Some history on work done by the Gallo and Montagnier groups, 1982-1985. 6 May 2004
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Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545

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Re: Some history on work done by the Gallo and Montagnier groups, 1982-1985.

The Perth group wrote:

“…
To claim an “infectious molecular clone of HIV-1”, the following must be done:
(a) Take the RNA from a particle which has the morphological characteristics of retroviruses and which has been shown to be infectious;
(b) Introduce this RNA or its cDNA into cells;
(c) Show that the DNA is transcribed into the original RNA and that the RNA is translated into proteins;
(d) Show that the cells release particles morphologically identical to those from which the RNA was obtained;
(e) Show that the particles have the same RNA as the original RNA;
(f) Show that the particles are infectious.

Is this correct? If not, where are we wrong?
…”

The Perth group is partially correct, but they obviously either do not understand much about retrovirology and molecular genetics, or they understand but wish to mislead others. I will attempt to explain some of their apparent misconceptions.

(a) As far as I know, it is not possible to use “molecular tweezers” of some type to retrieve a single RNA molecule from a virus particle that has been viewed under an electron microscope. There are many methods currently available for cloning retroviral genomes that were not available in the 1983-1984 time period when the French lab team lead by Luc Montagnier and the USA lab team lead by Robert Gallo were first characterizing their isolates of HIV-1 M group subtype B virus. I will thus focus on what these two groups did to come up with the first infectious molecular clones of HIV.

For obtaining HIV RNA, both groups used cell-free supernatant from HIV-infected T-cells. Both groups first did extensive analyses to show that they were able to culture and passage the virus on T-cells, and that the cultured virus, even after many passages was still producing virions which were strongly reactive with sera from AIDS pateints [2-26]. During this era before the first infectious molecular clone of the virus was available it had already become clear that the cultured but as-yet uncloned virus responsible for the AIDS epidemic in the USA and Europe, and it was even linked to AIDS cases in Africa [25].

Both groups made extensive use of density gradient ultracentfifugation, often retrieving the 1.16g/ml density band from one centrifugation and loading it onto a second gradient to “double” band it. For one example, the 1983 Barre-Sinoussi paper states that the virus could be propogated on normal lymphocytes from ether newborns or adults, that the virus had a buoyant density of 1.16 g/ml, and that identical cultures which had not been infected with cell-free virus remained negative for RT activity. They also state that they used methods which can distinguish between true retroviral RT activity and a similar activity present in DNA-dependent polymerases. RNA from this 1.16 g/ml band can be labeled with 35S or 32P and used as a probe to locate proviral genomes in genomic libraries or in cells infected with HIV or related viruses [1,36 etc.].

(b) While it is now possible to transfect cells with RNA or cDNA and observe the effects of the transfected molecule, it is my understanding of the history of this type of process, that such transfections were not yet common in the 1984-1985 time period. Instead the cDNA was inserted into a bacterial and/or mammalian cell cloning vector, prior to transfection into the mammalian cells. See [27] for an example from a virus other than a retrovirus.

(c) After transfection of cells with either complete viral cDNA, which can potentially make full infectious virus particles, or cDNA derived from spliced viral mRNAs which can make only some of the viral proteins but not infectious particles, the viral RNA transcripts can be, and were detected by Southern blotting and other methods. Likewise viral proteins were detected by serology and other methods.

(d) When complete infectious molecular clones of HIV-1 are transfected into cells, virus particles indistinguishable from those produced by other, uncloned isolates of HIV-1 are indeed produced. These virus particles produced from molecularly cloned HIV-1 genomes look identical to uncloned viruses by electron microscopy ( see [12 and 28-33] for a few examples). They react the same with sera from AIDS patients, but do not react with sera from healthy controls nor controls infected with other viruses [3,7, 40, etc…]. They produc proteins of the same molecular weights as the protein produced by uncloned viruses of the same type. They use the same cellular receptors for entry. Etc… (e) Cloned an uncloned HIV-1 viruses do indeed contain the same 9.7 kb genome, and produce the same genomic and spliced mRNA transcripts. It is also critical to note that it has been shown many times, that uninfected cells do not contain DNA or RNA sequences that hybridize to HIV-1 probes. For example, figure 3 in [36] shows that no signal was detected in uninfected H9 cells, HT cells or normal human thymus cells, thus clearly indicating that the cloned virus was exogenous and not some type of activated endogenous retrovirus. (f) Both cloned and uncloned viral particles are infectious [37-40]. Mutations can be introduced into the cloned viral genomes to reduce or eliminate this infectivity [39, 41-43, etc…].

The Perth group wrote:

“…
But where are the references that vindicate these [Brian Foley’s] claims? …”
And
“… Before the gene of the human insulin was cloned, a protein was isolated (obtained separately from all other human proteins) and was shown to have some specific properties. Subsequently, this protein was shown to be coded by certain sequences in the DNA obtained from human cells. Then the insulin gene was cloned. That is, when the DNA sequences were introduced in suitable cells, the cells produced insulin.
…”

Where are the references that vindicate the Perth group’s claims? How was human insulin “purified” to 100% purity, separate from all other human proteins? See [34,35] for a start. I am not an expert on insulin, but I believe that it was first studied in pigs and cows, and that the human gene was most likely identified by Southern blotting with a pig or cow insulin gene or cDNA. This would be analogous to identifying the HIV-1 genome by Southern blotting with a SIV genome or gene fragment, without purifying or “isolating” the HIV virions. HIV was cloned and sequenced before the first SIV was molecularly characterized. The only point I am trying to make here is that the techniques of molecular biology, whether they are applied to mammalian genes and proteins or viral genes and proteins, are powerful and effective.

REFERENCES:

1: Alizon M, Sonigo P, Barre-Sinoussi F, Chermann JC, Tiollais P,
Montagnier L, Wain-Hobson S.
Molecular cloning of lymphadenopathy-associated virus.
Nature. 1984 Dec 20-1985 Jan 2;312(5996):757-60.
PMID: 6096717

2: Sarngadharan MG, DeVico AL, Bruch L, Schupbach J, Gallo RC.
HTLV-III: the etiologic agent of AIDS.
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PMID: 6100648

3: Markham PD, Sarngadharan MG, Salahuddin SZ, Popovic M, Gallo RC.
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Competing interests: None declared