The methods used to clone HIV are the same ones used to clone other retroviruses. 5 June 2004
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Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545

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Re: The methods used to clone HIV are the same ones used to clone other retroviruses.

The Perth group wrote:
“… We are amazed that people like Christopher Noble take what we quote from other scientists' papers and ascribe them as being our “invention”. That is, ascribe to us formulations (“rules”) that other scientists have formulated. We have repeatedly pointed out this fallacy in this debate.
…”

The Perth group has indeed invented their “rules” for isolation of retroviruses. The paper they cite (1) as the root source of their rules, describes an attempt to seperate two or more viruses (Moloney-MSV, its helper virus Moloney-MuLV, and virions with one genome of each) based on differences in their sedimentation rates in sucrose gradient ultracentrifugation. The paper (1) does not state that any of these viruses was made 100% “pure” and free of all other substances. It states (page 239) “From electron micrographs (Figures 3b-d) these fractions contained mainly typical spherical C-type particles.”

Nowhere in the paper is the claim made that viruses must be made 100% pure before they can be characterized, further studied, or claimed to exist. Nowhere in the paper is it claimed that this method of separating virions from the majority of cellular components is the only method acceptable. Nowhere in the paper does is it claimed that any particular percentage of contaminating material will invalidate all other results.

Readers of the Perth group’s interpretations of papers should be careful to read the original papers themselves, in order to determine whether or not the Perth group is accurately representing the content of those papers.

The Perth group wrote:
“…
In 1975 the well-known retrovirologist John Bader wrote: “Although minor consistent differences between strains can be found, virions of RTV [retroviruses] have a characteristic buoyant density, and centrifugation to equilibrium in density gradients is the preferred technique for purification of RTV.”
…”

We have all repeatedly pointed out to the Perth group, that they are mistakenly interpreting the word “purification” in these papers, with the idea that the viral particles are made 100% pure with 0% contamination by any other material. Separation of materials by buoyant density in sucrose gradients can never assure 100% purity. It was the best method available and affordable in terms of labor and materials needed, in the early to mid 1970s for separating retroviruses from the majority of cellular debris, but it was not perfect. More to the point, perfection, or 100% purity is not needed, and never was claimed to be required. This method remains useful, and is routinely used in HIV research. However, other methods such as producing infectious molecular clones of retroviruses have allowed tremendous advances in viral studies since the mid to late 1970s.

The Perth group wrote:
“…
Barre-Sinoussi, Chermann and Montagnier themselves cited this paper in 1984: "For endogenous reactions using only the viral RNA as template primer, the virus [HIV] was concentrated and purified on sucrose gradient using isopycnic centrifugation (1)”. Reference 1 is their Spectra paper.
…”

I changed the reference number here to match refs listed below,

This is correct. The isolation and characterization of the first 5 or 10 infectious molecular clones of HIV-1 M group subtype B were carried out using EXACTLY the same protocols used for the isolation and characterization of the first complete genomes of Moloney-MSV, Moloney-MuLV, and many other retroviruses.

The Perth group has not totally invented the rules, they have only added on a few requirements of their own invention, in a rhetorical technique known as “moving the goalposts”. In order to claim that other retroviruses have been studied, but HIV has not, they attempt to convince people that a few trivial differences between HIV and other retroviruses result in failure to meet some fictitious “requirements”. The method of sucrose gradient centrifugation does indeed exist. The requirement that the resulting virus preparation be 100% pure in order to be useful for such purposes as obtaining RNA for use as a probe, does not.

The Perth group wrote:
“…
Q1. What method did Montagnier, the “discoverer” of “HIV”, use?
…”

Dr. Montagnier’s group of researchers used the EXACT same techniques to produce their first molecular clone of HIV-1 M group subtype B virus, as were used to produce the first molecular clones of the Moloney-MSV virus (2,3). They cloned the proviral DNA from infected cells to create the Lambda-J19 and Lambda-J81 clones. The Gallo labs used this method to create the Lambda-HXB-2 and Lambda-HXB-3 clones. The Gallo and Levy labs also used the same method that used to obtain the second molecular clone of Moloney-MSV (4). Beatrice Hahn used unintegrated circular viral DNA to create the Lambda-BH8, Lambda-BH-9, Lamda-BH10 clones (5). Paul Luciw used both integrated proviral DNAs to create the Lambda-8A, Lambda-8B, Lambda-9B and Lambda-7D clones, and unintegrated circular DNA to create the Lamda-7A clone (6,7).

