Re: Two questions to Brian Foley 29 April 2004
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Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545

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Re: Re: Two questions to Brian Foley

The Perth group wrote:

Two questions to Brian Foley

In his rapid response “Re: A second response to Christopher Noble's "What part of "infectious molecular clone" do you fail to understand?" 20th April 2004, Brian Foley wrote: “RNA [cellular RNA] splicing and polyadenylation occur in the nucleus, although there are also cytoplasmic version of polyadenylation”.

This fact has been known for a long time by many individuals including retrovirologists such as Robert Gallo (1,2). Because of this, it is difficult to understand why Gallo and Montagnier:

(i) banded their supernatants from cell cultures containing tissue from AIDS patients;

(ii) selected a poly(A)-RNA from the 1.16gm/ml band (without showing that the band contained retrovirus particles either in the pure or impure form) and called it “HIV” RNA (“HIV” genome);

(iii) introduced the cDNA of this poly(A)-RNA in cells and claimed that it proved molecular cloning of “HIV”. (3,4,5)

Is this true? If not, where are we wrong?

(i) Both the team of researchers lead by Robert Gallo and the team of researchers lead by Luc Montagnier used T-cell cultures infected with HIV-1 M group subtype B viruses obtained from AIDS patients. Both groups had been working for several years with T-cells and HTLV-I viruses, so they were much more adept than most other groups at keeping human T-cells alive in culture, and working with these T-cell tropic viruses.

(ii) Both groups did use sucrose density gradient centrifugation to separate viral particles in the cell culture supernatants from much but not all of the cellular debris and other components. These bands were thus “enriched” for viral particles, but not 100% “pure”. I have found one paper where the authors describe using an oligo-dT column to capture RNA from a 1.16 g/ml sucrose fraction [6]. Although they called this RNA “poly-A RNA” they did not show conclusively that it was polyadenylated. The genomes of lentiviruses are rich in adenosine and may have bound to the oligo-dT column without having poly-A tails added. At any rate, this RNA was not cloned, it was only labeled with 32P and used to probe HTLV-I and HTLV-II genomes in order to get an estimate of the sequence similarity between this new virus and the previously described T-cell leukemia viruses. The paper by Arya et al. discusses the various sizes of RNAs that they labeled, and speculates that the smaller ones represented spliced mRNAs while the largest, about 9kb in length, would be a reasonable size for a complete genome.

(iii) It is not possible to simply introduce cDNA into cells and claim that anything is “cloned”. In order for cloned DNAs to be propagated in E. coli cells, yeast cells, or human cells, they must be inserted into cloning vectors suitable for use in whichever cell type is chosen. Likewise, one cannot just throw any cDNA into a cloning vector and claim that this cDNA is “the” cDNA that was desired, many tests must be done to prove that the cloned DNA is indeed derived from the desired gene, whether it was a viral gene, a genome, or a cellular gene. Many methods, such as serological analysis southern blotting, restriction fragment length analysis and DNA sequencing can be used alone or in various combinations to prove that the cloning vector insert is in fact the desired gene or genome.

The papers (3,4,5) below cited by the Perth group do indeed discuss a few of the details of cloning and sequencing HIV genomes, but they alone are not sufficient to understand the full extent of the work done with these clones both before and after sequencing, to prove that they are indeed clones of HIV-1 M group subtype B viruses. For example, the Shaw paper (5) describes 5 different clones of HIV-1 genetic material. The Lambda-HXB-2 and Lambda-HXB-3 clones were derived from integrated proviral DNA, harvested from HIV-infected H9 cells, and their cloned inserts contain both HIV-1 proviral DNA and flanking human cellular DNA from the proviral integration site. These two clones had nearly identical restriction fragmentation patterns. The Lambda-BH-5, Lambda-BH-8 and Lambda-BH-10 clones were subgenomic fragments of HIV-1 because they were cloned using the SstI restriction endonuclease which cuts within the HIV-1 genome. The “BH” in the names of these clones presumably are the initials of Beatrice Hahn, because a second paper authored by Hahn et al [7] is cited as the source of descriptions about how these clones were created. I have not yet obtained a copy of that paper, so I cannot yet comment on how the clones were created.

In order to understand why it was that researchers became convinced that these cloned and sequenced viral gene fragments or complete genomes represented genomic material that must have come from the etiological agent of AIDS, one must read many more papers which describe other uses of these same clones. For example, in a Cell paper by Crowl et al [8] the authors discuss how the gene products of the env genes from several of these same clones (Lambda-HXB-2, Lambda-HXB-3, Lambda-BH-8, Lambda-BH-10, LAV and ARV-2) were recognized by the antibodies in the sera of several different AIDS patients, but not by antibodies in the serum of a healthy blood donor. Likewise a pair of papers by Sarngadharan et al [9,10] backed this up with further serological analyses.

