Re: Twelve simple questions to Brian Foley 2 June 2004
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Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545

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Re: Re: Twelve simple questions to Brian Foley

The Perth group wrote:
Banding in sucrose density gradient has been “the law” for “HIV” at least till 1997. (3) This has been the method used by Barre-Sinoussi, Chermann, Montagnier in 1983 and by Gallo et al in 1984. By this method they claimed to have obtained "purified" HIV.

It is clear that the Perth group understands that density gradient centrifugation is useful in the study of retroviruses. Their confusion seems to be in failing to understand that a band containing retroviral particles does not need to be 100% “pure” in order to be useful. The paper by Pablo Gluschankof (3) did not state that all of HIV research was invalidated by their finding some cell debris and/or cellular vesicles mixed in with their virus preparations, they only state:

These vesicles, which are released from both noninfected and HIV-1-infected cells, contain a selection of cellular membrane proteins similar to, but not identical to, those in virus particles. Contamination of gradient-enriched virus by vesicles of cellular origin has implications for the preparation and use of vaccine formulations derived from whole-virus preparation.”

Everyone agrees that HIV-1 and other lentiviruses are enveloped viruses. The envelope is composed of host cell membrane, containing several host proteins such as HLA-DR, as well as the viral Env protein. These host cell proteins, whether they are present on the viral particles themselves, or in contaminating material, can influence vaccine outcomes, as summarized by David Montefiori in 1995 (1). They do not invalidate all other HIV research. There is no “law” stating that virus preparations must be 100% “pure” to be useful.

The Perth group wrote:
Q1. Is it true that Montagnier in 1983 and Gallo in 1984 claimed to have obtained "purified" "HIV". That is, they cleared “HIV” of all extraneous elements and thereby obtained material containing nothing but “HIV”.

The paper by George Shaw (2) does use the word “purified”, but they do not state that this means the virus was 100% pure. Likewise, the paper by Mikulas Popovic (4) also used the statement “The yield of virus from H4/HTLV-III cells was assessed by purification of concentrated culture fluids through a sucrose density gradient…” but again I would not interpret that as a claim for 100% purity. The only point, really was to show that the reverse transcriptase activity peaked at the density of 1.16 g/ml which is typical for retroviruses. The 1984 paper by M. Sarngadharan (5) says that viruses “purified” from the supernatants of cultures by sucrose gradient centrifugation showed a gag protein with a molecular weight of 24 kiloDalotons, when the “purified” viral proteins were electophoresed on SDS-polyacrylamide gels. None of the papers by Montagnier’s group used the words “pure” or “purified” as far as I can see.

The only points worth making about all this are: 1) that even if the virus particles could be “purified” to 100% purity, they would still contain some cellular proteins carried by the cell membrane that envelopes the virus. And 2) that all retroviruses look pretty much alike so looking at them with an electron microscope does very little good, it is the serological and molecular characterization of the viruses that is critical.

The Perth group wrote:
Whenever researchers claim to have made a scientific discovery the duty of scientists is to critically analyse their evidence.

The “critical analysis” of evidence includes repeating the experiments, which is what the Levy lab did to reconfirm the serology of AIDS in the USA (6) and to obtain another HIV-1 M group subtype B genome, from another USA AIDS patient (7,8). People can “rethink” science all they want, but they will never contribute to the truth if they do not do a little research.

The Perth group wrote:
the particles could very well be HTLV-I.

Dr. Levy’s clone turned out to be another HIV-1 M group subtype B virus, and the complete genome accession number is K02007. Anyone can look it up and find that it has the Vif, Vpu, Tat, Rev and Nef accessory genes of the HIV-1 M group of lentiviruses. It is not a T-cell leukemia virus at all.

The Perth group wrote:
We have never presented an example of purified virus with evidence that it consisted of “a mixture of three or more viruses”.

The Perth group has never presented an example of ANY virus that ever met all of their conditions for proof of existence. The one paper they try to cite repeatedly is the 1973 Spectra paper by Francois Sinoussi (9), where the authors clearly stated that they were working with a mixture of replication-defective MSV and its helper virus MuLV. The whole point of that paper was an attempt to separate the viruses by density. It appears that they may have come close to separating the viruses, but with molecular methods which became available in the late 1970s, cloning the genomes of the two viruses became rather simple in comparison (10,11), so that the exact composition of the two viruses became apparent. At the time Francois Sinoussi wrote that paper in 1973, it was not yet known why the sarcoma virus was defective, nor that the MSV/MuLV combination would also package hybrid virions with one genome of each type.

No virus has ever been isolated and characterized by the method that the Perth group claims is required for proof of the existence of HIV-1.

