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DiscussionMethodology of virus recovery.

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DISCUSSION: Methodology of Virus Recovery
Pincus: On the basis of informal discussion, several
people have suggested a review of a few basic concepts
regarding type C viruses. The complex polymorphism
of endogenous type C viruses in many species has
become increasingly apparent in recent years. Many
criteria have been used in preliminary classifications,
including host range (e.g., ecotropic, xenotropic),
source of isolation (e.g., exogenous tumor, endogenous tissue), structural polypeptides (e.g., Class I, 11,
111). However, a comprehensive classification relating
structure and function is not yet available, and a
relatively detailed characterization must be provided
of each particular virus isolate.
It is also necessary to enumerate the technique
used to detect the presence of a particular type C
virus. Endogenous type C viruses have been detected
in at least three very different forms: 1) Copies of viral
genomes are found in normal tissues of all mouse
strains and many other species, using nucleic acid
hybridization. These genetic sequences are usually not
expressed, either as viral proteins or infectious virus.
2) Gene products of the “viral” genome are frequently expressed as viral structural proteins, in the
absence of complete infectious virus. A well-characterized example involves the GIx antigen, which is a
variant of the viral envelope glycoprotein gp70. Most
murine strains show low levels in the gp70 glycoprotein, as well as the major internal p30 protein, in
normal tissues. 3) The least common form of type C
virus expression involves actual infectious virus which
can be isolated from animals or tissue culture. Infectious type C virus appears after chemical and physical
stimuli (e.g., carcinogens, x-ray) in many mouse
strains; endogenous viruses are also isolated from
sporadic aging mice, with different incidences for various strains. However, infectious virus is found from
birth in only a few strains, e.g., A K R mice, which
express ecotropic virus and develop lymphoma, and
NZB mice, which express infectious xenotropic virus
and develop autoimmune disease.
New Zealand mice share many features found
in patients with systemic lupus erythematosus (SLE),
and spontaneous expression of infectious xenotropic
type C virus in these mice has provided a rationale to
search for a type C virus in SLE. However, as we have
heard here from Drs. Schwartz and Dixon. virus expression and autoimmune disease are not necessarily
Arthritis and Rheumatism, Vol. 21, No. 5 Supplement (June 1978)
correlated, and the possible role of virus expression in
autoimmunity remains enigmatic. Furthermore, there
is no conclusive evidence at present for the existence
of a unique human type C virus; all type C viruses
isolated from human tissue are identical in protein
structure to known primate viruses. Finally, the finding of altered immunologic responsiveness in NZB
mice and SLE patients, including elevated antibody
titers to most common human viruses found in sera of
SLE patients, raises the possibility that virologic phenomena may reflect rather than induce altered immune function. On the other hand, most of the immunologic abnormalities seen in SLE can be found with
clinical viral infection, supporting the “viral hypothesis” in SLE, although viruses other than type C viruses must remain under consideration.
Schwartz: May I ask a question about a point of nomenclature that has to do with the emphasis placed on the
distinction between exogenous and endogenous viruses? For example, the mouse ecotropic virus is put
in the category of exogenous virus. But in the mouse,
there are integrated viral genomes that segregate as
mendelian traits. These genes code for ecotropic viruses. Thus, there may be a point of confusion between true exogenous viruses like the feline leukemia
virus or the bovine leukemia virus and the mouse
ecotropic virus. By contrast with the former, the latter
are truly integrated and segregate as an endogenous
virus, like the xenotropic virus.
Todaro: I think we both agree that the mouse ecotropic
viruses are endogenous viruses. For virologists it is a
very important distinction; it makes a great deal of
difference in deciding whether one is dealing with an
agent coming in from the outside, the way a virus
does, or one is dealing with an endogenous agent
that becomes activated. The mouse ecotropic viruses
would be endogenous agents even though they may
move from cell to cell within the animal, and perhaps
recombine with each other. Feline leukemia and
bovine leukemia are more complicated. From the
epidemiology it is clear that the disease is a consequence of infection from the outside. Indeed, you
could also say the same thing about certain kinds of
murine viruses-that they too could be transmitted in
a horizontal manner.
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DISCUSSION
Paul: Dr. Aaronson, would you tell us more about the
mapping of genes for p30? Have any p30s been shown
to exist in polymorphic forms?
