Did Luc Montagnier Discover
By Djamel Tahi
Continuum Winter 1997
Text of a videotape interview performed at the Pasteur Institute,
July 1997. Please note: The answers by Luc Montagnier have been numbered
for easier reference to the analyses in the reply by
Papadopulos-Eleopulos et al.
DT: A group of scientists from Australia argues that nobody up
till now has isolated the AIDS virus, HIV. For them the rules of
retrovirus isolation have not been carefully respected for HIV. These
rules are: culture, purification of the material by ultracentrifugation,
Electron Microscopic (EM) photographs of the material which bands at the
retrovirus density, characterisation of these particles, proof of the
infectivity of the particles.
LM: No, that is not isolation. We did isolation because we "passed
on" the virus, we made a culture of the virus. For example Gallo said :
"They have not isolated the virus...and we (Gallo et al.), we have made
it emerge in abundance in an immortal cell line." But before making it
emerge in immortal cell lines, we made it emerge in cultures of normal
lymphocytes from a blood donor. That is the principal criterion. One had
something one could pass on serially, that one could maintain. And
characterised as a retrovirus not only by its visual properties, but
also biochemically, RT [reverse transcriptase] activity which is truly
specific of retroviruses. We also had the reactions of antibodies
against some proteins, probably the internal proteins. I say probably by
analogy with knowledge of other retroviruses. One could not have
isolated this retrovirus without knowledge of other retroviruses, that's
obvious. But I believe we have answered the criteria of isolation.
DT: Let me come back on the rules of retrovirus isolation which
are : culture, purification at the density of retroviruses, EM
photographs of the material at the retrovirus density, characterisation
of the particles, proof of the infectivity of the particles. Have all
these steps been done for the isolation of HIV? I'd like to add,
according to several published references cited by the Australian group,
RT is not specific to retroviruses and, moreover, your work to detect RT
was not done on the purified material?
LM: I believe we published in Science (May 1983) a gradient which
showed that the RT had exactly the density of 1.16. So one had a peak
which was RT. So one has fulfiled this criterion for purification. But
to pass it on serially is difficult because when you put the material in
purification, into a gradient, retroviruses are very fragile, so they
break each other and greatly lose their infectivity. But I think even so
we were able to keep a little of their infectivity. But it was not as
easy as one does it today, because the quantities of virus were
nonetheless very weak. At the beginning we stumbled on a virus which did
not kill cells. The virus came from an asymptomatic patient and so was
classified amongst the non-syncythia-forming, non-cytopathogenic viruses
using the co-receptor ccr5. It was the first BRU virus. One had very
little of it, and one could not pass it on in an immortal cell line. We
tried for some months, we didn't succeed. We succeeded very easily with
the second strain. But there lies the quite mysterious problem of the
contamination of that second strain by the first. That was LAI. (2)
DT: Why do the EM photographs published by you, come from the
culture and not from the purification?
LM: There was so little production of virus it was impossible to see
what might be in a concentrate of virus from a gradient. There was not
enough virus to do that. Of course one looked for it, one looked for it
in the tissues at the start, likewise in the biopsy. We saw some
particles but they did not have the morphology typical of retroviruses.
They were very different. Relatively different. So with the culture it
took many hours to find the first pictures. It was a Roman effort! It's
easy to criticise after the event. What we did not have, and I have
always recognised it, was that it was truly the cause of AIDS. (3)
DT: How is it possible without EM pictures from the purification,
to know whether these particles are viral and appertain to a retrovirus,
moreover a specific retrovirus?
LM: Well, there were the pictures of the budding. We published images
of budding which are characteristic of retroviruses. Having said that,
on the morphology alone one could not say it was truly a retrovirus. For
example, a French specialist of EMs of retroviruses publicly attacked me
saying: "This is not a retrovirus, it is an arenavirus". Because there
are other families of virus which bud and have spikes on the surface,
DT: Why this confusion? The EM pictures did not show clearly a
LM: At this period the best known retroviruses were those of type C,
which were very typical. This retrovirus wasn't a type C and
lentiviruses were little known. I myself recognised it by looking at
pictures of Equine infectious anaemia virus at the library, and later of
the visna virus. But I repeat, it was not only the morphology and the
budding, there was RT...it was the assemblage of these properties which
made me say it was a retrovirus. (5)
DT: About the RT, it is detected in the culture. Then there is
purification where one finds retroviral particles. But at this density
there are a lot of others elements, among others those which one calls
LM: Exactly, exactly. If you like, it is not one property but the
assemblage of the properties which made us say it was a retrovirus of
the family of lentiviruses. Taken in isolation, each of the properties
isn't truly specific. It is the assemblage of them. So we had: the
density, RT, pictures of budding and the analogy with the visna virus.
