV40 DNA cannot
replicate in the test tube.
Replication is also controlled by the
phosphorylation state of T-antigen. If we look at the
form of T-antigen that's most common in productively-infected cells or in transformed cells for that
matter, it's a highly phosphorylated form of T-antigen; in particular at two key seriene and one
threonine residues.
This form of T-antigen is not able to
replicate viral DNA, although it represents the bulk
of the protein in the infected cell. The form of T-antigen that's able to replicate SV40 DNA is an under-phosphorylated form which lacks phosphorylation at
these two key seriene residues. This is a minor form
in infected cells, but fortunately for biochemists
like us who want to study it, it's the major form
that's produced in recombinant baculovirus infected
insect cells.
The unphosphorylated protein has made any
cholase also inactive. So we have a lot of different
things going on. We have a multi-functional protein
whose activity is then being regulated more exactly by
its phosphorylation state.
I'd like to turn then to the early stage of
the infection when T-antigen is trying to prepare the
cell to support the viral infection. If you look here
you'll see a representation of a cell cycle in
eukaryotic cells. Most cells that T-antigen would
infect in an animal would be in a resting state.
And as soon as T-antigen concentration
builds up, presumably in this highly phosphorylated
form, it will have the effect of forcing the cell back
into the cell cycle, forcing it through the early G-1
phase of the cell cycle and into the S-phase.
It does this by circumventing some of the
signal transduction pathways that normally would
control cell growth. It does this through its
interactions with cellular growth control proteins
such as the Rb tumor suppressor protein, the p53 tumor
suppressor protein.
Also in 1978 it was first shown by Adolf
Gressman that T-antigen was sufficient to stimulate
cells to re-enter the cell cycle and progress into the
S-phase. And this experiment was reproduced in my
lab, shown here, either in secondary African Green
Monkey kidney cells or in CV-1 cultured cells.
The cells were serum-starved and then
treated with -- and were microinjected with SV40 DNA,
or treated with serum and then the kinetics of re-entry into the S-phase were followed. You can see
down here that T-antigen protein does this faster than
SV40 DNA.
There are a number of functions of T-antigen
besides binding to Rb and binding to p53 that are
involved in this growth stimulation functions. Each
of the mutations shown by these red bars will inactivate these growth stimulating functions but does not
affect the ability of T-antigen to replicate viral
DNA.
So it's possible to specifically knock out
these growth stimulating functions, and this is by no
means all of them. There's only four of them here.
They're interacting independently with cellular growth
control proteins.
To give you an idea of the importance of
these interactions, in the biological activity of T-antigen in stimulating cell growth and eventually
transforming the cells, bear in mind that not only
SV40 T-antigen interacts with these cellular growth
control proteins, but also other groups of viruses.
Adenoviruses and code early proteins that target
the same cellular proteins, and the human papillomaviruses -- which we know are risk factors in human
cancer -- are early proteins which target the same set
of -- at least some of the same set -- of cellular
growth control proteins.
So I'd like to stop there and hope that I've
prepared you to fit the biology, together with some of
the data that you're going to here over the course of
the next two days on SV40, as a possible human pathogen. Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr.
Fanning. Indeed, you have kept the time beautifully
and also prepared us for the next session of speakers.
Our next speaker will be Dr. Keerti Shah,
who is in the Department of Molecular Microbiology and
Immunology at Johns Hopkins, and will talk on SV40 as
an infectious agent in simians and humans. Dr. Shah.
Return to Table of Contents
DR. SHAH: This morning, while registering
for this meeting, I met a lot of people whom I had not
seen for 10 to 20 years and I said, we are all old-timers. One of them told me that I was the oldest of
the old-timers.
What I want to do is, I was going to review
some of the background for the infection of human
beings with simian virus 40, which was a contaminant
mainly in the inactivated Salk polio vaccine. And
what I was going to cover was the natural infection in
Rhesus monkeys in India who are the donors of the
kidneys in which the vaccines were made.
I'll describe a little bit of the experimental infections in these animals because that gives us
some insight into how the virus might be transmitted,
and then describe briefly the circumstances of human
exposure to SV40 and the early studies of finding out
if this virus was pathogenic for man or not.
And I had reviewed this topic in some detail
in 1976 -- and this citation is shown on the slide --
and almost everything I'm going to say is contained in
that review paper.
The three viruses which are very similar is:
one is the simian virus 40 of the Macacs, and the two
human viruses, BK viruses and JC virus, which are very
similar biology to the simian virus 40. The BCV and
JCV were identified in 1971; SV40 in 1960.
They're called polyomaviruses after the
polyomavirus of mice, which was the first virus of
this subfamily that was characterized. And there are
many polyomaviruses scattered in a large number of
species -- as shown there in rabbits and mice, in
parakeet, in cattle -- so that it is widely distributed. And each polyomavirus is very well-adapted to the
species in which it grows. So they're highly species-specific and no polyomaviruses is shared between two
different species.
The primary infections with these viruses
are almost completely harmless and after the virus
enters the body there is probably some multiplication
at the local site. Then there's a period of viremia
on the viruses in the blood and it reaches its target
organs by viremia.
The target organ in most instances is the
kidney. The viruses in their primary infection
produce viremia that reach the kidney. There is
probably some virus excretion in urine, and after that
the viruses remain latent in the kidneys indefinitely,
perhaps for the lifetime of the particular infected
species.
They are reactivated in times of immunological impairment and transplant patients, patients with
AIDS, are the ones in whom these viruses are found
very frequently. Viruses are found most frequently --
BK and the SV -- in the urines of these exposed
individuals.
This is the distribution of the Rhesus
monkey which provided the kidneys in which the
collections were made. And it's a Macacus species
which lives in north India. And we have done some
work on the infection in these Rhesus monkeys.
In south India there's another Macacus
species, a bonnet Macacus, which is not naturally
infected with SV40. But the Rhesus Macacus, whose
picture you see -- here there is a female with a baby
-- this is an angry male, and he looks as if he might
transmit something more than SV40. Actually in India,
very often these monkeys will bite individuals who are
nearby.
And many of these Rhesus live in ecological
contact with human beings as shown here in this temple
in Nepal, where they are there in large numbers. One
of the questions which I looked at at the time, was
whether the virus from the Rhesus monkey was transmitted to people naturally, and if this were to happen it
would occur in a country like India. And the first
NIH grant that I obtained was to study if simian virus
40 was responsible for any human cancers in India.
Only some of the Macacus species are
infected with SV40, and in the Rhesus Macacus only
about 20 percent of juvenile monkeys, and perhaps all
of the adult monkeys, have antibodies to the virus.
So it is not widely prevalent.
In some situations, as in a colony that was
established in the island of Cayo Santiago off Puerto
Rico, where the Rhesus monkeys were brought there in
1938, they came to the island with SV40 infections,
but then it was eventually lost -- the SV40 infection
was lost from this Rhesus colony. So this can occur.
Although the infection is not widely
prevalent in young Rhesus, when they are brought
together and caged together as they were in India
prior to their transport to the United States, and
then in the U.S. before they were used for vaccine
production, the antibody prevalence reaches practically 100 percent, because there is a great deal of
transmission from infected animals to non-infected
animals.
If you infect the Rhesus experimentally, it
is infected extremely efficiently whether the virus is
given subcutaneously or -- orally this virus was
introduced into the stomach of the Rhesus monkey, or
by the intranasal route. In all instances they have
a period of viremia, generally in the first week that
this virus is in the blood.
The period of virulia, virus in the urine
which is seen two to six weeks post-inoculation, in
this particular experiment the virus was not recovered
from rectal swabs and throats swabs. And then all the
animals, no matter how they were infected, they
developed very high titers of antibodies to --
neutralizing antibodies to the viruses, and they also
developed antibodies to the T-antigen that Dr. Fanning
described in the earlier presentation.
There are many other studies done at the
time simply to see where the virus is. And in a
number of studies in the African Green Monkeys it was
shown that the virus is excreted in the urine, it is
latent in the kidney, and there may be a lower-level
shedding of the virus during infection. In African
Green Monkey the virus was recovered sometimes from
throat swabs and stools.
SV40 does not produce much or less in the
Rhesus Macacus. It is extremely rare that it would
produce any less in the Rhesus Macacus. No tumors,
benign or malignant, have been ascribed to SV40, any
tumors in the Rhesus Macacus.
But just as JC virus and BK virus will
produce human disease in immunosuppressed people, so
does simian virus 40 produce disease in Rhesus
Macacus, especially when they're immunosuppressed.
When monkeys that have the immunodeficiency virus in
them, the simian virus 40 will produce an illness
which resembles PML, progressive multifocal leukenepalopathy, which is a degenerative disease of the
nervous system, demyelinating disease.
It also sometimes produces renal pathology,
renal tubular necrosis, which is very similar to that
produced rarely by BK virus. So with all of these
viruses, most of the illnesses occur only in immunosuppressed populations.
Now, the factors that determine how much
virus will be in the vaccines are listed here. First,
the source of cells that are used. Now, many of the
vaccines were produced in Rhesus cells, but some were
produced in cynomolgus cells, which is another Macacus
species.
The Cynomolgus Monkey is not naturally
infected with SV40. So if the cells were of Rhesus
origin, there's a greater chance of that being
contaminated than if it was cynomolgus cells.
The type of culture of the cells are grown
in a monolayer culture. They expressed simian virus
40 and replicated simian virus 40 very readily,
whereas in some instances the vaccines were made in
what are called the Maitland cultures, where the cells
are not in monolayer form, but they are in the form of
minced kidney tissues. This Maitland-type of culture
did not support the replication of SV40 as well as the
monolayer cells.
Then in many instances the kidneys pooled.
The number of studies then would show that if the
vaccine was made in a single -- all the cells derived
from a single animal, it has a low chance of being
contaminated with SV40, but if you pooled the kidneys,
then any one infected kidney would contaminate all the
rest, and then you have a much higher chance of
getting contaminated vaccine.
One of the big, major factor was -- especially in terms of live SV40 and only matter of
importance is live SV40, not inactivate SV40 --
depended upon whether the vaccines were live vaccines
or inactivated vaccines. In the live vaccines such as
the oral Sabin vaccine -- which are not inactivated --
the SV40 remained in high titer; whereas in the Salk
vaccine the formalin that was used to inactivate the
polio virus, also inactivates SV40 to a large extent.
So people would get, in the contaminated
vaccines, either live SV40 along with a good bit of
inactivated SV40, and they would get smaller amounts
of SV40 in the Salk vaccines than they would get in
the Sabin vaccine.
And very probably, although we are not
completely sure about this from the data that we had
available to us in 1976, only a proportion of the Salk
vaccines had contamination with SV40 because a large
proportion of the vaccines were made in the Maitland-type culture which do not replicate SV40 very well.
The most important exposure is the third
that I've listed here: licensed inactivated polio
virus vaccine. But in 1955 and 1961 the vaccine was
contaminated -- some lots were contaminated. As we
said before, being an inactivated vaccine it would
have low amount of live SV40, but 98 million people
had received the vaccine by 1961.
The live polio vaccine which would have
large amounts of SV40 in the United States, only the
experimental lots contained live SV40. By the time
the live polio vaccine was licensed it was required to
be free of SV40. So in the U.S. people were not --
not a large number of people were exposed to SV40 in
the live vaccine.
The live RS vaccine which is the first line
there, it's given to very few people and it's important simply to see what happens to SV40 when it is
given intranasally. The second one, the inactivated
adenovirus vaccine -- I think it's an error on my part
here because those were live virus vaccines that were
given to military recruits.
If you give the SV40 intranasally to
individuals, the virus excretion occurs in throat
excretions to some extent. There's a very low level
antibody response. With oral vaccine there is almost
no antibody response and the virus is recovered very
infrequently, suggesting that the infection is very
transient. There is no information with respect to
the subcutaneous vaccine, how often it is -- if the
virus is disseminated from people who are infected by
subcutaneous vaccine.
And I'll pass this over just to show, the
people who are most likely infected with SV40 vaccine,
the year of birth, 1941, '61 -- people born between
1941 and '61 -- have a high probability of being
infected. Those were born up to 1963 and later, those
are very small probability of being infected.
The are a number of studies -- this last
slide -- the number of studies done in the United
States to see if the virus had bettered in a city of
poor people -- and I have just listed them. I think
we may have a chance to discuss many of them in the
course of the two days.
But it is summarized at the bottom that
while the studies did not reveal any ill effect of
SV40, they did not have enough numbers, there was not
sufficient period of follow up. The most susceptible
would be infants who were infected in early life; not
many of them could be followed. So while the data did
not show any pathogenicity for SV40, there were -- all
of the studies had their limitations.
Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Shah.
We will continue now with SV40-like sequences in
choroid plexus tumors and ependymomas by Dr. Robert
Garcea who's at Children's Hospital in Denver,
Colorado. Dr. Garcea.
Return to Table of Contents
DR. GARCEA: Thank you. What I would like
to present today is the data from a New England
Journal paper in 1992. And I'd like to present it in
the context of exactly how the experiments were
carried out. And so it's an historical, sociological
presentation rather than a -- although I will give you
all the facts, I think you'll see -- I want to give
you the flavor of how we proceeded with these experiments.
My laboratory at that time was located at
the Dana Farber Cancer Institute, and our laboratory
is primarily interested in the structural biology of
papomaviruses and really not in translational work,
although I am a Pediatric Oncologist. In 1989 John
Bergsagel came to the laboratory to look for a post-doctoral fellowship and wanted to pursue whether there
was any link between polyomaviruses and human, in
particular, pediatric malignancies.
I didn't think any existed, but I was struck
-- and this paper had always stuck in my mind -- this
is from 1984, Brinster and Pometer, who described --
I think it was the second transgenic animal ever made
with the SV40 large T-antigen driven under its own
enhancer/promoter.
These animals developed very peculiar
tumors, all of them in the coriplexus of the animals.
And so I thought that this was quite a striking result
and therefore I told John to go learn PCR and examine
all the coriplexus tumors he could get his hands on at
the Children's Hospital in Boston, the presence of
polyoma-like sequences.
Now, coriplexus tumors are very, very rare
tumors. They're only three percent of all of childhood malignant intercranial tumors, and we estimate
that there's about 30 to 60 cases per year in the
United States. Very interestingly though, all of
these tumors are -- the majority of them occur within
the first year of life, and they are in the differential diagnosis of hydrocephalus in utero.
A related tumor that we decided to study
along with coriplexus tumors, because of the animal
data of tumorigenicity of SV40, and because the
ependymal cells are also a lining cell of the ventricular cavity of the brain, were ependymomas. Ependymomas are a little bit more common but again, still
strikingly rare.
So John went to the pathology department at
Children's Hospital, to the archival specimens. And
he decided to -- what I thought in my wildest hopes
was that maybe he would find BK or JC in these tumors
and in my less-than-wildest hopes I knew that we would
probably get PCR working in the laboratory.
John was to do a computer-assisted search of
the polyomaviruses and align the sequences and find
out where there were very close homologies between the
viral genomes to make PCR primers. And not surprisingly, what he came up with was a region just after
the splice site in large T-antigen, which is the Rb
binding site. This site is very highly conserved
among most of the polyomaviruses. And so John made
primers in this region.
And the strategy that we initially employed
was to -- these regions are identical for the forward
and reverse primers, for both the BK and the JC
viruses. I think we were trying to economize -- at
that time it was expensive to buy primers, and so I
think we were trying to be a little bit economical
here.
And so these primers at the end would
amplify either BK or JC, and the strategy therefore,
was to amplify the specimen and then probe with an
internal probe that was unique for either BK or JC.
And so what we did, John went to the
archives, scraped slides off -- just in passing and I
think we'll get back to this -- is that at least at
Boston Children's, before 1976 most of the brain
samples were fixed in Bouin's solution which has I
think, picric acid in it, and we found that the DNA
was highly fragmented. So most of our specimens came
subsequent to 1977.