All of these researchers made use of sucrose gradients for separating virions from the majority of cellular material. The probes made from the viral RNA do not have to be 100% pure. Probes need only to have more viral RNA than any other single species of RNA, so that they are able to light up the provirus-containing Lambda clones to a far higher degree than they light up the majority of the other Lamda clones, which contain cell-derived DNA without provirus. The partially pure or gradient-enriched virus was also used for other purposes such as western blots. For none of these purposes is 100% pure virus needed, whether it is HIV-1, HTLV-1, a mix of Moloney-MSV and Moloney-MuLV, or Moloney-MuLV alone.

The Perth group wrote:
“…
Q2. What is the evidence in the Montagnier 1983 study which convinced Christopher Noble “HIV” exists?
…”

The evidence presented in the 1983 paper with Francois Barre-Sinoussi as the lead author include:

1) The viral RT activity from HIV-infected PBMCs banded at 1.16 g/ml in sucrose density gradients.
2) PBMCs cultured in the same way, without added virus, were consistently negative for reverse transcriptase activity.
3) The viruses were observed budding from infected cells by EM.
4) The virus could be passaged in culture, both on PBMCs from adults and on cord blood lymphocytes from newborns.
5) The viral RT activity was clearly distinguishable from endogenous cellular activity, not only by banding at 1.16 g/ml, but also by its lack of inhibition by actinomycin D, its preference for Mg2+, and a lower pH optimum than the RT activity of HTLV-I.
6) The HIV virus did not react with monoclonal antibodies specific for HTLV-I, nor with polyclonal goat anti-HTLV-I antibodies. When whole serum from HIV-infected and HTLV-infected patients was used, there was some cross-reactivity between proteins with apparent molecular masses of p24 (HTLV-I) and p25 (HIV-1).

It is not very important whether or not the evidence in that single 1983 paper is enough to convince Christopher Noble that HIV exists. There have been several thousand other papers published before and after this paper which reconfirm the evidence that HIV-1 M group viruses are the cause of the majority of AIDS cases in humans, and that the HIV-1 N and O groups and several groups of HIV-2 viruses contribute very little to the pandemic.

For the past several years, the Perth group has been implying that Francois Barre-Sinoussi and Claude Chermann had created some “rules” for virus isolation in 1973. Because the Spectra paper (1) contained no such rules, I was under the impression that the Perth group was referring to some other paper or talk that I had not seen. In fact, the Spectra paper describes sucrose gradient separation of viral particles from the majority of cellular contaminants, but does not lay down any rules at all for “virus isolation”.

REFERENCES:

1: Sinoussi F, Mendiola L, Chermann JC, Jasmin C, Raynaud M.
Purification and Partial Differentiation of the Particles of Murine
Sarcoma Virus (M. MSV) According to their Sedimentation Rates in
Sucrose Density Gradients.
Spectra 4:237-243 (1973)

2: Reddy EP, Smith MJ, Aaronson SA.
Complete nucleotide sequence and organization of the Moloney
murine sarcoma virus genome.
Science. 1981 Oct 23;214(4519):445-50.
PMID: 6170110

3: Vande Woude GF, Oskarsson M, Enquist LW, Nomura S, Sullivan M,
Fischinger PJ.
Cloning of integrated Moloney sarcoma proviral DNA sequences in
bacteriophage lambda.
Proc Natl Acad Sci U S A. 1979 Sep;76(9):4464-8.
PMID: 159456

4: Tronick SR, Robbins KC, Canaani E, Devare SG, Andersen PR,
Aaronson SA.
Molecular cloning of Moloney murine sarcoma virus: arrangement
of virus-related sequences within the normal mouse genome.

PMID: 392518

5: Shaw GM, Hahn BH, Arya S, Groopman JE, Gallo RC, Wong-Staal F.
Molecular characterization of human T-cell leukemia (lymphotropic) virus
type III in the acquired immune deficiency syndrome.
Science 226: 1165-1171 (1984)

6: Luciw PA, Potter SJ, Steimer K, Dina D, Levy JA.
Molecular cloning of AIDS-associated retrovirus.
Nature. 1984 Dec 20-1985 Jan 2;312(5996):760-3.
PMID: 6096718

7: Sanchez-Pescador R, Power MD, Barr PJ, Steimer KS,
Stempien MM, Brown-Shimer SL, Gee WW, Renard A,
Randolph A, Levy JA, et al. Nucleotide sequence and expression of an AIDS-associated
retrovirus (ARV-2).
Science. 1985 Feb 1;227(4686):484-92.
PMID: 2578227

8: Barre-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S,
Gruest J, Dauguet C, Axler-Blin C, Vezinet-Brun F, Rouzioux C,
Rozenbaum W, Montagnier L.
Isolation of a T-lymphotropic retrovirus from a patient at risk for
acquired immune deficiency syndrome (AIDS).
Science. 1983 May 20;220(4599):868-71.
PMID: 6189183

Competing interests: None declared