Aaronson: Genetic mapping in the murine system has
been done by the use of temperature sensitive mutants
and defective sarcoma viruses that turn out to be
recombinants between a portion of helper virus and
some other, presumably transforming sequences. Dr.
Mariano Barbacid of our laboratory has defined the
gag gene order that I described.
Paul: What about the place within the genome of the
mouse where the p30 gene exists?
Aaronson: In the mouse, those types of mapping studies
have not yet been finished. It is known that the structural information for one type C virus in the AKR
mouse is in linkage group 1. Also, there is evidence
with other endogenous mouse type C viruses that
independently segregating inducibility loci contain
viral structural information. The other point to make
is that these viruses appear to be present within the
genome as complete viruses; certainly this seems to be
so in the mouse system. In other words, all of the
information for a given virus can be localized to a
discrete locus within the mouse genome.
Steinberg: Since the AKR virus may be an unusual
virus, can any of the other viruses be mapped? We've
heard that there are 10 to 20 copies per genome.
Would these be linked to a specific region of chromosomal material or are they randomly inserted in the
genome?
Todaro: That is not entirely clear yet. I mentioned one
set of experiments in the cat where we have done
crosses t o genome-negative cats. First of all, the F,
has half the copy number and then the backcrosses
seem to segregate all or none of the copies. This would
suggest that the viral genes are not randomly distributed throughout the genome. But this is a case where
there is a potentially infectious virus. It is worth
pointing out that in some of the other cases like the
primates, even though we can detect hybridizing sequences, we d o not know if in fact the whole genome
is there. It may be defective-missing a piece necessary for replication. Alternatively, the genome could
be scattered around the cellular genome such that the
possibility of making a whole virus would be extremely small.
Benacerrai: You have listed a variety of type C viruses
but you have not said which ones you favor. I wonder
if you would care to comment more precisely as to
which is more important. Can you also tell us what
approaches could be used to determine the evolutionary value of type C viruses for the host?
Todaro: I d o not think this is an either/or situation: The
genes can be helpful to the host in certain situations
and harmful in others. Genes that confer disease resistance would have a strong selective advantage to the
recipient. Certainly the resistance to infection is an
important one, and I would expect resistance in the
cases where the virus has been transmitted to a recipient host-for example, the baboon virus into a cat
host. The baboon virus, which grows in many
species, will not grow in domestic cat cells but will
grow in leopard cells, so we are now in the process of
growing hybrids and backcross cell cultures to see if
there are a limited number of integration sites or if
there is chronic expression, for example, of gp70,
which blocks the receptors. In chickens such expression serves to block exogenous infection. But in
the case where it is transmitted to a new species and
acquired by a new species, the selective value would
be that of conferring resistance to infection.
Benacerraf: Is it a domesticated pathogen?
Todaro: Yes. When the animals are first exposed to the
virus, the prediction is that it might be quite lethal and
that survivors are selected from among those able to
successfully integrate the virus. For example, the virus
from the Asian mouse is quite lethal to gibbons by
producing leukemias and lymphosarcomas. If left unchecked, the mouse virus might integrate into some of
the gibbons and would then offer resistance to superinfection, perhaps by occupying a limited number of
integration sites. That mechanism obviously could
not explain the whole process. Another part of it
could be the gp70 differentiation story. There is not
just one function, and I'm not sure whether one
should consider the function of the whole virus or
each of the individual components. The reverse transcriptase may also have a normal function; having
that gene might be useful for a cell whether or not it
has or expresses a whole virus.
Tan: I wonder if Dr. Todaro or other virologists would
tell us a little more about the proteins that are transcribed by the gag genes. I was interested in his saying
that p12 is an RNA binding protein. Is this a basic
type of protein or is it an acidic type of protein? In
trying to draw a comparison with the basic proteins
DISCUSSION
and acidic proteins of mammalian cell nucleii, is there
anything more known about the p12, p15, and the p30
types of these proteins? There are a number of autoantibodies in SLE that are specific toward not only
basic but also acidic proteins, as Dr. Kunkel pointed
out this morning.
Todaro: The p12 of mice leukemia viruses is an acidic
phosphoprotein; the p10 is a very basic protein that
will bind nonspecifically to a variety of cellular
DNAs.
Ziff: Is there an immune response to gp70 in the NZB?
On the one hand, we have Dr. Dixon’s data on the
failure to find serum antibody and on the other Dr.
Mellor’s description of staining for viral constituents
in the glomerulus.