So what John did was, for most of these
specimens which were on slides and from paraffin-embedded material, he precipitated the DNA. He then
analyzed with globin oligos to see whether the DNA was
intact, then he PCR'd with the BK and JC oligos, did
a southern blot, and he probed with a specific oligos
for either BK or JC.
And this is one of the original blots that
John got. It's what we call low stringency. Actually, the whole PCR reaction was of low stringency and
that cost us some consternation. And the other
problem was that John, at that point being a physician, didn't quite understand the necessity for
monitoring temperature in southern blots.
But what we found in some of the first
samples was that there was a -- for example, we could
get amplification in many samples of BK, for BK and
even of JC, but many, many times these bands were
hybridizing in both situations to both the BK and JC
probe. And just for interest sake, this went on for
quite a number of months, about three months, before
I became a little frustrated and I told John to stop
this and just sequence one of them so we could figure
out what was going on.
And this was when the first matrices
surprise happened. And that is, when John sequenced
two of these bands he found that they were neither JC
or BK but SV40. And there's a very characteristic
nine base pair change in this region that he sequenced
between those viruses.
And when we went back and looked at the
primers, indeed the primers that we had -- since this
region was so conserved, would have amplified SV40
under our conditions. And therefore, we went back and
we used those primers with now a probe that was more
specific for an internal region of SV40, and we used
higher stringency conditions in the southern blot at
52 degrees -- and John had realized the importance of
temperature by that time I think.
And most of our samples then, were amplified
and blotted with this SV40 specific probe, although
still -- and I think this is a topic for further
discussion -- some had also BK or JC sequences in
them, which we could not figure out why, but we were
still now stuck with the fact that we had sequenced
SV40 and that many of our samples were amplified and
being identified with a probe for SV40.
John then went back and made new primers.
The original primers were these right here after the
splice site, PY forward and PY reverse with the probe
in the middle.
John made now, another set of primers that
amplified across the intervening sequence, SV forward-2 and SV reverse, and also another set of primers that
amplified a very small, 107 I think, base pair region
that lie just outside the previous amplification.
So when John -- we were concerned here that
maybe there was some CDNA contamination in the
laboratory. Contamination of course, is going to be
a major issue in our discussion.
And so for example, he took some of the
tumor DNA -- now this is, we have found that -- we
were lucky in that our first set of primers amplified
about 170 to 180 base pair fragment, because from the
paraffin-embedded material it was very difficult to
get long amplifications, and so when we got some fresh
tumor specimens it was possible to do these longer
amplifications.
And this for example, is an amplification
with those long primers, with those primers that
amplified across the intervening sequence, and it
generated a band which could be specifically cut with
an enzyme that would only cut SV40 and not BK or JC.
So this is, besides sequencing, another way we
determine that some of our samples were probably SV40.
At that time when we got this result, I
wanted to verify the data with at least some immunohistochemistry. And so I called up Janet Butel and
along with Milton Finegold, the pathologist at Texas
Children's Hospital, we did immunohistochemical
studies of these tumors -- and this is an example of
immunohistochemistry with a large T-antigen of an
ependymoma, and you can see that there's some very
darkly-staining nuclei in this field, whereas the
others don't stain.
And this for example, is a coriplexus tumor
which is hard to see, but there are very darkly-staining nuclei here. And I think we'll get to the
problems with the immunohistochemical cross-reactivity
of different T-antigens, but this was the data that
Janet and Milton obtained.
We went back then, with the set of primers
that amplified that short region, and re-analyzed all
the specimens that we had in hand. And this is the
table of all the tumors we had at that time. And so
we had 20 coriplexus tumors, ten of which amplified
with those short, 107 base pair amplifying primers,
ten of 11 ependymomas amplified.
We put neuroblastomas -- we analyzed those
because transgenic animals with JC or BK also give
rise to neuroblastomas and so we were very curious in
that, also because it was a pediatric malignancy, but
we didn't find any sequences in those tumors.
These are other controls. We studied normal
brains, seven normal brains. We had a problem in the
beginning because it was difficult to find brains that
were not pickled for a month, and seven specimens were
capable of globin amplification.
One was positive for SV40, which we thought
was unusual. This was a 28-week, premature infant
that died shortly after birth. The neuroblastomas I
talked about, and we also examined normal blood.
Fifty were studied at time and they were all negative.
Subsequently we've studied several hundred and they've
been all negative.
So at that time in this paper, we concluded
that a segment of DNA corresponding to SV40 T-antigen
was amplified from these tumors, and we concluded that
perhaps SV40 or a related virus -- at that time we
didn't know whether it was intact SV40 since we'd only
amplified from one part of the viral genome, and maybe
some hybrid virus was involved here.
And the tumors that we were finding this in
were similar to those tumors that were induced in
experimental animals by SV40. And so the questions
that we had at that time were: is this a hybrid virus;
is there full length copies of DNA in these tumors or
are these episomal or integrated; where else are these
sequences found? And of course, cause and effect is
always a problem here, and I think that that's an
issue for discussion.
There were two very interesting patients in
our first study. One was a patient with Aicardi
Syndrome, and this is a very rare syndrome that has
agenesis of the corpus callosum in it. There's
associated other abnormalities and rarely there's
coriplexus tumors in these patients.
But perhaps more interesting, one of our
coriplexus patients was a member of a Li-Fraumeni
family, and this of course, most are aware is, the
proband is a young patient usually with a sarcoma, and
there's two 1st-degree relatives with cancer --
oftentimes breast cancer or osteosarcomas.
At that time, there were two kindreds
identified by Judy Garber and Fred Li, with coriplexus
tumors as part of the phenotype, and subsequently
several other kindreds have been found having coriplexes tumors as part of this syndrome. About half of
these individuals will have some mutations in p53, and
the other half I think, are still up in the air.
Because of the occurrence of one Li-Fraumeni
patient in our initial series, and because David
Malkin and Steve Friend were across town at Mass
General, I asked David for all of the Li-Fraumeni DNA
specimens he could give me in a blinded fashion. And
so we analyzed 163 Li-Fraumeni-related specimens --
and I'll talk about how they were related in a minute
-- because we were interested in trying to follow up
this one patient.
We'd also done some other controls. We
looked at Wilms tumor because it's a kidney tumor and
these virus seem to be trophic for kidneys. Subsequently, we looked at lung cancer because afterwards
as you'll hear, Michele Carbone had found the virus in
mesotheliomas and we were interested in other lung-related tumors.
So for the Li-Fraumeni specimens we analyzed
163 of these, and only 19 were positive. Now we were
amplifying for the large fragment -- that fragment
that amplifies across the intervening sequence. And
I think what I was struck most about this analysis --
and so my first surprise was finding the SV40 sequence, and my second surprise was in decoding the
specimens and finding that five were from osteosarcomas, four were from the blood from osteosarcoma
patients, and one was from a lymphocyte cell line made
from an osteosarcoma patient.
Now, all of these DNAs have been prepared by
David Malkin and Steve Friend. And so there was a
preponderance here of, half or over half of these in
a very large series were related to osteosarcomas.
And at that time I think I got my first call from
Michele Carbone telling me about the mesothelioma
data. And I told him about the osteosarcoma data and
he said, oh yes, we've seen that too.
And so at that point, that's when Michele
and I started to collaborate on the bone tumors which
he's going to tell you about. And we also told
immediately, Janet Butel, who's also got some data on
the osteosarcomas. So I'm not going to give any more
of that except as an introduction to their talks.
So this is a further breakdown of the Li-Fraumeni. There were three from unknown tumor types.
I would say in the 163 samples there were many, many
breast cancer specimens because this was part of the
Li-Fraumeni syndrome, and none of those were positive.
There were somatic sources, and this was
from blood or fibroblast cell lines that had been made
from these patients. But again, a number were related
to the osteosarcomas.
So that in short, is the introduction to
Janet's and Michele's talks, I think. And I think Dr.
Butel and Dr. Carbone will give more details on the
subsequent analysis of these sequences in brain and
bone tumors.
And I would just like to point out my
collaborators at the Dana Farber when we were there,
was John Bergsagel as a post-doctoral Fellow who
really, from an M.D. coming into the lab, did a
spectacular job. And I have to say that we didn't
publish the New England Journal paper for at least a
year of repeating all of these things over and over
again from John, and he was a very meticulous person.
Wendy and Kristie Johnson worked up the Li-Fraumeni specimens in my laboratory. We had a very
nice collaboration with the Baylor group, with Janet,
Milton, and John, and at the University of Chicago,
now at Loyola with Michele Carbone.
And thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr.
Garcea. Before we go on to the next talk, if Dr.
Mutti is in the audience, I understand he wants to
talk to me, and if he'll come up here we'll try to
settle the problem before he's announced to speak.
Our next speaker is Dr. Janet Butel from
Baylor College of Medicine. She will talk to us on
evidence for the presence of authentic SV40 in human
brain and bone tumors. Dr. Butel.
Return to Table of Contents
DR. BUTEL: Thank you very much. I'm going
to pick up the story now from where Bob Garcea left
off. We thought about his finding of the SV40 DNA
fragment in human brain tumors and two major questions
emerged: did the DNA represent authentic SV40, or was
it a new virus or some recombinant virus; and if in
fact it really was SV40, were there human-specific
variants that we could identify?
So in the next 15 minutes I'm going to
present some evidence that suggests very strongly that
authentic SV40 is present in at least a few human
tumors. And since I'm going to go over several types
of data rather quickly, I wanted to just summarize
what the main points were going to be.
And that is, by PCR we've been able to
detect SV40 fragments from four different regions of
the viral genome, and sequencing showed that it's
really SV40 DNA. The regulatory region structures are
typical of a non-duplicated enhancer region that's
found in some natural isolets.
We detected variability in the C-terminal T-antigen gene sequences. We isolated infectious SV40
from one human brain tumor, and as Bob mentioned
briefly, we have some similar results with bone
tumors.
So Bob sent us blinded samples of DNA from
some of the brain tumors for a follow-up study. And
this map shows the SV40 genome and it shows the
positioning of different sets of primers that we use
for PCR analysis. Here was the Rb proximal region
that Bob described.
We looked at the regulatory region of SV40
down here at the end of the T-antigen gene, and then
a region in VP1, the structural protein of the virus.
Our idea was, that if separated regions from the viral
genome could be detected, then most likely the virus
that was present in the tumor was SV40.
Source controls for the regulatory region
studies. We used a series of plasmas that John
Lednicky had constructed previously, and these
contained one, two, or three copies of the 72 base
pair region; that's the enhancer region. Lab strains
usually have a duplication in this region and some
natural isolets from monkeys have had a single copy.
This is a gel showing the PCR results using
these primers from the regulatory region. A number of
these blinded brain tumor samples were positive, and
the size of the fragment that was generated was the
same size as the fragment containing a single 72 base
pair region.
When John sequenced those PCR products, in
fact the sequence was exactly SV40 from each of those,
and in fact, they did contain non-duplicated enhancers.
This shows the results of PCR assays using
the VP1-specific primers. The same tumors that had
been positive for the regulatory region were also
positive and generated a VP1 fragment. And the
sequencing of those PCR fragments showed that it was
an exact match with SV40.
We then examined the samples using the T-antigen primers. Now, from everything we know about
SV40 we would predict that the full length T-antigen
gene ought to be present if infectious virus were
present or if T-antigen were involved in tumor
development. And using the primers from the C-terminus of the T-antigen gene, the same tumors that
had been positive for the other parts of the viral
genome were positive and yielded products.
When the products were sequenced there were
some nucleotide changes that were detected. This is
compared to strain 776 here at the top. Each of these
slides represent something slightly different about
the sequence and of the five brain tumors that were
sequenced, each one yielded a slightly different T-antigen sequence.
And some of those nucleotide changes would
result in amino acid changes when you look at the
predicted amino acid sequence for T-antigen. And
that's what this slide shows. There were some
substitutions, there were some deletions, and there
were some insertions in what was found in the brain
tumors.
We tried to isolate virus from the brain
tumor samples and we did succeed in one case. The
tumor DNAs were lipofected into monkey kidney cells:
both TC7 and CV1. Only one of the samples, sample
number 12, produced CPE. It took a pretty long time
for the CPE to show up -- six weeks. The cells were
extracted and John cloned the DNA and we called this
virus isolet SVCPC.
The virus induces typical CPE vacuolization.
We're starting to characterize the virus. Renee
Steward in the lab has done growth curves comparing
our Baylor wild type strain of virus with the human
tumor isolate, SVCPC, in both monkey kidney cells and
in human cell lines. This happens to be a renal tumor
cell line that we got from the American Type Culture
collection.
And the point is that both the wild type
virus and the human isolet grow very well in both
monkey and human cells. So it's clear that SV40 can
grow well in human cells.
The next question is, were there changes in
other parts of the T-antigen gene among different
virus isolets? So Renee sequenced the entire early
region, not only of SVCPC but of several other isolets
including two old human isolets, SVMEN isolated by Dr.
Kreig in 1984 from a meningioma, and SV40PML isolated
in '72 by Drs. Weiner and Shah.
There were a few nucleotide changes that
were found but no huge differences, and bear in mind
we're comparing here both monkey and human isolets and
viruses that were recovered over a span of about 35
years.
Now, when the nucleic acid sequence was
converted to the amino acid sequence, it was really
remarkable to discover how conserved the T-antigen
protein is. There were no changes in the protein at
all among any of these isolets until we got down to
about the last 90 amino acids, and then we saw a
cluster of changes. So on this basis we refer to this
as the variable domain of T-antigen and this is the
region where we're seeing some variation in the tumor
associated sequences.
Renee has just about completed sequencing
the late region of SVCPC. This slide summarizes the
results from the coding regions. If there's a
vertical line below this horizontal bar that means
there's an amino acid change, and we didn't find any
amino acid changes in the agnoprotein, in VP2 or VP3,
and a single change in VP1. So it's clear that these
human isolets are typical SV40.
Now, when I heard from Bob Garcea and
Michele Carbone that they were finding SV40 DNA in
osteosarcomas, we wondered first of all, could we
confirm that on an independent set of samples, and
then secondly, perhaps it would be possible to
identify a distinct bone tumor associated virus
variant.
So my pathologist colleague, Milton Finegold, obtained for us ten osteosarcoma samples from
St. Jude's Hospital, and this slide just summarizes
some of the patient information. I want to point out
that all of these patients were born 1965 or later,
after the use of the contaminated vaccine was discontinued.
The samples were provided to us blinded,
they were extracted -- John Lednicky will discuss some
of the important steps in processing these samples and
analyzing these samples by PCR during the panel
discussion. But we used the same four sets of primers
to analyze the osteosarcomas that we had used in the
brain tumor study.
This shows the PCR results using the primers
for the regulatory region of SV40. Five of the ten
osteosarcoma samples were positive and that they
yielded a product. And when John sequenced those,
each contained a single 72 base pair region -- that
is, a non-duplicated enhancer -- and the sequence was
typical SV40. We also used primers specific for JC
and BK regulatory region and didn't pick up anything
in these osteosarcomas.
This just summarizes the VP1 results from
the osteos. The same five samples were positive and
when the products were sequenced, again, there was an
exact match with SV40, VP1.
The T-antigen gene was also present in the
same five samples. We got positive signals with both
sets of primers: those from the Rb proximal region
and then those from the C-terminus of the T-antigen
gene. I'm just showing the amino acid calculated
sequence.
The take-home lesson is that again, that
each of the osteosarcomas contained a slightly
different T-antigen sequence when we looked at the
variable domain of T-antigen. Three of the tumors
yielded new sequences that we hadn't seen before, and
in two cases, it was a known sequence.
So finally, I want to end by telling how we
addressed the question of whether this variable domain
at the end of the T-antigen gene is highly mutable --
meaning it changes rapidly over time -- or in fact, is
it stable and the variation that we were observing
would reflect stable strain differences.