Much of this work done in the mid-1980s could not be fully appreciated until much later in the 1990s when samples of lentiviruses from all over the world had been collected and analyzed by other labs not connected with these labs run by Gallo and Montagnier. As late as 2003, researchers are still discovering details of HIV-1 biology that pertain to incidents in the early culturing and cloning of this large family of lentiviruses. For example, it has only recently been discovered why some clones of HIV-1 M group virus grow only on “permissive” activated CD4+ T-cells while other clones can be grown on non-activated or resting CD4+ T-cells [11,12].

The Perth group wrote:

Would please Brian Foley tell us what does he mean by “infectious molecular clone” of “HIV”?

The Perth group should not concern themselves with what I or any other individual would consider to be an adequate definition of an infectious molecular clone. I am not the world’s leading authority on this topic. I am only a peon or minion in the world of retroviral research. Instead the Perth group should be reading textbooks, such as “HIV and the Pathogenesis of AIDS” by Jay Levy, or more general virology and molecular biology textbooks, so that they can see that the issues they are raising pertain to all of biology and not just to HIV-1 and AIDS. They should go out and talk to a few AIDS patients and see if their “oxidative stress” theory makes sense to those who are afflicted with this condition. Can they find AIDS patients who did not have sexual or blood-blood contact with another HIV-seropositive person?

Why did the Perth group become so interested in AIDS and no other areas of virology? If they are convinced that retrovirology is off base, then why not question the slaughtering of chickens which test seropositive for the recently identified subgroup J of Avian Leukosis Viruses [13]? If they are convinced of fraud in the HIV retrovirology labs, why not take their case to court, instead of entering the political arena in South Africa? Is it because empty rhetoric is much more effective in politics than in courts of law, where truth and solid evidence are often requested?

Perth References

1. Edmonds M. A History of Poly A Sequences: From Formation to Factors to Function. (2002) p. 285-390. In: Progress in Nucleic Acid Research and Molecular Biology edited by Moldave K, Vol. 71. Academic Press.

2. Gillespie D, Marshall S, Gallo RC. RNA of RNA tumor viruses contains poly A. Nature: New biol 1972;236:227-231.

3. Wain-Hobson S, Sonigo P, Danos O, Cole S, Alizon M. Nucleotide sequence of the AIDS virus, LAV. Cell 1985;40:9-17.

4. Ratner L, Haseltine W, Patarca R, Livak KJ, Starcich B, Josephs SF, et al. Complete nucleotide sequence of the AIDS virus, HTLV-III. Nature 1985;313:277-284.

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. (1984) Science 226: 1165-1171

Foley References

6: Arya SK, Gallo RC, Hahn BH, Shaw GM, Popovic M, Salahuddin SZ,
Wong-Staal F.
Homology of genome of AIDS-associated virus with genomes of human
T-cell leukemia viruses.
Science. 1984 Aug 31;225(4665):927-30.
PMID: 6089333

7: Hahn BH, Shaw GM, Arya SK, Popovic M, Gallo RC, Wong-Staal F.
Molecular cloning and characterization of the HTLV-III virus associated with
Nature. 1984 Nov 8-14;312(5990):166-9.
PMID: 6095086

8: : Crowl R, Ganguly K, Gordon M, Conroy R, Schaber M, Kramer R,
Shaw G, Wong-Staal F, Reddy EP.
HTLV-III env gene products synthesized in E. coli are recognized by
antibodies present in the sera of AIDS patients.
Cell. 1985 Jul;41(3):979-86.
PMID: 2988795

9: Sarngadharan MG, Bruch L, Popovic M, Gallo RC. Immunological properties of the Gag protein p24 of the acquired
immunodeficiency syndrome retrovirus (human T-cell leukemia virus
type III).
Proc Natl Acad Sci U S A. 1985 May;82(10):3481-4.
PMID: 2582414

10: Sarngadharan MG, Popovic M, Bruch L, Schupbach J, Gallo RC.
Antibodies reactive with human T-lymphotropic retroviruses (HTLV-III) in
the serum of patients with AIDS.
Science. 1984 May 4;224(4648):506-8.
PMID: 6324345

11: Greene WC.
How resting T cells deMURR HIV infection.
Nat Immunol. 2004 Jan;5(1):18-9.
PMID: 14699403

12: Ganesh L, Burstein E, Guha-Niyogi A, Louder MK, Mascola JR,
Klomp LW, Wijmenga C, Duckett CS, Nabel GJ.
The gene product Murr1 restricts HIV-1 replication in resting CD4+
Nature. 2003 Dec 18;426(6968):853-7.
PMID: 14685242

13: Benson SJ, Ruis BL, Garbers AL, Fadly AM, Conklin KF.
Independent isolates of the emerging subgroup J avian leukosis virus
derive from a common ancestor.
J Virol. 1998 Dec;72(12):10301-4.
PMID: 9811780

Competing interests: None declared