Those are the four characteristics. (6)
DT: But how do all these elements allow proof that it is a new
retrovirus? Some of these elements could appertain to other things,
LM: Yes, and what's more we have endogenous retroviruses which
sometimes express particles - but of endogenous origin, and which
therefore don't have pathological roles, in any case not in AIDS.
DT: But then how can one make out the difference?
LM: Because we could "pass on" the virus. We passed on the RT
activity in new lymphocytes. H. We got a peak of replication. We kept
track of the virus. It is the assembly of properties which made us say
it was a retrovirus. And why new? The first question put to us by Nature
was: "Is it not a laboratory contamination? Is it perhaps a mouse
retrovirus or an animal retrovirus?". To that one could say no! Because
we had shown that the patient had antibodies against a protein of his
own virus. The assemblage has a perfect logic! But it is important to
take it as an assemblage. If you take each property separately, they are
not specific. It is the assemblage which gives the specificity. (8)
DT: But at the density of retroviruses, did you observe particles
which seemed to be retroviruses? A new retrovirus?
LM: At the density of 1.15, 1.16, we had a peak of RT activity, which
is the enzyme characteristic of retroviruses. (9)
DT: But could that be something else?
LM: No..in my opinion it was very clear. It could not be anything but
a retrovirus in this way. Because the enzyme that F. Barre-Sinoussi
characterised biochemically needed magnesium, a little like HTLV
elsewhere. It required the matrix, the template, the primer also which
was completely characteristic of an RT. That was not open for
discussion. At Cold Spring Harbour in September 1983, Gallo asked me
whether I was sure it was an RT. I knew it, F. Barre-Sinoussi had done
all the controls for that. It was not merely a cellular polymerase, it
was an RT. It worked only with RNA primers, it made DNA. That one was
sure of. (10)
DT: With the other retroviruses you have met in your career did
you follow the same process and did you meet the same
LM: I would say that for HIV it is an easy process. Compared with the
obstacles one finds for the others...because the virus does not emerge,
or indeed because isolation is sporadic - you manage it one time in
five. I am talking about current research into others illnesses. One can
cite the virus of Multiple Sclerosis of Prof. Peron. He showed me his
work a decade ago and it took him around ten years to finally find a
gene sequence which is very close to an endogenous virus. You see...it
is very difficult. Because he could not "pass on" the virus, he could
not make it emerge in culture. Whereas HIV emerges like couch grass. The
LAI strain for example emerges like couchgrass. That's why it
contaminated the others. (11)
DT: With what did you culture the lymphocytes of your patient?
With the H9 cell line?
LM: No, because it didn't work at all with the H9. We used a lot of
cell lines and the only one which could produce it was the Tambon
DT: But using these kinds of elements it is possible to introduce
other things capable of inducing an RT and proteins, etc..
LM: Agreed completely. That's why finally we were not very ardent
about using immortal cell lines. To cultivate the virus en masse - OK.
But not to characterise it, because we knew we were going to bring in
other things. There are MT cell lines which have been found by the
Japanese (MT2, MT4) which replicate HIV very well and which at the same
time are transformed by HTLV. So, you have a mix of HIV and HTLV. It is
a real soup. (13)
DT: What's more it's not impossible that patients may be infected
by other infectious agents?
LM: There could be mycoplasmas...there could be a stack of things.
But fortunately we had the negative experience with viruses associated
with cancers and that helped us, because we had encountered all these
problems. For example, one day I had a very fine peak of RT, which F.
Barre-Sinoussi gave me, with a density a little bit higher, 1.19. And I
checked! It was a mycoplasma, not a retrovirus. (14)
DT: With the material purified at the retrovirus density, how is
it possible to make out the difference between what is viral and what is
not? Because at this density there's a stack of other things, including
"virus-like" particles, cellular fragments...
LM: Yes, that's why it is easier with the cell culture because one
sees the phases of virus production. You have the budding. Charles
Dauget (an EM specialist) looked rather at the cells. Of course he
looked at the plasma, the concentrate, etc...he saw nothing major.
Because if you make a concentrate it's necessary to make thinly sliced
section [to see a virus with the EM], and to make a thin section it is
necessary to have a concentrate at least the size of the head of a pin.