So to do that we analyzed high and low
passages of two different strains of SV40 for which we
had a known history. And this study was possible
because I had frozen away in my freezer, some low
passage stocks that had been frozen down for more than
25 years. And I'm just going to show you the story
with VA4554.
This virus was received at Baylor in 1967.
It was passed a couple of times and a stock frozen
down in 1971. We reconstructed the history. We know
that Dr. Hilleman sent the virus to the Enders
laboratory in the early-60s.
Peter Tegtmeyher used this virus, manipulated it in all of his genetic studies. Then in the mid-70s he sent the virus to Judy Tevethia's lab where she
used it in her genetic studies. And then in the
early-80s Judy cloned the virus and sequenced it.
So John Lednicky went to this very low
passage stock of VA4554 that we had, he cloned out
isolets from that stock, and then compared the
sequence of these isolets with this sequence that Judy
had determined on this lineage of virus that had a
very different history.
John cloned out two types of viruses. One
had an archetypal regulatory region, one had a duplicated enhancer from the low passage stocks. The
sequence of each was exactly like the sequence that
Judy had determined for her virus.
And then finally, when we examined the
sequence at the C-terminus of the T-antigen gene, the
sequence was exactly what Judy had determined for her
virus. So our conclusion is that the sequence is
stable and we think that these variations reflect
viral strain differences.
So I've run out of time and in summary, this
just reiterates what I had said earlier was going to
be the take-home lesson. And these are the people who
have been involved in the work.
CHAIRMAN KIRSCHSTEIN: Thank you very much,
Dr. Butel. I want to repeat, if Dr. Mutti is here I
need to speak to him.
The next presentation will be by Dr. Michele
Carbone, who is at the Loyola Medical Center in
Illinois. Evidence for SV40-like DNA sequences in
human mesotheliomas and osteosarcomas. Dr. Carbone.
Return to Table of Contents
DR. CARBONE: Thanks. And first of all, I
would like to thank Dr. Lewis and Dr. Levine for
having invited me here. I did my post-doctoral
training with Dr. Lewis and I was hired by Dr. Levine
as a visiting associate in his lab, and he gave me the
possibility to start my career, so I'm particularly
grateful to the organizer of this meeting for what
they did for me.
Today I am going to talk about the evidence
that we have for the presence of SV40 sequences in
human mesotheliomas and in human osteosarcomas.
Tomorrow I will present our new data that suggests
that not only these sequences are present in the
tumors, but they may also in some instances, contribute to the transformed phenotype. And tomorrow we'll
also talk about, as before, oncogenicity in hamsters.
In order to start my -- this is SV40
viruses, small DNA tumor viruses. In our studies in
humans we are prompted by our findings in hamsters.
What we found when we injected SV40 intracardially
into animals, was that only particular tumor types
developed, specifically, mesotheliomas, osteosarcomas,
lymphomas, and on the -- sarcomas.
The reason that we injected the virus in the
heart was to expose more cell types to the virus and
to see whether every cell could be transformed by SV40
or only specific cells were transformed. Now, from
our tissue cultures -- not ours, but from tissue
culture experiments that other people did -- we know
that SV40 will infect more or less, every cell. Even
in humans it can easily infect karyotinocytes.
You can clearly see that the most common
cancer, at least those that developed in humans, never
developed in a hamster when you inject SV40. We have
never seen a carcinoma. I was particularly struck by
the fact that the mesotheliomas developed and so we
repeated the experiment injecting as before, into the
pleural space. And in that case, 100 percent of the
animals came down in tumors in three to six months.
Other investigators had found that when you
inject SV40 intracranially in animals, only ependymomas or ancyroid plexus tumors develop. So the
conclusion of this experiment is obviously, SV40 is a
virus that for some reason, even if it can enter
different cell types, will transform only particular
cell types. They must be more susceptible to transformation by this virus.
Again, I was particularly struck by this
tumor. This is how it looks histologically. This is
a mesothelioma. Mesotheliomas are a tumor with
incidences increasing inordinately. Today we have
2,000 or 3,000 cases in the United States. Actually,
I just heard from the -- that 4,000 are projected for
'97.
And this is a high number if you consider
that until 1950 or so most books of pathology denied
the existence of mesothelioma between they were so
rare in that many people so that they didn't exist at
all. So it's like going from zero to 4,000.
The reason for that is the use of asbestos.
At the beginning of the century asbestos was used
largely in all the western world, and it's obvious
that exposure to asbestos induced mesotheliomas after
approximately 20 to 50 years.
However, 20 to 50 percent of mesotheliomas
-- and that depends what study you look at -- are not
associated with asbestos exposure. Now, that's a big
number because obviously there is a large number of
mesotheliomas that are increasing from 1960 for which
we cannot account. So we ask it, could SV40 or a
related virus be related to the development of
mesotheliomas?
And this slide summarizes whether that time
was known as SV40 human pathogen. SV40 can transform
human cells in tissue culture. SV40 human transformed
cells induce tumors when injected into human volunteers. Millions of people were injected with SV40
contaminated adeno and polio vaccine and after 1963
vaccine should be SV43.
While I was trying to convince my -- at that
time I was supervising Dr. Levine's lab -- but I
couldn't convince them to look at these SV40 tumors
because obviously it was a very risky project. So
while I was struggling to convince somebody to work on
this with me, Dr. Garcea published in New England
Journal of Medicine that 60 percent of human ependymomas contained SV40-like sequences.
It's a study that was later confirmed by
other investigators, including a recent paper by
Martini, et al, in cancer research a couple of months
ago, and from the laboratory of Dr. Butel where Dr.
Lednicky isolated infectious SV40 from one of these
tumors indicating that at the least, in that case, the
SV40 life sequence was infected as we thought.
So eventually I was able to convince Dr.
Procopio that had done his post-doctoral training at
the NIH and was a tenured professor in Italy, to come
to the NIH and do this work together to see whether
there was any SV40-like in these mesotheliomas.
We used the same technical approach that had
been used by Dr. Bergsagel in his studies and the
reason is, as Dr. Garcea indicated before, that the Rb
binding domain should be there if the T-antigen is
doing something, because the Rb pocket binding domain
is that region of the antigen that binds Rb, p107, Rb2
or p130. So the cell are proteins that must be
inactivated by the antigen in order to receive
transformation, therefore, that would be the region
that you most likely would expect to find.
We started a great collaboration with Dr.
Harvey Pass, that at that time was the Chief of
Thoracic Oncology here at the National Cancer Institute. Harvey has an incredible collection of mesotheliomas and he candidly gave access to us to his
collection and also he worked very closely with us
during these experiments.
And this is a summary what we found; that
is, 29 of these 48 mesotheliomas that Harvey gave us
tested positive with primers that amplified the Rb
pocket binding domain of T-antigen. Sequence analysis
confirmed that those sequences, the type that amplified with the specific proper SV40, were in fact SV40,
and the arrow points to that unique region of SV40
that Dr. Garcea already talked about so I'm not going
to repeat it.
Immunoperoxidase staining indicated that
some mesotheliomas cells contained a nuclear antigen
that strongly reacted with the monoclonal antibody
against, as before, the T-antigen. So we were seeing
either as before, the T-antigen or something very
close to it.
And actually, if you look at that again you
can see that only the tumor cells stain and in fact,
that the reactive fibroblasts that are around the
tumor cells do not stain. And immuno-precipitation
studies from frozen tissue precipitate a 90 kilodalton
protein with the monoclonal antibody against the
antigen that reacted in western blot with the monoclonal antibody against the antigen.
And this was the conclusion of that paper,
we found SV40-like DNA sequences in 29 of 48 mesotheliomas stasis and demonstrated the antigen expression
in 11 of 14 specimens. The associated lung did not
contain SV40 sequences although they contained
asbestos.
We suggested that an SV40-like virus may act
independently or as a co-carcinogen with asbestos.
Moreover, the selective T-antigen expression by
mesotheliomas and not the surrounding pulmonary
barenchyma, may have diagnostic and therapeutic
implications.
At that time I gave the code to Bob Garcea
and from what we knew in animals, there was another
possibility for finding SV40 among the many human
cancers was obviously osteosarcoma. And the other
question was, are there other tumors in humans that
may contain SV40 sequences?
So we started a collaboration -- me, Bob,
the laboratory of Dr. Pass, and the laboratory of Dr.
Procopio in Italy -- and we studied 345 different
human samples including 159 bone tumors and sarcomas
for SV40-like sequences by PCR.
Only after all four laboratories completed
the analysis was the code identifying the origin of
the bone tumor specimen broken and the results
compared. And these are the results that we published
in Oncogene last summer.
Fifty-three of 159 bone tumors and sarcomas
-- that is exactly one-third -- tested positive. Of
the 186 known bone tumor sarcoma samples only one out
of one neurofibromatosis type, one was positive. And
the other 185 were all negative.
Ten samples, all from bone tumors, gave
conflicting results, meaning that at least one
reaction in one of the four laboratories was positive.
These samples were considered negative.
And these are some of the results that we
obtained. In this particular study we used a different set of primers that are the long primers that Bob
referred before. And they are indicated up there --
I don't have a stick to indicate them -- but you can
see a hoop at the top line. They are indicated by
SV42 and SV-rev.
They amplify the same RV pocket binding
domain that we used to amplify before that is indicated by the primers before SV-rev. But they also
amplify the intraregion of the antigen. And the
reason to include the intraregion was that while you
want to find SV40 T-antigen there so that's why you
put the Rb pocket binding domain there, it would have
been nice to find some mutations, because there is
always the question when you do PCR, people will ask,
are you sure that there's no contamination?
Well, this is what we found. This is the
574 page paper that you expect to amplify with this
parameters. And the last time in the right under the
H letter that indicates hamster, is a hamster osteosarcoma. So you can see that in addition to the 574
base pair band there are other bands with different
molecular weights.
Particularly prominent is the band around
300 or so base pair. When Dr. Rizzo, that is my
research associate, sequenced those bands that were
hybridizing with the probe specific for SV40, the
sequence indicated that that was SV40 T-antigen, but
that there were deletions within the intraregion. And
we never found deletion outside there; we never found
deletion in the Rb pocket binding domain.
For some samples we also tested the other
region of SV40 genome to see whether they were there,
and for example for those two samples indicated with
the numbers 103 and 105 that were positive for SV40-like sequences, in panel B we are amplifying the
capsid throat in PV1 and in panel C we're using two
sets of parameters that amplify the carboxyl terminal
domain of the antigen.
So for at least these samples a lot of the
SV40 genome was there. We also sequenced the regulatory region of SV40 and we found 272 base pair.
This slide points to an intriguing coincide
that comes out from these studies. And these are the
tumors associated with SV40 in humans. Ependymomas
are induced by SV40 hamsters and in humans contain
SV40-like sequences. The same is true for choroid
plexus tumors, the same is true for mesotheliomas, the
same is true for bone tumors, the same is true for
sarcomas.
The last one, true histiocytical lymphomas
that are induced by SV40 hamsters, are so rare in man
that many people doubt that they exist at all.
And this is the conclusion for, this talk at
least. The significance of SV40 and SV40-like
sequences in human tumors is presently unclear.
Specifically, it is unknown from what source these
sequences originate; whether these sequences contribute to tumor development. And again, I am going to
present some data suggesting that in some cases they
can, and Dr. Weiss has been very kind to offer me to
present this data at the beginning of this round table
tree tomorrow afternoon.
And whether these sequences can be used as
targets for designing new immunotherapeutic and
molecular approaches for the management of malignancies expressed in T-antigen. And while that can seem
futuristic or too much optimistic, I think that in
fact, it's a very exciting area.
This is one of the best immunoperoxidase
that I have, of course, but this is a human mesothelioma and you can see the staining of these mesothelioma
cells that clearly distinguish them from the surrounding non-malignant cell. Now obviously the presence of
a unique antigen on a tumor cell gives you, at least
in theory, the possibility to attack those cells, and
we currently are working on this hypothesis.
Thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr.
Carbone. Our next speaker is Dr. Allen Gibbs who will
present SV40 DNA sequences in mesotheliomas. Dr.
Gibbs is from Llandough Hospital the United Kingdom.
Return to Table of Contents
DR. GIBBS: First of all, thank you. I want
to thank the sponsors for inviting me to present the
work that has been conducted in Cardiff in this. I'd
just like to point out that the pronunciation of the
hospital is "thrandock" and not "thrandough", but I
will excuse the American pronunciation.
I'd also like to acknowledge Dr. Bharat
Jasani -- he was also at this meeting -- who really is
responsible for the actual methodological approach of
the study.
Why did we conduct this study? Well first
of all, we had seen the paper by Dr. Carbone on the
finding of SV40-like sequences in mesothelioma; that
my particular hospital has had a long association with
examining mesotheliomas and I have an archival store
of several thousand mesotheliomas. And these go back
to the days of Chris Wagner who worked in my hospital
and in fact, was the person who put the association
with asbestos and mesothelioma on the map in 1960.
And we were particular interested, really,
in looking at our archival material because a considerable proportion of that has been well worked up,
both diagnostically and from the point of view of
exposure data, and that includes a very detailed
mineral analysis on the lung tissues.
So we were interested in trying to exploit
this material, looking at the possible role of SV40
and asbestos in causation of mesothelioma. So this
was a, basically a pilot study to examine frozen and
archival, surgical and post-mortem mesothelioma
tissues for SV40 DNA-like sequences, and we were using
the method of PCR.
I'm not going to talk about the Tab expression using immunohistochemistry now, but it may
possibly come up in one of the later sessions.
The materials and methods that we started
with are cases. There were nine pleural mesotheliomas. I also took nine adenocarcinomas that were
metastasized to the pleura, and nine reactive pleura.
These varied from reactive eosinophilic pleuritis to
non-specific chronic inflammation of the pleura.
All the cases had formalin fixed and
paraffin embedded material, but in addition I mentioned to, as we went along, obtained four mesotheliomas which we had frozen down. These were complementary to the four other surgicals -- three surgicals and
one post-mortem.
There were three post-mortem cases and six
surgical biopsy cases when it came to the mesotheliomas. The breakdown was eight males and one female.
The age range varied from 38 to 73 with a mean of
58.8, and there was a mixture of histologies here.
There were both the epithelial, biphasic and the
sarcomatous types. All of these had a history of
exposure to asbestos.
We basically used the same technology that
Michele Carbone used in his paper, and we employed the
Bergsagel primers, the direct known as the SV40,
specific DNA sequences which was 107 base pair, and
then one -- sorry, 105 -- and then the 172 base pair
sequence that recognized the papomaviruses BK, JC,
etc.
And for controls we had some SV40 transfected human thyroid cells, one positive control and
one negative, and the positive contained one copy per
cell. And employed the controls both on frozen
material and on formalin fixed, paraffin embedded
material.
This shows the gel electrophoresis on the --
here's the mark here which tells you the size of the
protein that's found. This was one of the mesotheliomas which was negative. This was the positive thyroid
transfected cells, and then these other four were the
four positive mesothelioma cases.
This shows the controls of three positive
controls; again the marker here. This was the frozen,
positive control and these two were both fixed in
different ways, and they were positive, too.
So to summarize the results of our PCR
analysis on these cases, we found the SV40 specific
segment detection in four out of the nine mesotheliomas: one out of three post-mortem cases, three out of
six surgical biopsy cases. We didn't find any present
in the reactive pleurae and we didn't find any in the
adenocarcinomas.
Just to go into some more detail on the
actual mesotheliomas showing the concordance, or
discordance between the SV40 and the papomavirus
sequences, that the SV40 here was consistently
present, both in the frozen and the paraffin material
for each case that was positive, but that we got some
cases which were negative to the SV40 which had the
other sequence.
And again, these show the same thing for the
extra cases. These were all just the paraffin
embedded surgical cases.
This is just to compare our study with the
published literature on studies of mesothelioma that
-- Michele Carbone has already gone into this; that
there's a somewhat similar rate in the two studies.
A recent study by Strickler in fact, failed to find
any SV40-like sequences in 50 cases, and then there's
another study by Cristaudo that found positivity in 72
percent; again on archival material.