Pincus: The controversy stems in part from the different
methods used to detect viral antibody. There is evidence for naturally occurring antibody in sera of
many mouse strains to whole intact and disrupted type
C virus preparations. However, most murine sera that
bind to whole virus preparations do not bind isolated
viral proteins in radioimmunoassay. The explanation
for this apparent discrepancy is not known. The
whole virus assay may have greater sensitivity and it is
possible that antigenic determinants that are bound
with low avidity to natural antibody might be altered
during protein purification. On the other hand, a nonviral protein adsorbed onto the virus and reactive
with normal serum may be detected using radioimmunoprecipitation of whole virus.
Ziff: But others might say that the immunoglobulin
binding to virus is nonspecific.
Pincus: There may be a specific explanation for reactivity with nonviral proteins. Rabbit antisera prepared
to fetal bovine serum (independent of virus) show
significant binding to whole virus preparations. It is
possible that a complex of viral protein with a fetal
bovine serum component may be involved in this type
of radioimmunoprecipitation reactivity. As noted before, reactivity is generally not seen with isolated specific viral proteins.
Warner: Dr. Nowinski showed that by using the naturally occurring mouse antibody in a precipitation assay, and analyzing the precipitated material by polyacrylamide gel electrophoresis, the viral p15 and p30
components were present.
Pincus: This is correct. If an immunoprecipitate of normal serum with whole radiolabeled virus is analyzed
on polyacrylamide gels, these proteins are demonstrated. However, I reiterate that one cannot demonstrate immunoprecipitation by normal sera of the isolated p30 or p15E proteins purified prior to
immunoprecipitation. It’s a question of the order in
which analysis of viral proteins is performed. This
problem pertains directly to the question of a unique
human type C virus. Normal human immunoglobulins are similar to normal murine immunoglobulins,
in that both show the property of precipitating whole
mammalian type C virus preparations. The human
reactions fulfill many criteria for specificity, including
lack of reactivity with uninfected cells or with avian
type C viruses, which are immunologically noncrossreactive with the mammalian viruses. The situation is
complex, and not fully understood at present.
Schwartz: In the radioimmunoassay you described, Dr.
Aaronson, I assume you take the bulk tissue and
grind it up and make an extract and then test that
preparation for antigen?
Aaronson: Yes, you grind it up and sonicate the extract.
You can perform immunoassays on the extract, but
you can’t stop there because of the problem of nonspecific reactivity. Further purification procedures are
required.
Schwartz: It is certainly conceivable that within that
mass of tissue there will be differential expression of a
putative antigen on a minority of the cells. If, for
example, only 3% of the cells in the tissue express that
viral associated antigen, would you be able to detect
this in so few cells in the tissue?
Aaronson: The question pertains to how you do the
assays and what you do to ensure that you aren’t
losing something. I think that in any kind of system of
evaluation you have to develop appropriate positive
controls. In many endogenous virus systems, the approaches which we frequently described have worked
in isolation of antigens that can be shown to crossreact with the p30 protein of some known type C
virus.
Black: There is evidence that mice develop immune
responses both to their ecotropic and to their xenotropic viruses, with respect to both humoral and cellmediated immunity. In other words, there’s no apparent tolerance to their own endogenous virus. It may
be difficult to distinguish from the immune response
whether the virus is endogenous, where the genetic
information is vertically transmitted and integrated into
the DNA, or exogenous viruses, and where the trans-
DISCUSSION
mission is horizontal. This question relates to the two
viruses that were supposedly isolated from human
leukemia. One is a simian sarcoma virus from the
woolly monkey and the other is the baboon endogenous virus. Humans do not have information in their
DNA to code for such a simian sarcoma virus; so if
we do apply this criterion, it implies horizontal transmission, like any classical infectious agent. Therefore
one should develop antibodies to an acquired virus as
one does with any infectious agent.
The baboon infectious agent, as George Todaro outlined, is an endogenous xenotropic virus in
the DNA of the baboon. The proviral DNA is present
in a complete copy in the baboon. Thus, it’s fair to
ask, do humans have an endogenous virus? So far, the
only evidence that we do is that we have in our DNA
about 10% to 25% of the sequences of the baboon
endogenous virus and the immune response data that
I have discussed.