So enormous amounts of virus are necessary. By contrast, you make a thin
section of cells very easily and it's in these thin sections that
Charles Dauget found the retrovirus, with different phases of budding.
DT: When one looks at the published electron microscope
photographs, for you as a retrovirologist it is clear it's a retrovirus,
a new retrovirus?
LM: No, at that point one cannot say. With the first budding pictures
it could be a type C virus. One cannot distinguish. (16)
DT: Could it be anything else than a retrovirus?
LM: No.. well, after all, yes .. it could be another budding virus.
But there's a ... we have an atlas. One knows a little bit from
familiarity, what is a retrovirus and what is not. With the morphology
one can distinguish but it takes a certain familiarity. (17)
DT: Why no purification?
LM: I repeat we did not purify. We purified to characterise the
density of the RT, which was soundly that of a retrovirus. But we didn't
take the peak...or it didn't work...because if you purify, you damage.
So for infectious particles it is better to not touch them too much. So
you take simply the supernatant from the culture of lymphocytes which
have produced the virus and you put it in a small quantity on some new
cultures of lymphocytes. And it follows, you pass on the retrovirus
serially and you always get the same characteristics and you increase
the production each time you pass it on. (18)
DT: So the stage of purification is not necessary?
LM: No, no, it's not necessary. What is essential is to pass on the
virus. The problem Peron had with the multiple sclerosis virus was that
he could not pass on the virus from one culture to another. That is the
problem. He managed it a very little, not enough to characterise it. And
these days to characterise means above all at the molecular standard. If
you will, the procedure goes more quickly. So to do it : a DNA, clone
this DNA, amplify it, sequence it, etc..So you have the DNA, the
sequence of the DNA which tells you if it is truly a retrovirus. One
knows the familiar structure of retroviruses, all the retroviruses have
a familiar genomic structure with such and such a gene which is
DT: So, for isolation of retroviruses the stage of purification is
not obligatory? One can isolate retroviruses without purifying?
LM: Yes .. one is not obliged to transmit pure material. It would be
better, but there is the problem that one damages it and diminishes the
infectivity of the retrovirus. (20)
DT: Without going through this stage of purification, isn't there
a risk of confusion over the proteins that one identifies and also over
the RT which could come from something else?
LM: No .. after all, I repeat if we have a peak of RT at the density
of 1.15, 1.16, there are 999 chances out of 1,000 that it is a
retrovirus. But it could be a retrovirus of different origin. I repeat,
there are some endogenous retroviruses, pseudo-particles which can be
emitted by cells, but even so, from the part of the genome that provides
retroviruses. And which one acquires through heredity, in the cells for
a very long time. But finally I think for the proof - because things
evolve like molecular biology permitting even easier characterisation
these days - it's necessary to move on very quickly to cloning. And that
was done very quickly, as well by Gallo as by ourselves. Cloning and
sequencing, and there one has the complete characterisation. But I
repeat, the first characterisation is the belonging to the lentivirus
family, the density, the budding, etc.. the biological properties, the
association with the T4 cells. All these things are part of the
characterisation, and it was us who did it. (21)
DT: But there comes a point when one must do the characterisation
of the virus. This means: what are the proteins of which it's composed?
LM: That's it. So then, analysis of the proteins of the virus demands
mass production and purification. It is necessary to do that. And there
I should say that that partially failed. J.C. Chermann was in charge of
that, at least for the internal proteins. And he had difficulties
producing the virus and it didn't work. But this was one possible way,
the other way was to have the nucleic acid, cloning, etc. It's this way
which worked very quickly. The other way didn't work because we had at
that time a system of production which wasn't robust enough. One had not
enough particles produced to purify and characterise the viral proteins.
It couldn't be done. One couldn't produce a lot of virus at that time
because this virus didn't emerge in the immortal cell line. We could do
it with the LAI virus, but at that time we did not know that. (22)
DT: Gallo did it?
LM: Gallo? .. I don't know if he really purified. I don't believe so.