So the conclusion to the study was a simple
one. That we found SV40 DNA-like sequences in some
British mesothelioma cases. Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Gibbs.
The next talk will be shared by two speakers. First,
Dr. Luciano Mutti who will speak on SV40 on mesotheliomas. Dr. Mutti is at the Fondazione San Maugeri in
Italy. And the second part of the talk will be
presented by Dr. Antonio Giordano from Jefferson
Medical College, who will talk on a retinoblastoma
family in mesothelioma. Dr. Mutti.
Return to Table of Contents
DR. MUTTI: First of all, let me thank the
organizer of this fine workshop for my invitation. My
work has been -- I share my work with Dr. Giordano
from Philadelphia. And very briefly, as you can see,
SV40 has been able as stated, to induce some tumors on
hamsters, and as far as mesothelioma is concerned,
injection of SV40 in pleural space, able to induce in
100 percent, mesothelioma, and while injecting in
peritoneal space, is able to induce it near 50 percent
of the animal.
In humans, raw data showing that SV40-like
sequences are detectable in mesothelioma cells in
about 60 percent of cases, and the T-antigen is
detectable in a good number of cases. And SV40 has
been so considered as a powerful cofactor in addition
to asbestos fiber, for example, in inducing this
tumor.
We studied this population of ten patients
with this type of size of neoplasperitoneoma and nine
of pleural mesothelioma, and with this type of
histology. Some of these patients had not been
exposed to occupational asbestos fibers, but had been
exposed to an environmental exposure of a -- they
lived in an area wherein a plant working on asbestos
items had been working for at least 20 years.
So pollution of at least free fibers for
meter cubed has been, nowadays, even now, detectable
in this area. And these patients had been an occupational exposure for this period.
And there is another of these patients that
had been exposed both to occupational and environmental exposure, because they lived in this plant -- they
worked in this plant and they lived in this area.
Very briefly, we found SV40-like sequences
in three out of ten patients we studied. And as you
can see, one patient had both occupational and
environmental exposure and the other two, only
occupational exposure.
So we can see that there are some implications of these conclusions because SV40 can be
considered actually as a tumorigenic factor, but it's
important for us to assess -- to state that SV40 could
be considered as an inducer, as a tumor, as an
antigen, for mesothelioma.
So far it's not been possible to induce an
effective neuro-response in mesothelioma cells, but
there are a lot of studies that can demonstrate that
a self antigen, a tumor or self antigen of mesothelioma cells do exist.
And one of the most important strategies to
increase the new response is to increase the self-antigen expression, and with perspective in treatment
of the -- of mesothelioma cells or mesothelioma is,
without any doubt, to find out tumors that are
dangerous, such as the antigen against it's possible
to induce an immune response and a T-mediated response.
Just very brief, so I'll leave my talk to
Dr. Giordano because he carried out another study
about these patients, studying especially every family
protein.
CHAIRMAN KIRSCHSTEIN: Dr. Giordano.
Return to Table of Contents
DR. GIORDANO: Okay, so thank you, Dr.
Mutti, actually for allowing me to present some common
data that we carry out in collaboration. In the first
light --
CHAIRMAN KIRSCHSTEIN: Before you start, I
need to make an announcement. Dr. Chanock has an
urgent message at the message center.
DR. GIORDANO: Okay. So very quickly, the
retinoblastoma families is formed by three members, Rb
107, and the Rb2 p130. The Rb2 p130 is the family
member cloned a few years ago in my laboratory -- we
code Rb2 and then code for under 30 kilo often. The
03 family member, okay, they share a pocket region,
so-called pocket region because the functional domain
that is a point of the structure is tackled by
different genus tumor viruses like the virus C1 as is
before T-antigen. Is seven of papomavirus and do it
as a function of domain because this is how this
family or protein go through the cell cycle machinery.
So once we cloned Rb 2 we went on in the
collaboration with Dr. Knudsen at Fox Chase Cancer
Center. And with Dr. Knudsen we marked the Rb 2 on a
chromosome 16, 12.2. And that start actually, our
interest in the mesothelioma, because 16.2 by Dr.
Testa's group at Fox Chase has been found also being
the region found deleted in 16 out of 25 mesothelioma
tested by Dr. Testa.
So we went on and carried on a series of
experiments. And as a first characterization that we
did, we found that Rb 2 is important to growth
suppressor gene. When you applied to different tumor
cell line the gene, basically you express in appropriate way, you achieve a growth suppressive property
that is pretty dramatic in several tumor lines: like
osteosarcoma, like glioblastoma, like nasopharyngeal
cancer.
So by immunochemistry, okay, immunochemistry
we went home and first test a -- cell line cancer
tissue. And what do we found? We found that in line
cancer actually, there is -- we divide in different
histological groups. We found very interesting,
retinoblastoma family in line cancer, especially the
Rb 2 P 130, as in vast relationship, okay, with the
aggressiveness of the disease. More aggressive is the
tumor, undetectable is the Rb 2 gene.
So again, we went on and start the collaboration with Dr. Mutti and we start to test the
mesothelioma lines in the tumors, actually, from
tumors that also we obtain from different sources.
If you see here, we just use the primers
that amplify the regional T-antigen that is required
to tackle the retinoblastoma family. And we found the
sequences of T-antigen in four of the patients of the
nine patients that we tested.
But more interestingly, when we performed
immunohistochemical analysis, when we perform immunohistochemical analysis on these mesothelioma, alike up
in the line cancer, we find 11 of the Rb family do not
change. So there are high, medium high level of the
Rb family in all the mesothelioma tissue that we
tested.
So T-antigen, sequence of T-antigen we found
in these patients, and when we extract actually, the
protein from this fresh tissue using antibody --
monoclonal antibody, this T-antigen -- we find the
protein the right size, okay, in which T-antigen
migrate. This one is a cos cell line that -- this
place, SV40 T-antigen, and this one a cell line HLA
60. This does not see any DNA tumor virus. So there
is no expression of T-antigen.
So Rb family highly expressed in all
mesothelioma lines. So a mechanism that the work
probably we could predict, I mean, just considering
the data by several laboratories, that T-antigens and
other genus tumor virus binds in a physical association with the original blastoma family, lead us to an
experiment in which we took the source of T-antigen in
these patients having mesothelioma, and we ask if
there is any physical association with the original
blastoma family.
As you can see here, okay, the Rb, the p107,
and the pRb 2 physically interact; they interact in a
physical pro-interaction with those three family
members. So no wild conclusion from this of course,
but clearly there is no blastoma family, okay, member
in -- that in line cancer for instance, and don't see
intermediate cancer in the screening of studies we
did, correlates, especially the Rb 2, P 130, correlates with the aggressive disease in mesothelioma do
not change.
And one of the mechanisms that probably we
suggest is that known mechanism suggested by several
laboratories that T-antigen by binding these original
blastoma family does not allow them to perform the
suppressive property. Thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, DRs. Mutti
and Giordano. Our next speaker is Dr. Mauro Tognon
from the University of Ferrara, Italy, who will speak
on SV40 and mesotheliomas.
Return to Table of Contents
DR. TOGNON: Thank you very much, the
organizer, for inviting me to this interesting
meeting. And congratulation also, to the Chairperson
for the very nice pronunciation of my name.
Probably the people are already tired to
listen to the PCR amplification of the Rb pocket
domains of the T-antigen, but in our investigation we
did exactly the same thing. And in these experimental
control we have in the first lane, the BK, the second,
JC, and the SV40. One other, 72 base pair were
amplified by the primer that is being named P4 and P5.
And as you can see ethidium bromide staining, we have the amplification of this region, and
because this region is common to the three different
polyomaviruses, we set up an experiment with the
internal liogprobe that recognized specifically the
three different polyomavirus.
We used a very high stringency condition
just to be sure that this time we did the experiment
with these two primers that amplify the Rb pocket
domain of the large T-antigen which is common to the
three polyomaviruses. All the time we have clear
results by hybridization. In this case, we used the
recombinant plasmid DNA that contains the entire
genome of the three viruses.
In these experiments we analyze different
human brain tumors and in particular I would like to
say about the experiment, because we always start with
500 nanograms of human genomic DNA, and all the time
we extracted DNA in -- what can I say -- in old
fashioned way, we tried to avoid all the time to
centrifuge or to precipitate the DNA with ethanol.
And the reason is that in our experience, if
you precipitate the DNA with the ethanol, or you
extracted DNA with the commercial kit, you lose the
signal after the hybridization with the internal
olioprobe.
So probably this makes sometimes the
difference, is a possibility, between the high level
internal percentage that we found in our human brain
tumor and we can see a little bit later, also in the
normal brain tissue, of the positive sample that we
found.
In this case the control was always present.
This is the 172 base pair. This is the only possibility because it's the control to see the amplified
bands in the ethidium bromide staining, but in the
other samples that are under analysis, the amplified
bands never appear. In this case we amplify the DNA
for 35 cycles.
The results come out only with the internal
olioprobe that is specific in this case for SV40. As
you can see, there are in these typical experiments,
some primary tumors that come from ependymoma, across
the plastispapilloma, plasticytoma, and neoblastoma.
The neoblastoma was negative and also the sponsoblastoma was negative.
We analyzed also the normal brain tissue
from people that died by car accident and we extracted
the DNA in the same fashion, and the bands that
amplify the control is always present. You can see
the hybridization band that comes out after three or
four days of exposure, our radiography, but none of
the six samples that were analyzed in the experiment
were positive for the SV40 sequences.
Okay, this is another experiment that we set
up to investigate the presence of SV40-like sequences
in the peripheral blood cells. As you can see, after
the amplification and hybridization two of these
samples are positive and indeed, we have the results
of peripheral blood cells that sometimes are positive
for these sequences, even if the percentage of
positive sample in peripheral cell of normal individual.
This is not blood that comes from the
patients but we took 70 different blood samples from
the blood bank of the general hospital at the University of Ferrara.
Because we found the sequences specific for
SV40, both in the tumor sample and in the peripheral
blood cells of normal individuals, we think that
probably there are sometimes contamination of the
blood of our biopsies that come from the neurosurgeon.
And so we investigate the same kind of
tumors, starting this time from the tumor cell line.
All these samples were from glioblastoma cell lines.
We analyzed, if I remember correctly, 18 glioblastoma
cell lines. Nine were set in the lab of the Department of Pathology at the University of Verona, and
they never worked with SV40, and the other nine were
purchased from European culture collection.
And surprisingly, when we found that the
results of our data after the hybridization with the
internal olioprobes, comes out that practically all
the Italian tissue cultures cell lines from neoblastoma were positive and none were positive from the
tissue cultures that we purchased in England.
This is another data that comes from the
experiments we did in the same fashion with other
tumor -- primary tumors from the -- primary brain
tumors. And in this instance we isolated the RNA from
the tumor cell line, the glioblastoma cell lines, and
among four different glioblastoma cell lines, three
were positive for the messenger RNA. Even in this
case, the experiments were set up with the specific
primers from the Rb pocket domain.
This RT-PCR has been recently conduced, not
only with the glioblastoma cell line but also with the
osteosarcoma cell line. It's a new experiment that we
set up recently. And some new data is now out from
the normal tissue from people that are not affected by
any kind of disease. These are three samples that
come from buffy coats.
These are three different cell lines that
were obtained from the Institute of Medical Genetics.
Also these people -- of the University of Ferrara --
also these people never work with SV40. And these are
three samples that come from the sperm fluid in
another University, University of Modena.
As you can see, these experiments is only
representative of the sample that I analyzed. Some of
them are positive, some other are negative, and at the
end I'll show you the table that summarizes all this
data.
When we try to specify if the Rb pocket
domains that the large T-antigen sequences are
specific of SV40, we clone in sequence at the beginning from a glioblastoma primary brain tumor, a
glioblastoma, the SV40 sequences, and they came out --
it was practically the same sequences of the SV40 wild
type.
The difference that was also pointed out
during the talk of Dr. Bob Garcea, is that the nine
base pair insert that are present in BK and JC are
always absent in the SV40 sequences, and also there
are some other differences among the other bases that
we sequence. So by comparison and by checking in the
databank the sequence, we know there is a real SV40 --
I'm sorry -- okay, got it.
These are different sequences that were
obtained from other primary brain tumors. This is an
ependymoma and the sequence is the wild type SV40.
These two come from two different glioblastoma cell
lines, and we found a mutation in this position. The
sequence is A, C, G, T.
And this is another primary brain tumor, a
neoblastoma, that has two different mutations. Both
are transitions in the first base of the triplets and
at the end when we checked the amino acid we found
that there are a substitution of the amino acid in
this position.
Okay, after checking the specificity of the
sequences for SV40, we checked also in two of the
blastoma cell lines, the presence of the large T-antigen. And in this case we used a specific monoclonal antibody that react specifically with the large
T-antigen SV40, and at the same time we compared these
data with a polyclonal antibody that recognize SV40
and the BK virus T-antigen.
As you can see the results are similar in
terms of positive data, but if you check here -- this
is the control of BK transformed cell that is called
T-53 -- this light doesn't react with the monoclonal
antibody, specifically SV40 and the polyclonal
antibody reactor, and we can see in the nuclei the
positive fluorescence.
On the top we have the cos cell that are
transformed with the large T-antigen, and expressed
the large T-antigen in the nucleus, and as you can see
even here, the monoclonal antibody, specifically SV40
react, and the polyclonal antibody that recognize both
the large T-antigen, react with the same cells.
In a previous area of experiments we did
more or less the same experiment in searching for the
homology, not only for the SV40 but also for the BK.
Actually, this was a first series of experiments and
also in this case we used the internal olioprobe
specific for BK to discriminate along the other
polyomavirus sequences that eventually are present.
In this case, the same sample that I showed
you before, ependymomas and plastispapilloma, so now
to be positive for the BK, the same for the three or
four blood cells, and the same data are for the RT-PCR
in the glioblastoma cell line.
When we sequence the BK positive for the
large T-antigen Rb pocket domains, it turns out in 12
different samples, ten from the tumor samples and two
for normal tissue, that all the sequences were the
same compared to the BK virus Dunlop strain. We
couldn't find any mutation in the 12 different
sequences that we performed in 12 different samples.
This tells me, I have to show you the table
that summarizes -- wait a second, I go back for a
second. No, it's just I want to show you this
transparency. Okay. This is the table that summarizes the data for the detection of the specific BK virus
DNA, and detection of SV40 DNA. And as you can see,
all the primary brain tumor that was positive for the
BK DNA were also positive for SV40 DNA in terms of
much more positiveness for the BK with respect to the
SV40 DNA.
Similar data were obtained also for the cell
lines, and these tell us that the positive doesn't
depend on the blood -- contaminated the samples, and
we have some new data compared to the previous
publication directed to the primary bone tumors, and
the data are very similar to those that we had before
from Dr. Carbone, Dr. Garcea, and Dr. Butel.
Approximately the five percent of the
osteosarcoma are positive. We analyzed also some huge
tumors, and it turns out that 33 percent are positive.
And once again we analyzed those of the cell line and
the osteosarcoma are 43 positive for SV40, but all of
them are positive for the BK. And interesting, these
giant cell tumors, these rare tumors, 80 percent are
positive for SV40. And the small osteosarcoma is a
particular kind of osteosarcoma -- 36 percent are
positive for SV40.
Also some other tumors came out to be
positive, but they are less of an extent for SV40 and
positive also in the 42 percent for BK. But interesting, the normal brain tissue is quite different in
terms of original data compared to the results that we
heard before by other speakers.
And we have for example, the normal brain
tissue, only one out of 13 is positive for SV40; the
bone, none are positive; but the peripheral blood cell
in general, that we obtained from density gradient
centrifugation, 23 percent are positive for SV40.
But if you take the B and TD 4 side that are
cell lines, in this case transformed by the ABV, you
see that the SV40 is approximately 11 out of 15
samples were positive; 73 percent is quite high. Even
the TD4 side contains the SV40 and as we published
already, the nine out of 20 sperm fluids were positive
for SV40.