Schwartz: But there is a problem here, and I think it’s an
important one. First of all we don’t know what the
hypothetical agent of lupus might be. The tenor of the
whole morning session was that a type C virus would
be the best candidate. Of course, there’s no proof of
this. Therefore, the kind of positive control you mentioned may be an antibody, rather than an antigen. In
an “unknown” system a viral antigen could be expressed in a chronically infected individual on only a
minority of cells. In fact, Dr.Viola referred to that
this morning when he talked about subacute sclerosing panencephalitis and indicated that only certain
parts of an infected brain may contain the virus,
whereas other parts of the brain could be completely
normal. My point is that maybe the approach of
taking the whole spleen, for example, and making an
extract of it and then looking for an antigen is not
going to be fruitful because you will lose 1%, 5%, or
10% of cells that have the antigens you are seeking.
Dixon: Knowing the sensitivity of your test, one should
be able to calculate the threshold-approximately
what proportion of cells would have to be positive, or
how many specific molecules per 1Og cells would be
needed for reliable detection.
Schwartz: If you are talking about a low fraction of
antigen-positive cells, it seems to me that detection of
antibody to the putative antigen would be a far more
sensitive technique.
Phillips: I want to make a comment regarding the specificity of the measles antibody reaction This is old
s55
data now that I don’t think we should have to reiterate. We showed 4 years ago that you can adsorb
reactivity specifically with measles antigen and not
with control culture antigen; the antibodies are very
specific and they are elevated.
Aaronson: I think that lupus provides a very important
test system from the perspective of a virologist. Here
we can try to determine whether there may be aberrant reactivity against endogenous viral proteins,
whether p30, p10, pl5, or gp70. It’s a tedious screening process but it seems worth it to try to find evidence
of immune reactivity against type C viral proteins.
Patients with this disease may make immune responses against their endogenous viruses. So those
kinds of studies are both worthwhile and in progress.
Dixon: You were very clear about the failure to find
antibodies against type C viruses in humans with a
variety of diseases. How about the antigens? With
your best assays have you found any of these viral
antigens that you can correlate with lupus?
Aaronson: As of now, we have looked at five lupus
kidneys and the results have been negative. In terms
of looking at affected tissues of a number of disease
states, we’ve looked at over 100 or so-which is quite
a number when you have to d o the kind of purification procedures that we believe are necessary. I want
to stress that I believe that endogenous viruses will be
found in humans just as they have been found in so
many other species. But at the moment the evidence is
not convincing.
Phillips: Let me speak briefly about the point on the
baboon endogenous viruses because the experiments
that we’ve done to isolate the virus were specifically
aimed at this possibility, and failed to reveal infective
virus. The particles are there microscopically, whether
they are type C virus or not. But we don’t get baboon
virus out. So I think that the question is moot. One
can almost say with today’s knowledge that the baboon virus is not there.
Ziff: Dr.Black, why did you use placental material in
your study of immunity t o type C virus in humans?
Black: We looked in pregnancy for immune reactivity to
a possible endogenous virus because 1 ) viruses are
seen in placentas; 2) the baboon virus itself was isolated from a baboon placenta; 3) Gross and Feldman
have found several type C viruses in rat placentas.
When the viruses are seen in the placenta, they are
seen at the maternal-fetal junction. Of course, this is
S56
where one might expect to have an immunologic reaction-on the maternal or host side against the paternal antigens of the fetal graft in the placenta. Thus the
expression of endogenous type C viruses might result
from a host versus graft reaction; we have demonstrated that viruses can become activated from such
reactions.
Winchester: Dr. Black, in the cell-mediated immunity
that you see at this point in pregnancy-with the
virus-infected fibroblast system vigorously controlled
for considerations of histocompatibility-have you,
for instance, been able to use the fibroblasts of maternal origin to show that there is absolutely no possibility of the cellular response resulting from a weak
DISCUSSION
transplantation antigen enhanced by viral infection?
Furthermore, for considerations raised by the Doherty-Zinkernagel experiments, there should be some
histocompatibility between target cell and effector.
Black: No, we haven’t done that. We attempted to
utilize a system where effector and target cells share a
histocompatibility antigen, but we haven’t used the
mother’s own fibroblasts. However, the reaction is
virus-specific because with the uninfected fibroblast
we get no reaction and the same effector lymphocytes
give no reactivity with similar cells infected with another type C R N A virus, the simian sarcoma virus.
Also, adsorption of effector lymphocytes with disrupted virus absorbs the reactivity.
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