The Perth group wrote:
All along this debate, Brian Foley has been telling us that the existence of the “HIV” “infectious molecular clone” proves that “HIV” has been 100% “purified”.

All along, I have been attempting to get through to the Perth group, that infectious molecular clones virus are the only way to really study retroviruses. If you only use the techniques that were available up until 1973, such as gradient centrifugation and EM, you don’t really know what virus or mixture of viruses you are working with. Serology and DNA sequencing tell us a whole lot more. I have never said or implied that a cloned virus proved that the virus had been made 100% pure before the clone was obtained.

The Perth group wrote:
However when we requested him to give us just a few references we found to our utmost surprise he declined with an emphatic “No”.

I have already provided the Perth group with many papers which prove that infectious molecular clones of HIV-1 and many other lentiviruses have indeed been created and studied. The Perth group denies that any of them meets their criteria for “proof” and they are correct, because the “proof” they are looking for is impossible. No virus has ever met those criteria, and no lentivirus ever can meet them.

As the two Spectra papers repeatedly cited by the Perth group point out, very few retroviruses are “ideal” for purification by ultracentrifugation. It turns out that lentiviruses have several characteristics that make them less than ideal for the procedure that the Perth group claims to be required.

The Perth group wrote:
Q4. Since the "proviral DNA" was obtained by using the cDNA of the poly(A)-RNA as a probe doesn’t this mean that the "proviral DNA" is a transcript (reverse) of the poly(A)-RNA and thus that its ultimate origin is poly(A)-RNA.

Q9. Since lHXB-2 was obtained using the cDNA of poly(A)-RNA as a probe does it not mean that lHXB-2 is a transcript (reverse) of the poly(A)-RNA and thus that the ultimate origin of lHXB-2 is the poly(A)-RNA from the 1.16g/ml band.

If the Perth group does not understand the difference between a lambda phage genomic library and the probe used to probe it, they should take a class in molecular genetics before attempting to convince the world that they know more about how virology should be done, than the virologists who are working in the field.

I actually suspect that at least several members of the Perth group know exactly what the probe was, and how the clone was made. I suspect that they are just attempting to sow confusion here.

Briefly, retroviruses carry two single-stranded RNA genomes in the virion. The retroviral reverse transcriptase uses a cellular transfer RNA primer (the Lys-3 tRNA for all lentiviruses) (12). The retroviral integrase enzyme then inserts the proviral DNA into the genome of the host cell, where it can remain dormant for the life of the cell, or can become activated to produce new virions, if it is not defective. The ultimate origin of the proviral genomes in the Lamba-HXB-2 and Lamda-XHB-3 was not any 1.16 g/ml band. The probe used to screen the genomic library WAS derived from one or more 1.16 g/ml bands.


1: Montefiori DC.
New insights into the role of host cell proteins in antiviral vaccine
AIDS Res Hum Retroviruses. 1995 Dec;11(12):1429-31.
PMID: 8679285

2: 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)

3: Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ.
Cell membrane vesicles are a major contaminant of gradient-enriched
human immunodeficiency virus type-1 preparations.
Virology 230:125-133 (1997)

4: Popovic M, Sarngadharan MG, Read E, Gallo RC. Detection, isolation, and continuous production of cytopathic retroviruses (HTLV-III) from patients with AIDS and pre-AIDS. Science. 1984 May 4;224(4648):497-500. PMID: 6200935

5: Sarngadharan M.G., Popovic M, Bruch L. Shuepbach, J. and Gallo, R.C.
Antibodies Reactive to Human T-Lymphotrophic Retroviruses (HTLV-III)
in the Serum of Patients with AIDS.
Science 224:506-508. (1984)

6: Levy JA, Hoffman AD, Kramer SM, Landis JA, Shimabukuro JM, Oshiro LS.
Isolation of lymphocytopathic retroviruses from San Francisco patients
with AIDS.
Science. 1984 Aug 24;225(4664):840-2.
PMID: 6206563

7: 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

8: 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

9: 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)

10: Van Beveren C, Galleshaw JA, Jonas V, Berns AJ, Doolittle RF,
Donoghue DJ, Verma IM.
Nucleotide sequence and formation of the transforming gene of a
mouse sarcoma virus.
Nature. 1981 Jan 22;289(5795):258-62.
PMID: 6256659

11: Shinnick TM, Lerner RA, Sutcliffe JG.
Nucleotide sequence of Moloney murine leukaemia virus.
Nature. 1981 Oct 15-21;293(5833):543-8.
PMID: 6169994

12: Kleiman L.
tRNA(Lys3): the primer tRNA for reverse transcription in HIV-1.
IUBMB Life. 2002 Feb;53(2):107-14. Review.
PMID: 12049192

Competing interests: None declared