The new and the last -- when you work with
the PCR of course, you think sometimes you contaminate
your sample. So this is a new analysis what we did
very recently by PCR in human tumor and tumor cell
lines from DNA that was extracted ten years ago, and
in the laboratory that never worked with any kind of
virus.
And as you can see from the results, the
percentage of positive samples for SV40 are more or
less the same that was entered in the previous table,
both for the primary tumor and for the cell line.
So in conclusion, it seems to me that there
are some different regional distributions of the SV40
sequences. And compared, as I say, to this country or
in England, we have much more positive samples from
the normal tissue compared to other data.
I have also a couple of more results I would
like to tell to the people, that are obtained recently
in the Institute of Microbiology by Professor Barbanti-Brodano, that unfortunately today is not here
because he got a recent operation. And we tried to
rescue the viruses of the SV40 by transfecting with
lipofecting or lipofit -- I mean DNA from brain
tumors, brain tumor cell lines, and from preferable
cell experiments.
The only two isolates that we rescued so far
were from preferable cell sample from the dysplasia of
a vulva sample that was even in this case, obtained
from a collection of a large DNA sample that was at
minus 80 since 1985.
Now we are characterizing it to isolate it.
The first analysis -- I mean the first sequence
analysis seems to indicate that to isolate it at the
two SV40, and in particular, the difference that we
see as a variant are in the origin of replication.
This data are just preliminary so we have to control
a little bit in detail, these results.
Thank you very much.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr.
Tognon. Our final presentation today is by Keerti
Shah: A Search for SV40 in Human Mesotheliomas.
Return to Table of Contents
DR. SHAH: We were also very intrigued by
the finding of Dr. Carbone about the presence of SV40
in mesothelioma tissue, and we attempted to reproduce
that. And this has been written up and published in
this journal in 1996. We were not able to detect
simian virus 40 sequences in pleural mesothelioma.
We also looked at serum specimens from
mesothelioma patients and osteosarcoma patients for
antibodies to simian virus 40. And we were not able
to detect any evidence of SV40 neutralizing antibodies, the results documented in this paper.
And more recently -- something which is not
in this paper -- we tried to look for simian virus 40
in urine specimens collected from immuno-compromised
and non-immuno-compromised patients. We looked for BK
virus, JC virus, and also simian virus 40, and we were
not able to detect any simian virus 40 sequences in
these urines.
So this is the mesothelioma study. There
were 50 pleura-mesothelioma samples collected at the
Armed Forces Institute of Pathology over a number of
years -- this was from 1987 to 1992 -- and obtained
from many different hospitals and clinics in the
United States.
The patient's diagnosis was between -- age
year diagnosis was between 43 and 88, and median age
was 68 years. These were archive samples so we tested
the paraffin sections with proteinase K and we
amplified them with two sets of primers for SV40, and
another set of primers for beta globin to see if the
DNA was suitable for PCR.
The two primers for SV40 we used were
described by Dr. Garcea. They amplified 103 and 205
base pair regions of the T-antigen for SV40. And they
are shown here in illustration.
This is what we found. These are three
identical filters. The top one is with beta globin
probe, and these are amplified with the beta globin
primers. And most of the specimens we were able to
amplify beta globin easily. There were some failures
-- one here, one here -- so in 48 out of the 50
mesothelioma tissues we were able to amplify beta
globin so that the DNAs were thought to be suitable
for PCR studies.
These are the same identical filters here
and here they are tested with an SV1 probe, a probe
that was described by Dr. Carbone. These are all
mesothelioma samples. None of them show up SV40
sequence. And we have done a titration of SV40 cos-1
cells.
Cos-1 cells have one copy of the SV40 genome
per cell, and these are 300 cells, 30 cells, three
cells, and actually less than one cell. So with this
particular primer path we were able to amplify one
copy of this SV40 genome just by this particular data
with the second set of primers; again 300, 30, and
three SV40 copies.
So that you -- we thought that our technique
was quite sensitive but we were not able to detect any
SV40 genomes. So we also looked at 105 serum specimens: 35 were derived from mesothelioma, 35 from
sarcomas, and 35 controls, matched to the mesothelioma
patients. Then 97 -- and these were tested in SV40
plaque neutralization assay in BSC-1 cells -- 97 were
completely negative and five were partially protective.
They did not completely neutralize the
simian virus 40, but reduced the number of plaques.
And they were scattered in the three groups. There
were three out of 34 mesothelioma, one out of 33
osteosarcoma, and one out of 35 controls.
Then we looked at the urines and we collected -- these are 165 urines provided to us by Dr.
Strickler and Dr. Gater -- and they came from a study
of homosexual men, some of whom were HIV positive --
seven urines which were HIV-positive men and 78 from
HIV-negative men. The median age was 38/39; ethnicity, they were 91 percent white.
And this time we thought that if we do not
have good positive controls for SV40 no one is going
to believe our data. So we took SV40 cos cells and
spiked normal urine with the cells, and these tubes we
sent back to the NCI where they were processed with
the other urines.
And then when we received these 165 urine
specimens they also contained other tubes marked
similarly, which could not be distinguished but which
contained 17 specimens which came from the spiked
urine which were the SV40 positive urines, and
contained approximately 300 cos-1 cells or 300 copies
of the SV40 genomes.
These are the SV40 data. There are -- 17
urine specimens that we got were positive for SV40 and
there were -- each of the 17 of the positive controls,
the urine specimens that were spiked with SV40. The
other 165 urine specimens, none of them contained
SV40, but the prevalence of BKV and JCV was quite
high. About 50 percent of the urines contained either
BKV or JCV and some contained both.
This is the data from the -- for BKV and JCV
from the urine specimen. In the HIV negative urines,
or urines from HIV negative people -- only 2.3 percent
were positive with HIV negative, and it increased with
SV40 positivity and the degree of immunosuppression,
something that has been reported before.
The SV40 prevalence was very high in both
HIV negative urines as well as HIV positive urines,
and we did not see any much greater increase in
prevalence from what was already a very high prevalence in the HIV negative individuals.
So we could not detect SV40 sequences in
mesothelioma tissues although we did 50 tissues. From
the results of previous speakers we should have picked
up at least 20, 25 positive specimens. We did not
find serological evidence of SV40. One would think
that if these people were developing tumors because of
an SV40 infection, one would expect some evidence of
SV40 infection, and the antibodies to SV40 would be a
very good sensitive measure. Plaque neutralization
test is very specific; we did not find this.
Now, this is just a beginning study. At
least in this group of patients, immuno-competent
patients and immuno-compromised patients, we did not
find any virus in the urine.
One of the puzzles in all of these studies
is that all these patients in whom SV40 has been
found, many of them have been born after the vaccines
were cleared of SV40. So this suggests that SV40 must
be circulating in human communities.
And to get some evidence for that, whether
SV40 is circulating in human communities, we looked at
these urines from groups with generally shared lots of
polyomaviruses like BKV and JCV. And so we did not
find evidence of that also. Thank you.
CHAIRMAN KIRSCHSTEIN: Thank you, Dr. Shah.
This concludes the first session, a very interesting
session. There will now be a coffee break until
11:15.
(Whereupon, the foregoing matter went off
the record at 11:03 a.m. and went back on
the record at 11:30 a.m.)
CHAIRMAN BRIEMAN: I'm Rob Brieman. I'm the
Acting Director of the National Vaccine Program
Office, and I'd like to welcome you back for the next
session.
I would like to say that the NVPO is very
happy to sponsor this meeting and I think it is
providing the opportunity to look at very interesting
data and to examine the implications of the information presented from a public health standpoint.
The first speaker for this session is Dr.
Kristina Dorries from the University of Wurzburg in
Germany.
And I'm sorry. Before we begin, let me just
say that if anyone has additional data that they would
like to present in a short presentation -- either
positive or negative information -- in time for the
next panel, or the panel discussion which will be this
afternoon and led by Dr. Fried who's standing here --please see him either during this session or immediately following to arrange that presentation.
So again, our first speaker will be Dr.
Dorries.
Return to Table of Contents
DR. DORRIES: Thank you very much for the
invitation, to the organizers, and we will proceed now
to the human polyomaviruses, BK and JC virus, and I
will try to summarize some of the essential molecular,
biological, and pathogenic features of both viruses.
A common infection of BK virus is early in
childhood with zero conversion rates of more than 90
percent in the young. In contrast, JC virus is coming
a little bit later up and there we have zero conversion rate of 70 to 90 percent in the adult population.
According to PCR analysis, a virus persists
in nearly 100 percent of young adults and both viruses
persist in kidney epithelium. They are transmitted,
at least in part, by urinary excretion. Both viruses
are discussed to be involved in tumor induction in
men.
These have the viruses -- and both viruses
in common with SV40, but additional DNA homologies and
protein homologies are followed by similar morphology
of virus particles and by the same replication
strategies of the three primate polyomaviruses.
This is a genome of JCV and you'll see small
T-antigen and large T-antigen coding in the early part
of the genome, VP1, 2, and 3 in the late part, and
there is an open reading frame for the adenoprotein.
These DNA sequences are very homologous
between the two viruses, however the TCRs, the
transcription control region is different.
This is the non-coding control region with
the origin of DNA replication. That is conserved with
T-antigen mining sites 1 and 2, in the whole family of
the primate polyomaviruses and the transcription
control region is shown from SV40. And we have here
heterogeneity of single and double promoter elements
that are present in different -- that might be present
in different organs.
The conserved first in hearts or promoter
element contains binding sites for cellular factors
that are gliocell specific or associated to the basic
activation -- cellular activation -- and are associated with signal transcription elements.
The cause of human polyomavirus is not quite
clear. We don't know the entry site, but primary
infection always is followed by live, long persistence
of the viruses. We don't know whether the infection
is latent or low copy infectioned, but we can find
viral DNA, episomal DNA in the affected organs.
Under limited changes in the immune state,
as induced by pregnancy or older age, we find a
temporary activated infection with short episodes of
virus production and messenger RNA viral proteins that
can be detected. This situation is asymptomatic.
In contrast, in the clinical overt state of
infection under severe immune deficiency as induced by
lymphoproliferative diseases, immunosuppressive
therapy, or AIDS, we find an unrestricted virus grows
with efficient virus production and the lysis of viral
target cells.
Polyomavirus associated human disease are
only described in the central nervous system for JC
virus. It's progressive multifocal leukoencephalopathy, and for BK virus there is strong association with
the urogenital system. However, recently there is a
systemic disease described that involved tubular
lynenphorytis and disthicia pneumonitis and the
subacute meningeal encephalitis for BK virus.
This is summarized here on this slide but I
would like to concentrate on the cell type specificity. Cells of the connective tissue are clearly
associated with BK virus infection at epithelial cells
as fibrocytes of the three organs, and specifically in
the meningeal encephalitis we find epidermal cells
associated with BK virus infection and interestingly,
astrocytes.
The astrocytes are in contrast to JC virus
infection from the glio type of cells -- oligodendrocytes predominantly are infected.
This is only shortly in in situ hybridization of the epidermal layer on the ventricular system.
This is -- the brown color is BK virus DNA specific
hybridization, and this is a double immuno staining of
GFAP astrocyte specific protein, and in the nuclei
polyomavirus specific antigen is found.
This BK virus associated disease is a rare
disease. It was described once in Germany, and we
might now have a second case. However, this is
completely different to JC virus associated PML as
found now in steadily increasing numbers.
The characteristics of PML is disseminated focus
cytolytic infection of oligodendrocytes in cortex and
white matter of the brain, and this is followed by the
loss of myelin sheath.
This is a cartoon of a typical PML lesion
with the oligodendrocytes filled with virus particles
and the rim of the lesion, and at the center you see
reactive astrocytes and giant cells in mitosis. This
is believed to be a semi-permissive JC virus infections and there are cases where virus particles are
found in these cells.
Additionally, it might be associated with
transforming infection. Limited infiltration of
lymphoid cells is described recently in predominantly
in AIDS associated cases.
Here you'll see in situ hybridization of
typical PML lesion. You'll see the highly concentrated virus DNA by the black grains. It's radioactive
hybridization. This is at rim of such a lesion with
less concentrated DNA in freshly-infected cells.
In contrast, in the periphery we have only
an attenuated infection. These are epithelial cells
surrounding the renal tubules, and although this cell
contains not only DNA but also a viral antigen and an
electromicroscopy virus particles where detected, you
see that this is persistent, infected in the activated
state -- a more or less attenuated infection.
A molecular characterization of the virus
DNA population in these different sites of infection
in the brain revealed presence of heterogenous TCR,
transcription and control elements, that are repeated
at the GSP prototype from Wurzburg, and in the kidneys
you find the single, so-called archetype elements.
In the time before AIDS, lymphoproliferative
diseases were closely related. About 62 percent of
all cases in '84 were the basic diseases for PML, but
now in the AIDS era we discuss between five and ten
percent of AIDS patients with neurological symptoms
might eventually die by PML. And therefore, several
laboratories are now interested in factors or situations that might lead to the induction of disease.
And I summarized several factors that are
discussed in the moment that as, first, virus-dependent factors and host-dependent factors. In case of
virus-dependent factors, conditional viral dissemination is discussed that probably primary -- accidental
primary infection of the effector organs, the central
nervous system under immunosuppression as leading to
disease.
The second possibility is that the genetic
heterogeneity of the promoter/enhancer elements is
involved in pathogenic -- in the induction of the
disease, and this might be associated with the
neurotropic selection of highly active, cell specific
transcription elements.
From the host-dependent factors, genetic
predisposition can be assumed, but what is obvious is
the control of virus host interaction by the immune
system really plays an essential role in the activation processes.
The first possibility was that primary
infection might only happen under immunosuppression.
Under this condition, no involvement of the central
nervous system in persistent infection should be
expected and therefore we analyzed cellular DNA from
non-PML patients. And altogether I think we analyzed
70 patients by PCR analysis. And here, from several
regions of the genome these were hybridized with JC
virus-specific probes and the products were sequenced.
What came out is summarized here. This is
a T-antigen amplification product that hybridized with
JC virus DNA. And interestingly, from the same --
hybridization from the same material revealed that not
only JC virus is present, but also BK virus is
present. And these probes are species-specific for
both viruses.
From this data we can say now that probably
JC virus is persisting in the central nervous system
and reaches the central nervous system long time
before the induction of disease.
And that is a requirement for the selection
of neurotropic lysal-specific TCR variants that might
be selected by cytolytic -- might be selected by
activation processes in the persistent state.
And to analyze this we characterize the
variants that were present in the different ethnification products by cloning PCR amplification products
and sequencing them afterwards.
And altogether we found seven different TCR
subtypes. From the TCR type 1, three subtypes in TCR
type 2, two subtypes type 1. Repeating the tatabox
elements type 2 leaves tatabox element out and repeats
only conserved sequences that are following.
What was interesting is that we found single
elements -- these are so-called archetypes -- we found
double elements here and here. And here, those are
other duplicated elements. And additionally, we found
triplicate from TCR type 1 DNA.
This was a finding that was astonishing but
what really was new is that W1 and W6 were the
dominant subtypes that we found in persistently
infected brain tissue. W1 is similar to the mat 1
isolet from medicine, the American isolet, and the TCR
type 2 is similar to the GSB isolets. The predominant
type altogether was med types -- American subtypes.
Then we thought that possibly the situation
might -- that these rearrangements were -- came up
under persistent infection and we studied then a
kidney tissue. And interestingly we only could find
W1, double enhancer promoter elements in kidney tissue
of eight different patients. Only the single W4,
single archetypes are found in only low percentages.
From these data we only can assume that the
different rearranged elements are present in the human
population and are not rearranged anew in each
patient.
This brings us back to the third possibility
that changes in the virus host interactions due to
severe breakdown of the immunological surveillance are
the real requirement for the induction of clinical,
overt CNS disease.
