INTERVIEW LUC MONTAGNIER
Did Luc Montagnier Discover HIV?
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. Totally. (1)
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
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 retrovirus?
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 "virus-like".
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.
DT: But how do all these elements allow proof that it is a new retrovirus?
Some of these elements could appertain to other things, "virus-like"...?
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. (7)
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 difficulties?
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.
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 Iymphocytes.
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. (15)
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 characteristic. (19)
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 HIV.
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.
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 contradiction?
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. (26)
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 difference?
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 bands.
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.