I believe he launched very quickly into the molecular part, that's to
say cloning . What he did do is the Western Blot. We used the RIPA
technique, so what they did that was new was they showed some proteins
which one had not seen well with the other technique. Here is another
aspect of characterising the virus. You cannot purify it but if you know
somebody who has antibodies against the proteins of the virus, you can
purify the antibody/antigen complex. That's what one did. And thus one
had a visible band, radioactively labelled, which one called protein 25,
p25. And Gallo saw others. There was the p25 which he called p24, there
was p41 which we saw... (23)
DT: About the antibodies, numerous studies have shown that these
antibodies react with other proteins or elements which are not part of
HIV. And that they can not be sufficient to characterise the proteins of
LM: No! Because we had controls. We had people who didn't have AIDS
and had no antibodies against these proteins. And the techniques we used
were techniques I had refined myself some years previously, to detect
the src gene. You see the src gene was detected by immunoprecipitation
too. It was the p60 [protein 60]. I was very dexterous, and my
technician also, with the RIPA technique. If one gets a specific
reaction, it's specific. (24)
DT: But we know AIDS patients are infected with a multitude of
other infectious agents which are susceptible to ...
LM: Ah yes, but antibodies are very specific. They know how to
distinguish one molecule in one million. There is a very great affinity.
When antibodies have sufficient affinity, you fish out something really
very specific. With monoclonal antibodies you fish out really ONE
protein. All of that is used for diagnostic antigen detection. (25)
DT: For you the p41 was not of viral origin and so didn't belong
to HIV. For Gallo it was the most specific protein of the HIV. Why this
LM: We were both reasonably right. That's to say that I in my RIPA
technique...in effect there are cellular proteins that one meets
everywhere - there's a non-specific "background noise", and amongst
these proteins one is very abundant in cells, which is actin. And this
protein has a molecular weight 43000kd. So, it was there. So I was
reasonably right, but what Gallo saw on the other hand was the gp41 of
HIV, because he was using the Western Blot. And that I have recognised.
DT: For you p24 was the most specific protein of HIV, for Gallo
not at all. One recognises thanks to other studies that the antibodies
directed against p24 were often found in patients who were not infected
with HIV, and even in certain animals. In fact today, an antibody
reaction with p24 is considered non specific.
LM: It is not sufficient for diagnosing HIV infection. (27)
DT: No protein is sufficient?
LM: No protein is sufficient anyway. But at the time the problem
didn't reveal itself like that. The problem was to know whether it was
an HTLV or not. The only human retrovirus known was HTLV. And we showed
clearly that it was not an HTLV, that Gallo's monoclonal antibodies
against the p24 of HTLV did not recognise the p25 of HIV. (28)
DT: At the density of retroviruses, 1.16, there are a lot of
particles, but only 20% of them appertain to HIV. Why are 80% of the
proteins not viral and the others are? How can one make out the
LM: There are two explanations. For the one part, at this density you
have what one calls microvesicles of cellular origin, which have
approximately the same size as the virus, and then the virus itself, in
budding, brings cellular proteins. So effectively these proteins are not
viral, they are cellular in origin. So, how to make out the difference?!
Frankly with this technique one can't do it precisely . What we can do
is to purify the virus to the maximum with successive gradients, and you
always stumble on the same proteins. (29)
DT: The others disappear?
LM: Let's say the others reduce a little bit. You take off the
microvesicles, but each time you lose a lot of virus, so it's necessary
to have a lot of virus to start off in order to keep a little bit when
you arrive at the end. And then again it's the molecular analysis, it's
the sequence of these proteins which is going allow one to say whether
they are of viral origin or not. That's what we began for p25, that
failed ...and the other technique is to do the cloning, and so then you
have the DNA and from the DNA you get the proteins. You deduce the
sequence of the proteins and their size and, you stumble again on what
you've already observed with immunoprecipitation or with gel
electrophoresis. And one knows by analogy with the sizes of the proteins
of other retroviruses, one can deduce quite closely these proteins. So
you have the p25 which was close to the p24 of HTLV, you have the
p18..in the end you have the others. On the other hand the one which was
very different was the very large protein, p120. (30)
DT: Today, are the problems about mass production of the virus,
purification, EM pictures at 1.16, resolved?
LM: Yes, of course. (31)
DT: Do EM pictures of HIV from the purification exist?
LM: Yes. of course. (32)
DT: Have they been published?
LM: I couldn't tell you...we have some somewhere .. but it is not of
interest, not of any interest. (33)
DT: Today, with mass production of the virus, is it possible to
see an EM, after purification, of a large number of viruses?
LM: Yes, yes. Absolutely. One can see them, one even sees visible
DT: So for you HIV exists?
LM: Oh, it is clear. I have seen it and I have encountered it. (35)
Notes: Go here
for the reply by the Perth Group.