However, we have not mentioned under these
conditions recent results that were found in lymphocytes. First it was described that V cells in PML
tissue were actively infected by JC virus and in
addition, later on, peripheral blood leukocytes were
analyzed for the presence of JC virus DNA.
And as you can see here again, it has T-antigen PCR hybridization -- with T-antigen specific
probe revealed that almost all healthy individuals
were carriers of JC virus DNA. To a certain extent we
also detected BK virus DNA. However, the concentration is lower than the JC virus concentration,
although the primer is similarly sensitive for both
viruses.
This opens up now, a complete new field of
possibilities, how the virus could interact with host-related mechanisms and will bring us further I think,
in the next time.
To summarize, the conditions that are
associated with the pathogenesis of PML under immunocompetence, we find an asymptomatic JC virus infection
of the central nervous system, life-long persisting
viable JC genomes with highly active TCR types are
present in the central nervous systems, a growing
virus load in advanced stages of life was detected,
and also in individuals undergoing changes of the
immune state as induced by systemic tumors carry an
enhanced virus load.
A virus infection is probably controlled by
tight immunological surveillance, and the severe
changes of immunological virus/host interaction and
probably comes to a 2-step situation. First, we find
an activation of viral expression in peripheral
organs, we find a growing virus load by the activation
event and peripheral tissue and in the cells, and this
really probably is followed by an enhanced seeding of
virus to the central nervous system.
And a second all simultaneous step we find
an unlimited activation of viral expression in the
central nervous system, and then CNS tissue is
destructed by cytolytic multiplication, and additional
enhancement of the virus load by infected lymphocytes
may happen also.
And then possible interaction with the viral
transactivators should be discussed. In vitro
experiments with cytomegalovirus and HIV early
antigens have shown that the early antigens are
interactive with JC virus, and it could be that under
these conditions we find an enhancement of virus
replication.
Thank you.
CHAIRMAN BRIEMAN: Thank you very much, Dr.
Dorries for that elegant presentation that was, I
might also note, precisely timed -- perfectly. I need
to announce again that Dr. Chanock needs to check at
the desk because he has an urgent message.
The next speaker will address the question,
"is BK virus a co-factor in human cancer?", and it's
Dr. Michael Imperiale of the University of Michigan.
Return to Table of Contents
DR. IMPERIALE: I'd like to thank the
organizers for inviting me to talk. What I'd like to
do during my talk is sort of move into the realm of
molecular biology a little bit.
Okay, so you've already heard a little bit
about BK virus but I just want to repeat a few salient
points here. The first is that there's a subclinical,
persistent infection in about 80 percent of the
population. BK virus encodes a T-antigen which is
about 75 homologous to SV40 T-antigen which you've
heard a lot about this morning. However, that
homology is much higher in the transforming domains
that Ellen Fanning referred to in her talk.
It's been shown that over-expression of T-antigen or a co-expression of T-antigen with an
activated ras gene can transform cells -- this is both
in vitro and also in transgenic animals.
And finally, as you heard before, BK virus
has been associated with various types of tumors and
also with hemorrhagic cystitis in amino-compromised
patients.
So about two years ago what we wanted to set
out to do in my lab was to ask the question regarding
these tumors in an indirect sense and that is, does BK
virus have the tools that it would need to be involved
in human cancer?
This is just a table from a paper that was
published in Virology in 1995 from the group in
Ferrara, where they showed that BK viruses associated
with various urinary tract tumors -- and indeed, in
some of these tumors they found that they could detect
viral sequences by southern blotting rather than by
PCR, which implicated that there might be at least a
higher amount of viral DNA in those tumors.
Okay, so the first thing that we wanted to
look at was the interaction of viral T-antigen with
the Rb family of proteins. This is just to remind you
that these proteins inhibit cell cycle progression.
They do this by binding to family members of the E2F
transcription factors and block E2F function.
The next slide just talks a little bit about
E2F. It's a cellular transcription factor. It's
actually a family of heterodimeric proteins, and the
function of these transcription factors is to regulate
genes that involved in S-phase progression.
And the next slide is just a model of what's
going on here. So this is a normal cell. Up here at
the top, Rb and p107, p130 are bound to E2F. When the
cell receives a signal to divide the proteins become
phosphorylated; this results in release of E2F which
is now able to activate transcription of its target
genes which then drive the cell into S-phase.
In the model that's been worked out for SV40
T-antigen is shown down here at the bottom, which is
that T-antigen binds to the retinoblastoma protein and
its other related family members. This has the same
end effect as a mitogenic signal up here, which is
that the E2F is released and the cell can now enter
the cell cycle.
So we wanted to ask whether BK virus T-antigen was functioning in a similar manner. The
first thing that we did was to just look and see
whether T-antigen could complex with the Rb family
members in cells -- and these are a series of immuno-precipitation assays where we immuno-precipitate with
antibodies against T-antigen, run the proteins on a
gel and blot and probe for the three different Rb
family members. And you can see that we find complexes of T-antigen with Rb, p107, p130 in the cells that
are expressing T-antigen.
Now, what I need to point out here is that,
in order to see this we have to use 100 times as much
protein from these BK virus T-antigen expressing
cells, as from cos-1 cells which express SV40 T-antigen. And the reason for that is that there's a
hundred-fold less T-antigen in those cells.
So this is a T-antigen blot. We have to
load 100 times as much protein to see the T-antigen as
we do here. So any effects that BK virus T-antigen is
having here as compared to SV40 T-antigen, these
effects are occurring in spite of the fact that
there's a hundred-fold less protein there. And that's
something I'd like you to keep in mind as I go through
this.
The next slide on the right is just a whole
cell lysate blotted with antibodies against these
proteins. And if you look at the normal cells
compared to the cells expressing BK virus T-antigen,
you can see that there's less Rb, p107, p130. What's
left is mostly in the hypo- or under-phosphorylated
form. And I think Jim DeCaprio is going to talk more
about this tomorrow, so I'm not going to address that
issue.
But what I want to point out here is that
what this is saying is that most of what's left in the
cell here is in this form, which should be the growth
inhibitory form, and also most of what's there is not
bound to T-antigen because there's so little T-antigen
present in the cell. So the question is, are we
getting an induction of E2F activity?
So to look at this, what we did was to take
a gene that expresses a CAT reporter, under the
control of E2F DNA binding site, and transfect that
into cells, and the results have shown -- the next
slide on the right -- so these are just CAT activities
of these different cell lines.
You can see when we transfect this reporter
gena to normal kidney cells we don't get any activity.
If we put it into cells expressing BK virus T-antigen
we get an induction of activity. SV40 T-antigen we
also get an induction.
Notice that the induction only differs by
about 4- or 5-fold even though again, there's 100-fold
difference in the amount of T-antigen in these cells.
And these are just transient transfections where we've
done the same sort of assay showing the induction by
BK virus T-antigen compared to another protein that
interacts in the system, which is the adenovirus C1A
printing.
So in spite of the fact that there's very
little T-antigen binding to these proteins that we can
detect and that most of the protein that's there in
the cell is in this form here which should be bound to
E2F, it looks like we're getting induction of E2F
activity.
So the question we wanted to ask is, what
does the E2F look like in these cells? What we've
done then is to look at E2F binding activity by doing
DNA band shift assays. The left side here are just
straight DNA band shift assays, making extracts either
from normal monkey kidney cells, cells that are
expressing BK virus T-antigen, or cells expressing
SV40 T-antigen.
What I want to show you on this side here is
that, this is the free E2F, so this is the theoretically, transcriptionally active form of E2F. And
although this is a little bit overexposed so that you
can see some of these other bands, there is an
induction of the amount of free E2F in the cells
expressing the viral T-antigens as compared to the
normal cells.
And I should point out, this is not due to
differences in the number of cells that are in
different portions of the cell cycle.
Now, the right side of the -- I'm sorry, the
left slide over here shows that even though T-antigen
is present in these cells, there's a significant
amount of E2F that's still bound to Rb. And that's
shown here. You can see that there's a little bit of
E2F Rb complex that's left here, and this side of the
slide just shows how we identify the complex, as this
complex goes away if we add antibodies against Rb;
this complex goes away if we add antibodies against
p107.
This is to show that there are Rb E2F
complexes still present in the cell. What we've done
here is to use antibodies against the Rb protein to
immunoprecipitate and then release any E2F that was
present, bound to that Rb, and put that into the band
shift assay.
And you can see that there's still E2F
activity that's associated with Rb, both in the BK
virus T-antigen expressing cells and in the SV40. And
again, this is about equivalent, despite the huge
difference in the levels of T-antigen in these two
different cell lines.
Finally, the last slide on the left. So the
question is, if we're getting induction of E2F
activity, are we getting induction of cell growth? So
these are some growth assays of these cells. If we
take these cells and grow them in ten percent serum
you can see that all of these different cell lines
grow well.
However, if we put these cells into medium-containing load serums -- so this would be an assay
for serum independent growth which is a hallmark of
transformed cells -- we can see here that two different parental monkey kidney lines do not grow well in
low serum, whereas the cells expressing BK virus T-antigen or SV40 T-antigen both grow in low serum.
So what this tell us then, is that BK virus
has the ability to induce cells to grow. So this
would be one of the things that one might expect for
something that might be involved in tumorigenesis.
The next thing that we wanted to look at
then, is the interaction of T-antigen with p53, and
this is just a little cartoon here to show you how p53
works. Normally, if a cell is getting ready to divide
and that cell receives DNA damage, there's an induction of p53 levels, and that results in arrest of the
cell before it enters S-phase. That allows the cell
to either repair the damage or if the damage cannot be
repaired then that cell will be induced to undergo
programmed cell death or apoptosis.
Now, in the presence of SV40 T-antigen what
happens is, T-antigen binds to p53, the cell receives
DNA damage, there's no induction of p53, no G1 arrest,
and the cell can go on to divide with damage, or if
there's enough damage there will be mitotic failure
and cell death.
So we wanted to ask what was the interaction
between BK virus T-antigen and p53. The next slide on
the right is just an immunoprecipitation similar to
what I showed you before. For the Rb proteins, if we
immunoprecipitate with antibodies against T-antigen,
we bring down all of the p53, or over 95 percent of
the p53 protein that's present in the cell.
So this says that even though there's very
low levels of T-antigen present in the cell, it is
able to bind most, if not all, of the p53 in those
cells. So the question is then, is it having any
effect on p53 function?
So what we did then, was to take normal
cells or cells that are expressing T-antigen and
irradiate them and look at the response to DNA damage.
The next slide on the right is just an amino blot --
oops, on the left, sorry -- it's just amino blot where
we've looked at either the induction of p53 or one of
its important target genes, p21, upon irradiation.
So the left side of this slide shows the
normal cells; increasing amounts of irradiation we get
an induction of p53 levels; and we get a concomitant
induction of p21 levels.
In the cells that are expressing BK virus T-antigen, first you can see that there are higher
levels of p53 to start with. This is because T-antigen binding stabilizes p53. However, there's no
further induction of p53 upon irradiation and there's
also no induction of p21 upon irradiation.
The next two slides just show some cell
cycle analysis of this cells, the normal cells on the
left, the transformed cells on the right, and you can
see that when these cells are irradiated, the normal
cells, one gets mainly arrest here in the G1 phase of
the cell cycle. If you look at the cells expressing
SV40 T-antigen however, one does not get arrest in G1;
instead, one mostly gets arrest in G2 M-phase of the
cell cycle.
So what this says then is that BK virus T-antigen is interfering with p53 function and removing
its ability to block cells in the G1 phase of the cell
cycle upon DNA damage.
Okay, finally then, we did an experiment
based on some old experiments that were published for
SV40 many years ago, where people showed that SV40
infection can induce chromosomal damage. And so we
wanted to ask whether BK virus can also induce the
same sort of chromosomal damage.
So what we did then, was to take some
primary human fibroblasts and infect them with either
BK virus or also JC virus, and look at their chromosomes. And the next slide on the left shows the
result.
This is a karyotype from normal cells here.
These are the karyotypes from either BK virus-infected
cells or JC-infected cells. And in both cases you can
see that we're getting chromosome fragmentation, there
are translocations. And moreover, in the presence of
BK virus, we're also seeing this thickening of the
chromosomes which is indicative of endoreduplication.
And this actually makes sense when one looks
at the cell cycle analysis I just showed you; that the
cells may be getting hung up in M-phase. So BK virus
is able to induce DNA damage in these cells.
And what I want to point out here is that we
did this at a rather high multiplicity of infection so
that we could get enough karyotypes -- enough infected
cells to actually see a karyotype. And we assume that
BK virus may be inducing a lot of undetectable
chromosomal damage, even at lower multiplicities of
infection that could potentially be occurring during
a persistent infection.
So the next slide on the left then, I'd like
to just sum up by asking a question -- and let me
state from the outset, I don't know what the answer to
this question is -- but the question is, is BKV a co-factor in human cancer?
As I showed you, it can induce DNA damage,
it inhibits the p53 response to this damage so if
cells receive damage then they're unable to arrest in
G1 before that damage can be repaired, and moreover,
it's inducing cellular DNA synthesis so it's inducting
E2F activity. And the question is, can this lead to
increased mutation rates which then potentially could
lead to carcinogenesis.
And so I think I'll stop there, and just on
the next slide acknowledge the people who have done
this work. In my lab, Kimya Harris, Joan Christianson, and Eugenia Chang. And our collaborator in the
Department of Human Genetics, who's done the karyotype
analysis, Tom Glover. Thank you.
CHAIRMAN BRIEMAN: Thank you, Dr. Imperiale.
That was very impressive. The next speaker from
Allegheny University for the Health Sciences, is Dr.
Kamel Khalili who will speak about JC and BK sequences
in human tumors.
Return to Table of Contents
DR. KHALILI: I guess what I'm going to do
today is focus my talk on JC virus. Can I have first
slide, please?
This slide just demonstrates that the JC
virus is not another SV40 or humana SV40, and it has
its own personality and character. Kristina Dorries
very elegantly laid out some of those characteristics,
and I'm going to bypass some of these slides which I
have initially, to describe a JC viral control region
or lytic cycle and then demonstrate that.
In the next slide -- this is a virus which
infects greater than 92 percent of human populations
and as you heard from Kristina's talk, perhaps 100
percent PCR. And it's a virus which has no animal
reservoir, and perhaps more importantly, it's a virus
that we can talk about. When you talk about an AIDS
epidemic you cannot ignore JC virus.
It's a virus which used to be rare and the
disease of the virus was rare but not any more. And
perhaps I should mention that greater than ten percent
of AIDS patients which no logical disorders, which by
itself is greater than 70 percent, come down with PML.
So suggesting that the virus has a chance to
become reactive replicates in cells. The cells of the
virus is replicating oligodendrocytes.
This is oligodendrocyte which is responsible
for formation of myelin sheaths around the axon. In
fact, oligodendrocytes sends a processes that -- this
is an electromicrograph of normal brain -- shows that
the oligodendrocyte sends a process, circulating
processes, and these processes form the layers, very
compacted layers, around the axon, an important event
for the insulting axon.
Now, cytolytic destruction of these oligodendrocytes upon reactivation of endogenous JC virus,
all the JC virus which gets to the brain by B cell or
any other vehicle, results in the demyelinating
disease of PML.
As Kristina mentioned, the JC control region
sits between the viral early and late genome, and it
has a unique characteristic. It has a 298 base pair
repeat sequence and old genome viral DNA replication.
It's a bidirection promoter of single to SV40. In one
hand it shoots for the early gene expression; on the
other hand, the other sides controls late gene
expression.
Now, if you really go through the sequence
you do see that there is not much release sequence
homology or similarity between the JC control region
and SV40 control region. That's one characteristic of
the virus counts.
We spent about eight years -- over the last
eight years -- trying to understand what confers the
tissue specificity to the viral gene expression. We
knew that early gene expression is make or break for
the virus. So if you reduce immunosystem, virus gets
opportunity to replicate, but it does not replicate in
every cell. It replicates in all oligodendrocytes,
the cell which I showed previously.
So there is two barriers in the viral gene
expression: first is immune system and the second is
its cell-type specific barrier. The latter was easier
to address. We have the similar cell lines and we
went on through the standard biochemistry to understand that one of the sequences within the viral
control region, which, upon interaction with cellular
factor, turn on early gene transcription.
Again, Kristina demonstrates a slide summarizes all the transcription factors which could bind
to a JC control region. And through those studies, we
and others learn that perhaps interaction of the
protein some were inducible, some were tissue specific
factors.
So the control region, per se, is not --
it's essential but still does not put the virus
through the lytic cycle. So it seems that the
interplay between protein upon the interaction plays
more important role in this event.
So this slide is about a 3-year old slide.
It just shows some of the proteins that we and others
purified by standard cold room chemistry and cloned
the genes, and demonstrated that association of the
protein, for example in this case, YB-1 with the JC
control region, is important to induce viral gene
expression couple-folds, but sufficient -- it wasn't
sufficient to put the virus through the lytic cycle.
Then once other proteins were purified, we
realized that the communication of this protein with
each other perhaps plays a more important role.
Now, another feature of the virus which I'm
going to little bit talk about that, is the TAR
region. TAR is a sequence which first identified in
HIV genome, and this region, located downstream from
the HIV transcription initiation site, and it's
responsive to tat protein, a protein from HIV which is
important for the viral replication.
Now, a higher incidence of PML among AIDS
patients suggests that perhaps in addition to immune
system, there is some direct communication between
HIV-1 and JC Virus in the brain parenchyma.
So we asked the question of whether or not
HIV regulator protein included in tat could activate
JC promoter in the choleal cells. The answer to that
question was yes, and we demonstrated that this
activation of the viral promoter, JC promoter for HIV
tat was mediated through the sequence, TAR log
sequence which is located in the leader of the late
gene transcription -- very reminiscent to the structure described in HIV promoter.
In addition, it was demonstrated that tat
could bind to another cellular protein; a protein
which initially identified based on this ability to
transcribe, stimulate JC promoter, and as a result of
this interaction, augment expiration of the viral JC
virus.
So you see that we learn much more by
studying JC virus in terms of the mechanism of viral-viral interaction and the cell type of specific
transcription.
Now, let me move on, on the next slide. So
the question was JC virus, under proper conditions,
proper environment, produce a T-antigen, and once the
T-antigen is produced such as other papomavirus,
induce viral DNA replication, late gene transcription,
and certainly the destruction, the virus causes PML.
Now, what happens if you block replication
event so you still produce T-antigen under certain
circumstances, and what happened to that T-antigen?
Does T-antigen, the absence of replication that's
supposed to be crossed, could induce the tumor?
So the first clue for that came from the
animal studies, hamster model, where the JC virus
cannot efficiently reproduce. In the replication does
not happen because of the primary unit of DNA prolimerase is species-specific, cannot turn on JC viral DNA
replication in hamster.
Therefore, if you take a JC virus, inject in
the brain of a newborn hamster, what happens after two
months, you get tumors. Eighty percent of the
injected animals come down with a tumor. This was
demonstrated by a number of people, and also later on,
Sid Hoff and others demonstrated that intercerebral
inoculation of the JC virus in our monkey, the
squirrel monkey, induces glioblastoma multiformity.
A very devastating brain tumor which we still don't
know about the pathogenesis of the tumor and also we
don't basically have a cure for this tumor.
So this slide illustrates the gross section
of the brain of the newborn hamster. This is a tumor
for an open injection of JC virus and this is a normal
area. Now, if you take the tumor results here and
stain it for T-antigen, you basically get 100 percent
staining of the T-antigen in almost every cell.
There's a series of slides which -- studies
that we did, and I'm not going to go through that --
and we will learn that these cells do have their own
characteristic in terms of the cell growth and other
factors; factors which are important for control of
the cell proliferation.
And even the extent of the T-antigen in a
variety of the cells were different. We cloned these
cells; we are in process of analyzing it -- how low,
high, and medium dose of the T-antigen in the various
cells could utilize the different pathways which leads
to the formation of the tumor.
And at the same time, we are utilizing the
hamster model as a measure for studying formation of
the tumor. This is a very unique model. You can
inject JC virus in the hamster brain with notion that
80 percent of the injected animals come down with the
tumor. So what you could do is, you could monitor
development of the tumor; things that you cannot do in
any other animal species, and clearly we cannot do in
human.
So how you can do it in vivo, we have taken
this hamster model and in during MRI on the living
animal, we are monitoring formation of the tumor from
the time that injected to the time that becomes
significantly big and close to kill the animal.
So in parallel we are devising histopathological studies to understand the interplay, because
some of those regulatory factors that Mike talked
about, that E2F for example, cyclins during the
formation of the tumor, and at the same time we are
trying to see that whether or not the ring value stage
of the tumor, the partner of the T-antigen changes.
We know that the T-antigen binds to p53 and
Rb. These experiments are done in cell line, but we
can utilize this study, animal model, to do experiments in whole animal model.
Now, another model is the transgenic mice,
that expression of the T-antigen induced tumor in the
transgenic mice. And analysis of the tumor showed
that a tumor is formed in the abdominal area. It was
NSC-positive, normal crest origin tumor.
And when you do a histopathological analysis
of this tumor formed upon expression of JC in trans-genic mice, you see that they're highly differentiated
cells that -- tumor cells, basically infiltrates
between the wall of the colon and the muscle cells
here. These are all the tumor cells and these are all
T-antigen-positive cells.
Now, what we are trying to do, we are trying
to use these transgenic mice to study the importance
of some of the cell cycle regulators in whole animal
model information of the tumor. First we demonstrated
that T-antigen was expressed in RT-PCR, in almost in
every tissue of this experimental animal.
But when you look at the Weston blot you see
that the detectable level of the T-antigen was
obtained only in the tumor but not in any other tissue
derived from this animal.
So in the next slide here, we looked at the
interaction of T-antigen with p53 tumor suppressor,
our favorite experiment as Mark demonstrated, by
coming and precipitating with one antibody, probe the
Western blot with another antibody. The take-home
message is yes, p53 associates with the tumor and the
T-antigen associates with p53 in the old 2-reciprocal
experiment.
And the working model was is that if the JC
virus T-antigen associated with p53 takes away p53
from the loop as a result on same target to p53, p21
WAF, a protein which could suppress function of
cyclin's associated kinases and the complex which
eventually could phosphorylate Rb and liberate E12,
could be other function in this case.
And what we know is, each WAF could regulate
number of other cell cycle genes and put the cells
into the rapid proliferating stage, and at the same
time, T-antigen could associate with Rb, another way
that liberates E12.
Now, we really went on through every step of
this and examined every step in this tumor. For
example we asked, what happened to WAF gene in this
tumor tissue? We realized that there is no WAF gene,
basically WAF protein in the tumor cells. And then we
asked, if you don't have WAF, what happens to all the
cyclin and CDKs?
What I'm going to do in the next slide just
showed two examples of that. This is cyclin E and
this shows the level of cyclin E, and this shows that
it's a kinase activity.
And the partner to cyclin E is CDK2 -- the
level of cyclin is CDK2 and its kinase activity. So
you see that it is highly active in these tumor cells.
What happens if the cyclin E, CDK2 is very active?
After the previous pathway -- in the next slide -- we
examined the Rb phosphorylation and you see Rb,
unphosphorylated Rb which is supposed to be the single
band in the tumor, becomes two bands. The top band is
phosphorylated.
If the Rb phosphorylated dose E12 liberates
from the Rb -- next slide -- it does, and it all
regulates itself and you see massive amounts of E12.
And if E12 is functional or not -- yes it is, because
it activates PCNA -- it's on the same target and
expressed in the tumor.
So you see that we can utilize this model to
really go through all those cascade of events that has
been described in the cell cycle, and identify the
regulatory factors which really is important for the
pathogenesis of the JC-induced tumor.
Now, what I'm going to do is, we switched to
the human samples, and we all learned that JC virus
could be associated with a number of tumors in humans,
we learned this morning, and these are studies done
previously by many other people.
Recently, maybe two years ago, a 61-year-old, immunocompetent, HIV-negative -- this is very
important clinically -- individual came to neurology
service and he had a multiple grand mal seizure.
This individual, after MRI, showed that the
hypointension area in the left frontal lobe of the
brain -- after about a year, gallilinium staining
shows a new enhancement which sits right in the center
of the previous enhancement, suggesting that the tumor
is formed here.
Now, if you see that the tumor mass was so
big that it pushed the lower, the left anterior -- the
formal -- this structure, and you see that this
structure, which is ventricular, should be like this.
But the tumor is pushing this toward the right lobe.
Now anyway, so we got the samples from these
patient, and the RNA protein DNA was extracted and we
did RT-PCR for the presence of T-antigen DNA which was
there, and the RNA was utilized for the primary
extension for expression of JC virus RNA, and the
results is here. Basically says yes, early RNA was
expressed.
And then when we look at the protein by
Western blot, here is a T-antigen of the JC in the
tumor specimen. And if you do immunohistochemistry
you see that the T-antigen is present in 50 percent of
the tumor cells.
This slide shows the staining of cells with
Chi-67. It's a self-proliferating marker showing that
the cells are highly proliferating and this is Luxor
blue staining showing that the level of the myelination -- which shows they're heavily myelinated.
Now, if that T-antigen doesn't belong to a
JC virus, we amplify the control region of this virus,
and after sequencing they turn out to be member of a
JC virus -- it's a mat-4 strain of the virus which has
a characteristic of 98 base pair and 79 base pair
sequence.
Now, if the virus is there, why does it
cause PML? Why cannot replicate and destroy the
oligodendrocyte? We thought initially that it might
be a mutation, replication which does not respond to
T-antigen. As a result, you create a situation like
hamster; that you produce T-antigen in the brain but
DNA replication does not take place. T-antigen
triggers a cascade of events, and lead to the formation of the tumor in this patients.
Clearly the T-antigen was intact. That was
a wrong assumption. Then we learned from SV40 there's
another protein in the leader of the late gene --
adenoprotein.
We learned that if you introduce mutations
in the AUG of the adenoprotein -- this is worked on by
Tom Shank many years ago and also some of the mutants
that Jim Pepper has created, the mutation which has a
T-antigen deletion of the carboxy terminal of a SV40
T-antigen -- what happens is you do not get a viral
protein, late protein, be transported to the nuclei
and form the virions.
And when we look at the adenoprotein of the
JC virus we found that there's a mutation -- three
nucleotides right at the initiation site which put the
adenoprotein on a frame. At present we don't know
that's the cause, that's the important event that put
the virus through the other pathway, the tumor pathway
or not. So that's a question that we're asking.
So I'm going to stop here, but before ending
I would like to -- well, Dr. Shah mentioned something
very interesting this morning. He mentioned that this
meeting reminds him about 10, 15, 20 years ago when
the people, the polyomaviruses were gathering and
talking about this system. And indeed it does. It
resembles like at those meetings that we used to go
to, Cold Spring Harbor and talking about polyomavirus,
mostly on SV40.
But there's one person that's missing in
this today, and that's a person who did a lot of work,
a lot of contribution on SV40; things that we learned
on enhancer regulation of SV40, and the T-antigen.
This person is George Khoury who left us about ten
years ago. In 1987 he died of lymphoma, and I think
it's very appropriate that at this meeting we just
remember him and his contribution.
Thank you.
CHAIRMAN BRIEMAN: Thank you very much, Dr.
Khalili. Our next speaker is Dr. Richard Frisque of
Penn State University, and the title of his presentation if, "Rearranged and chimeric primate polyomavirus
genomes".
Return to Table of Contents
DR. FRISQUE: Well, I'd like to offer my
thanks also, to the organizers for the opportunity to
speak. The focus of my laboratory is on the pathogenic and oncogenic potential of JC virus. Now, much of
our work has relied upon the biological and molecular
comparisons with related viruses BK and SV40.
This slide shows the comparison of the JC
and SV40 genomes, and these are very similar. They
share 69 percent sequence identity. JC and BK share
even higher: 75 percent sequence identity. The
organizations as you can see, are essentially identical.
There are three regions as already have been
pointed out. I'll just quickly do that again. The
early region which includes this T-antigen that we've
all been talking about. In the case of JC there are
five different proteins produced, now we know, in the
early region, based on alternative splicing mechanisms.
The late region includes the capsid proteins
and then this control region -- or the regulatory
region as I call it -- is also present; the third
region. It includes the replication origin as well as
the signals for transcription.
Now although many biological similarities
occur between these three viruses, JC is distinct. It
has a prolonged lytic cycle; it is very inefficient at
transforming cells in culture; and its expression is
restricted to only a few cell types.
Over ten years ago we asked the question,
what makes JC biologically distinct? Which regions of
the genome, which sequences are important? And we
began making in our first approach, chimeric virus
between JC, BK, and SV40.
Now, our prediction was that if we replaced
some of the JC sequences with those of SV40 or BK, we
might make a more active virus and identify some of
those sequences that contribute to JC's unique
biology.
Now, in terms of relevance this workshop, I
think that if co-infections to occur in the human host
with one or more of these viruses, that in fact, if a
combination can occur between these three viruses then
perhaps we might generate more viable, perhaps more
active, recombinance.
Alternatively, or maybe in addition, we also
may see complementation occurring, where one virus
could complement the growth of the second co-infecting
virus. So that's the relevance then, to this workshop.
This slide shows you the first of the
chimeric genomes that we've produced. These are what
I call regulatory region chimeras. What we do is, we
take the coding region of one virus -- in this case,
JC -- replace its regulatory sequences with those of
BK or SV40. And similar things were done with the
other two viruses.
Now, if you can see it with this color --
I'm not sure how it looks back there -- but what we
first did with these chimeras was to look at their
transforming behavior in rodent cells. And what we
found was that when we had the same coding sequences
linked to the various regulatory regions, in all cases
SV40 was always more potent, JC was always by far, the
weakest at transforming cells.
Similarly, if we took viruses that had the
same regulatory region and different coding regions,
that again SV40 was always the most potent, JC was
always the weakest. And in fact, the coding sequences
seemed to have more of an influence upon this restricted behavior of JC than even the regulatory
sequences.
Now, we've also done some tumorigenicity
studies. This has been done with transgenic mice, and
as others have shown as well, the regulatory sequences
of JC, SV40 types of chimeras, the regulatory sequences are influencing the location of where the tumor
occurs, whereas the coding region -- again the T-antigen, primarily -- is involved in the tumor
induction itself.
Well again, these same chimeras are shown in
this slide, but in this case now, we're looking at
lytic behavior in terms of DNA replication and the
ability to produce viruses themselves, and also
looking at host range effects.
And what we found that, somewhat to our
surprise, that those constructs that had JC regulatory
sequences in either SV40 or BK coding sequences, that
in fact, these were viable viruses -- viruses produced. And in fact, they were more active than wild
type JC itself.
In addition, when we took one of these
chimeras, JC SV40, we found that its host range was
expanded over that of wild type JC. In fact, these
not only grew in human cells but they also grew in
monkey cells as well.
Finally, the last surprise from looking at
lytic behavior of these chimeras is shown over here on
the right, when we had constructs that had JC coding
sequences linked to BK or SV40 regulatory sequences,
which are more potent, that in fact these were dead
viruses.
So if I could have the next slide, I would
like to then look at that last point in more detail.
Here what we've done is make another kind of chimera.
In this case we've made chimeric replication origins.
We've made chimeric regulatory regions to see if we
could find out why the JC coding sequences in the
particular T-antigen was unable to interact productively with the origin of SV40 and BK.
Now what we've done is, we've put the
regulatory sequences of the various viruses onto a
plasmid that does not contain any other sequence
information for viruses, for the viral proteins. So
just the regulatory sequences are on this plasmid.
And as we've shown already with the JCT
protein, when these constructs were put into cell
lines expressing JCT protein, both BK and SV40 were
unable to -- their origins not replicate in the
presence of JC T-antigen.
What we found from the chimeras was, when we
had a JC on the early side of the replication and
origin, core origin, that SV40 sequences on the late
side, that in fact again, JC's T-antigen was not able
to productively interact with those sequences. In
other words, sequences on the late side of the core
origin were responsible for JC's T-protein's ability
to distinguish between the two origins.
And in fact, we've gone through the site-
specific mutational analysis now, where there's three
nucleotide differences within that small core region
within the AT-rich region, that allows JC to distinguish between the two origins.
On the other hand, all the replication of
origins, chimeric or wild type, were able to interact
with the SV40 T-antigen as shown on the right.
On this slide I'm showing you a new set of
chimeras. In this case we're producing chimeras that
had T-antigens that were exchanged for sequences in
three locations: either at the amino terminus, in the
central region of the T-protein, or the carboxy
terminus.
And in this slide I'm showing you a regulatory region -- and in this case, SV40's regulatory
region, although we've also made chimeras with the
JC's regulatory region as well. I'll just show you
the data with the SV40 origin and transcriptional
control signals.
And what we've found is, when we look at
transformation in rodent cells, that as expected, JC's
T-antigen if it was wild type, transformed very
poorly. By replacing the amino terminus with SV40
sequences, we did not see much of an increase in
transformed behavior. However, as we started to
replace carboxy and central regions of the T-antigen
with SV40 sequences in place of the JC, transforming
behavior went up until we used the wild type SV40 T-antigen which is very potent.
Now in addition, in studies done in collaboration with Dr. Frank O'Neill, we did some immortalization studies with these chimeras, in human cells.
And what we found was that most of these chimeras
could not immortalize human cells. SV40 does immortalize human cells, but ten percent of transformed
human cells become immortal.
What we were surprised to find was that
constructs that had JC or BK at the carboxy terminus,
these were also able to immortalize human cells and in
fact, it's a much higher level about 50 percent of the
lines that were looked at.
Well, in following with the way I've been
going here, we've also taken these same kind of T-antigens and looked at their ability to stimulate DNA
replication. Again, those are the same constructs as
I showed you on the last slide using the SV40 replication, origin, and transcription signals.
And what we found was that the constructs
that failed to interact with the SV40 origin for
replication -- for DNA replication -- were those
constructs that had JC within the central region of
the T-antigen. So those, in human cells, were unable
to replicate, thus identifying the part of T-antigen
which was able to distinguish between the two replication origins: JC or SV40.
We also found that two of the constructs,
those that had SV40 sequences at the carboxy end,
again were able to replicate in monkey cells as well
as human cells. So expanded host range involved as
expected, the carboxy terminus of T-antigen.
Well, I just wanted to summarize the
chimeric data at this point and again try to show you
some relevance to this workshop. If in fact, co-infections do occur, we have the question of whether
or not recombination can occur between these viruses.
And if so, if a recombinant does occur, what would we
predict, based on some of this data and some others,
is the phenotypes would be relative to wild type SV40.
And I would like to suggest as possibilities, that the recombinant would probably show reduced
transforming behavior, but perhaps higher or lower
mortalizing potential. We'd expect probably, DNA
replication activity would be reduced as well as virus
yields, we'd expect that the host range would perhaps
be more restrictive than SV40, and we would expect
that the recombinant itself might actually be more
active than wild type JC.
Alternatively, we can look at co-infections
as a possibility of complementation occurring. Again,
based on the data that I've shown you, we would
predict that JC probably would not enhance SV40 growth
if they co-infected the same cell. On the other hand,
we might predict that SV40 would enhance JC growth.
I'd like to switch gears at this point and
tell you about some experiments that we've been doing
looking for JC's presence in human tissues, again as
you've all heard, by PCR analysis and then followed by
sequence analysis.
We have not found any evidence yet for
recombination occurring between these three viruses.
What we do see, considerable rearrangement occurring
within the promoter/enhancer elements for transcription. And that's what I want to tell you about next.
What I'm showing you here is JC, and we're
looking at the regulatory region, in particular, the
transcriptional control region of the promoter/enhancer signals. In humans there are two types of
JCs that can be found: this archetype that you've
heard about, and what I call the rearranged form of
JC.
Now archetype is shown at the top in the
schematic, and what's unique about the archetype
genome is that it has a single copy of the promoter/
enhancer region; there are no large tandem duplications. On the other hand, I show you four examples of
rearranged forms of JC's transcription control region.
These all came from different PML patient's brains.
And what you can see is that, unlike the
archetype, there are differences in rearrange between
each other as well as with archetype. And they
involve deletion events usually involving the 66 base
per block of information in archetype shown here, and
sometimes its 23 base pair block of information is
missing as well.
Following these deletion events, then
duplication and sometimes even triplications occur,
such that these sequences here are duplicated relative
to archetype. What we believe is happening is that
archetype, which is found in the kidney and in urine
of healthy people, circulates in the population. We
become infected by that.
Within our body we believe that these
rearrangements may occur involving deletion events and
duplication events. And these rearranged forms have
been primarily found in brain, sometimes in PML
kidney, and in lymphocytes.
In our first PCR experiments that we
conducted about seven years ago, we first started by
looking at PML tissues. Obviously, we were expecting
to find JC in those tissues. And what I show you here
are the tissues of brain and kidney from five different PML patients.
Over here, the brain specimens. Lane 7s in
each case are the positive controls; lane 1 are the
negative controls. These are the five brain specimens; these are the five kidney specimens; and as
expected high levels of JC were found in these
tissues.
When we went ahead and sequenced these
isolets -- these PCR products, what we found was that
these in fact, were rearranged forms of JC. As well
in the kidney, we found primarily rearranged forms as
well, although we have found archetype in some PML
kidney.
More surprising at the time when we published this was that in fact, JC was also present in
normal brain tissue. In other words, we've had 18
different specimens shown on this slide in which
people had died of things unrelated to neurological
complications. And in fact, these are the positive
controls; negative controls are shown in the first
part of each panel. And these are the positive
samples that we've seen when we were doing PCR
analysis and then blotting the PCR products with the
JC-specific probe.
So JC we found, was present in greater than
50 percent of normal brain specimens that we looked
at. When we sequenced these, as I said -- I believe
I said -- these are rearranged forms. When we looked
at normal kidneys, on the other hand, normal kidney
was always archetype JC.
Well, at the same time when these initial
studies were being done we did have access to five
tumor specimens. These were five different patients
with medulloblastoma. So we also looked for the
presence of JC in those tumor samples and in fact, we
found high levels of JC in each one of those five.
This here in lane 8 represents a normal
brain -- one of our best normal brains in terms of the
amount of JC that was present. You can see the
difference in the amounts that were present. Again,
this is a positive control and a negative control.
Now, more recently our PCR analysis has been
conducted on a single PML patient. What makes this
patient interesting we believe, it is unusual in terms
of the way PML occurs. This occurred in a 5-year-old
child. PML usually occurs later in life.
This child had severe combined immune
deficiency. And so in addition to being young, we
believe also that the PML arose following a primary
infection rather than in most cases of PML where it
occurs following the reactivation of an earlier
persistent infection.
In addition, most cases of PML you can find
JC in the brain, in the lymphocytes, and in kidney.
When my grad student, Jason Newman, began looking at
this patient, we had specimens from eight different
tissues. And he found JC present in each one of
these: brain, coeliac plexus, spleen, kidney, lymph
node, liver, cardiac muscle, and lung. So JC was
present in all of these, and this is just a southern
blot to confirm the identity as JC.
Now, Jason was using primers that would lie
outside of the regulatory region of JC in a highly
conserved region for all JC isolets looked at so far.
And so the PCR primers he was using would allow to
amplify either archetype or rearranged forms of JC if
they were present.
What Jason found -- this is again, the
comparison, we're all going to compare with this
archetype strain called CY. CY is a strain that was
isolated by Dr. Yogo in Japan a number of years ago
and I'm using that as a reference.
What Jason found was that in all the kidney
clones that he looked at in sequence, they were
identical to CY except for a single change at a
hotspot of rearrangement for archetype at position
217. He also found, in cardiac muscle, archetype-like
JC. That is, there are small deletions or intermediate size deletion without duplication. He also found
in lung, another archetype-like strain.
This slide again -- with comparison to CY
shown at the top -- this is the other tissues that he
looked at. And what was he found was that there were
multiple rearrangements occurring within these JC
isolets. In fact, this is again unusual relative to
most PML patients that are looked at.
Usually when you look at a rearranged form
in a PML patient there's only one or maybe two types
of rearranged forms. Here, we found multiple kinds of
rearranged JC, and this is actually only a subset of
what he found.
Now you also notice that in some cases the
same clone was found in multiple tissues. In other
cases you found that in the same tissue -- brain,
brain, brain -- you found multiple clones. So there's
a lot of rearrangement going on here.
Now as I said, the primers that Jason was
using were laying right outside the regulatory region
so they would amplify whatever was predominantly
there. We wanted to see if archetype might be present
in tissues other than kidney, since that really hadn't
been looked at too closely.
This slide was constructed following JC's
work with archetype-specific primers. In other words,
he had two pairs of primers that he used, whereas one
member of each pair lay within the 66 base pair
sequence which would be archetype sequence that was
present. Rearranged forms essentially in this
patient, were always losing this region. So it would
be specific for the presence of archetype JC.
And what Jason found, was in the brain and
the lymph node that he could find the archetype JC or
archetype-like variance within brain and lymph node,
and this is the first time this has been shown.
So if I could have the last slide -- just
some conclusions from this work with the pediatric
patient that I've just been talking about. We believe
that the immune status at the time of exposure -- this
child has severe combined immune deficiency --
probably contributed to this widespread distribution
of JC that we see in her body, and to the extent of
rearrangement of this transcriptional control region.
The data I believe, shows us that rearrangements can occur early, perhaps after primary infection. This is in contrast to the past where we've
been thinking that it usually occurred following the
reactivation of a persistent infection. So at least
sometimes, rearrangement occurs quite soon.
We believe lymphocytes are probably involved; that whether they're involved directly in
rearrangement process we're not sure yet -- we may
hear some more information about that next; and we
believe that they certainly are involved in seeding
the virus to secondary sites of infection.
As already mentioned, we do not know where
the primary site of infection is, but lymphocytes may
take it, wherever that is, to these other tissues that
I've been showing you.
Finally, we would suggest that because we
see different predominance of archetype and rearranged
forms in different tissues, that in fact, this might
indicate that the replication potential of these two
forms differs in the differing tissues.
Now, given everything I've shown you so far,
as I said, we have not seen anything to resemble
recombination between JC, BK, and SV40 from these
kinds of studies. I should add quickly, that we
really haven't really looked that hard until recently.
So in terms of whether or not rearrangement occur,
that question is open.
Well, and partly for what I've shown, I
think rearrangements certainly do occur; they occur in
JC as well as in BK and SV40, leading to these
archetype to rearrange the types of transitions that
we see, so that this a highly plastic region of the
genome that might be interesting to look at further.
Thank you very much.
CHAIRMAN BRIEMAN: Well, thank you, Dr.
Frisque. You covered a lot of material within the
time limit and I think that was very nicely done.
The next speaker is Dr. Maria Chiard Monaco
of NIH, and she's been given a very limited time
period to address the question of JCV infection of
peripheral lymphoid tissue and the implications for
viral latency.
Return to Table of Contents
DR. MONACO: Good afternoon. First I want
to thank Dr. Lewis to give me the possibility to
present my data today. As we heard from the previous
speakers, JC is a DNA virus of the papomavirus family
and as Dr. Khalili mentioned before, more than 80
percent of the human population has been infected by
JC virus and few conversion occurs during childhood.
JC is thought as a neurotropic virus but as
we can see from these slides, JC can also infect cells
-- non-neural cells as well. Back in 1988, Dr. Sid
Hoff and Dr. Major found that JC virus DNA associated
to capitalize a lymphocyte from two PML patients in
the bone marrow and spleens. And we expounded a host
range of JC virus, focused our attention particularly
on cells of lymphoid tissue.
In this slide we have a nested PCR amplification from two AIDS patients: patient 1 is not PML,
and patient 2 was at the onset with PML. And this
line marker, there's normal B cells contains DNA from
a B cell isolated from peripheral blood mononuclear
cells of JC virus-negative individuals.
Patient 1 and patient 2 -- in the lane of
patient 1 and patient 2 we have DNA extracted from
unfractionated peripheral mononuclear cells, or B and
T subpopulation isolated by cell sorting technique.
And we found no evidence of JC virus DNA in
the T cells from the PML patients and in the unfractionated peripheral mononuclear cells, or B and T
cells from the non-PML patients. So this data shows
us that PML can infect the peripheral lymphocytes in
vivo, in particular, this population.
So next question we asked was, whether
progenitor cells could be susceptible to JC viral
infection. And to answer this question we looked at
two progenitor cell lines, KG1 and KG1-A. These lines
are derived from a patient who presented with leukemia
but eventually developed acute myelogenous leukemia.
But these two lines, CD34 antigen, that is a marker
from stem cells.
As we can see in these slides we have in A,
T-antigen-positive cells, and in B, T-antigen-positive
cells detected with two different monoclonal antibodies, and in C we have some hybridization with the KG1-A positive cells using a biotinylated JC virus probe.
And the interesting feature of these cell
lines is that when they are treated with formalizers
they can differentiate -- KG1 can differentiate into
a macrophage -- a mature macrophage. Instead, KG1-A
cells are not affected by this treatment. So for this
reason, we infected both KG1 and KG1-A untreated, and
we saw susceptibility of these cells by JC virus.
When we infected KG1 treated, PML-treated
cells, differentiating cells with microphage-like
characteristics, these cells were no longer susceptible to JC virus infection. So this data clearly
demonstrated that cells with monocytic lineage are not
susceptible to JC virus infection.
And we confirmed the susceptibility of some
cells, of precursor cells, with the primary CD 34,
human parameter CD34 cells as we can see in these
slides.
Previously, we described some of the
interaction between tonsillar thermo cells, in particular B lymphocytes. Thermo cells are an important
constituent of lymphoid organs, so we want to evaluate
their involvement in the polyomavirus infection.
In this slide we have a representative field
of tonsillar thermo cells infected by JC virus. The
percentage of thermo cells infected by positive for JC
virus T-antigen and viral DNA, was only 2.5 times
lower than the percentage of human fetal iligos cells
that are recognized as the most susceptible cells to
JC viral infection.
So if JC virus occurs by respiratory route,
tonsillar thermo cells, because they have relatively
high susceptibility to JV viral infection, and for the
natural interaction with the progenitor cells and
lymphocytes are ideally positioning to be a site for
initial infection and possibility to disseminate
virus.
We are testing this working hypothesis by
examining tonsillar tissue from children and other
donors for the presence of JC virus DNA, and so far
we've found 20 percent of these tonsils positive for
JC virus DNA. We don't know yet in which cell type
the virus, the DNA was sequestered, but this data are
additional evidence that tonsils or other lymphoid
organs could be a reservoir for the virus, or also
initial site for primary infection.
This work has been done in Gene Major's lab
and I want to thank him for his work. I also want to
thank Blanche Korfman, Peter Jensen, Dala Galanti,
Cathy Connor, for their help. Thank you very much.
CHAIRMAN BRIEMAN: Thank you, Dr. Monaco.
Now, before going off to lunch in what, for those of
you who haven't been here before, I think you'll find
to be a very atypically excellent cafeteria for, you
know, government cafeterias, let me remind you that we
will return here at 1:40 for a panel-audience discussion that will be moderated by Dr. Mike Fried. And
the topic is, "Issues related to the detection of SV40
DNA in human tissues". And again, if you have
additional data, either positive or negative, regarding detection that you'd like to have presented,
please contact him. Thank you.
(Whereupon, a luncheon recess was taken at 1:00 p.m.)
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