Twelve simple questions to Brian Foley 31 May 2004
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Eleni Papadopulos-Eleopulos,
Biophysicist
Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso, Sam Mhlongo, Todd Miller, Christian Fiala

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Re: Twelve simple questions to Brian Foley

Twelve simple questions to Brian Foley

 

In his rapid response, "Re: Eight simple questions and a repeated request to Brian Foley", 25 May, Brian Foley wrote:

"The Perth Group wrote: 

Q1.  Is it true that neither group published EMs of the 1.16g/ml band to confirm purification.

…"

Yes.  It is true"

 

We are pleased that Brian Foley agrees with us that both Montagnier and Gallo claimed their 1.16g/ml band was "purified" HIV but published no EMs to prove purification.

 

Brian Foley wrote:  "You have been told numerous times that proving that a 1.16g.ml band is 100% pure virus is not a requirement for studying retroviruses.  You repeatedly claim that it is, yet you have never shown anything but a rumour of a 1973 talk given by Francoise Barre-Sinoussi and Jean Claude Chermann, to back up your claim.  Do you have a transcript of that talk?  Do you have any evidence that this talk became the law for retrovirology?"

 

The “transcript of that talk” given by Francoise Barre-Sinoussi and Jean Claude Chermann is not "a rumour" but a paper published in a scientific journal. (1)   It is posted HERE.

 

We have mentioned this reference many times including in this debate.  Barre-Sinoussi, Chermann and Montagnier also referred to this paper.  In a paper published in 1984, they wrote:  "For endogenous reactions using only the viral RNA as template primer, the virus [HIV] was concentrated (7) and purified on sucrose gradient using isopycnic centrifugation (8)”.(2)      Ref. 8 is their Spectra paper.

 

On 26 June, 2003 Brian Foley sent us an email with the request to fax him two papers, one of which was the Spectra article.  Which we did and received a thank you note from him on 27 June 2003.

 

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.

 

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”.

            Yes or No?

 

Q2.      If purity is not a "requirement for studying retroviruses"  why have both Montagnier and Gallo claimed to have proven the existence of "HIV" , that is, its proteins and genome by purifying it?

 

Q3.      If purification is not necessary then is Montagnier mistaken in his view that “analysis of the proteins [and thus of the genome] of the virus demands mass production and purification.   It is necessary to do that.”

            Yes or No?

 

 

Brian Foley wrote: 

"The Perth Group wrote:

"…

Q2.  Is it true that Montagnier's and Gallo's viral RNA [and thus] proviral DNA and viral proteins" used for "Southern, northern and western blots" originated from their "purified" virus which may or may not have contained any particles with the morphology of retroviruses

…"

No. It is not true.

 

The proviral DNA came from HIV-infected cells. As far as I can tell from reading the papers, that virus was never passaged through a density gradient.

 

In some cases, poly-A RNA was extracted from density-enriched viral particles, and used for labelling probes for southern and northern blotting".

 

 

Since the proviral DNA is integrated into the cell genome and has no specific features it cannot be obtained by being “passaged through a density gradient” either of the cells or the cellular DNA.   Let us remind Brian Foley how the "proviral DNA" was obtained.  In our rapid response: "Eight simple questions and a repeated request to Brian Foley", 24 May, we wrote:  “In their paper entitled:  "Complete nucleotide sequence of the AIDS virus, HTLV-III" ["HIV"], Ratner, Gallo and their colleagues wrote:  "Clone HXB2 was derived from a recombinant phage library of Xbal-digested DNA from HTLV-III-infected H9 cell cloned in lJ1 (ref. 53)"(4).

In ref. 53 by Shaw, Gallo et al, the authors wrote:  that the "H9/HTLV-III DNA" [the DNA of the "infected" H9 cells] was screened with an HTLV-III cDNA probe (8) to obtain molecular clones of full-length integrated provirus [emphasis ours] with flanking cellular sequences…Figure 1 illustrates the restriction maps of these two clones, designated lHXB-2 and lHXB-3" (5).  Ref. 8 is a paper published by Arya, Gallo and their colleagues.  There one reads:  "The virus particles were purified from supernatant fluids of HT cells, clone 9 (H9) infected with HTLV-III (HTLV-IIIB) by centrifugation through a sucrose density gradient at equilibrium…The particles were lysed with sodium dodecyl sulfate (SDS), digested with proteinase K, and directly chromatographed on an oligo(dT) cellulose column.  The resulting polyadenylate [poly(A)]-containing RNA was used as template to synthesise 32P-labelled complementary DNA (cDNA) ["HIV" genome] in the presence of oligo (dT) primers" (6) [emphasis ours].”

 

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.

            Yes or No?

 

 

Brian Foley wrote: 

"The Perth Group wrote:

"…

Q3.  Is it true that the only proof they gave for the existence of the "HIV" proteins was a reaction between some proteins in their "purified" virus and antibodies present in the AIDS patients' sera

…"

 

No.  It is not true.

 

Serological evidence was only one of several lines of evidence".

 

 

Q5.      Would Brian Foley please tell us what other proof, in addition to the "reaction between some proteins in their "purified" virus and antibodies present in the AIDS patient's sera", he can find in Montagnier's 1983 paper and Gallo's 1984 papers, that is, the papers in which the "HIV" proteins were defined?

 

 

Brian Foley wrote: 

"The Perth Group wrote:

"…

Q4.  Is it true that a reaction between an antigen and antibodies present in an animal or human serum if not proof that the antigen is present in the animal or human and thus that the antibodies are directed against the antigen.

…"

 

Yes.

 

Serological evidence is only one of several lines of evidence used."

 

 

Q6.      If the reaction between an antibody and an antigen is not specific how was it possible (even if we assume that the antigen was “HIV”) for Montagnier and Gallo to conclude from this reaction that their patients were infected with “HIV”?

 

Q7.      What were the other "several lines of evidence used" by them?

 

 

Brian Foley wrote:

"The Perth Group wrote:

"…

Q5.      Since Montagnier and Gallo did not have proof for "purified HIV", were their "Southern, northern and western Blots" valid.

…"

 

Yes.”

 

We are amazed at such a response from Brian Foley.   The poly(A)-RNA (“HIV” genome) originated from the “purified” “HIV”.  The “proviral DNA” was a reverse transcript of this RNA.  That is, the nucleic acid probes originated from the “purified” “HIV”.   Brian Foley agrees with us that the poly(A)-RNA is not specific to retroviruses.   Just because a protein bands at 1.16g/ml it doesn't mean it is viral.  The “purified” virus either did not have any particles with the morphology typical of retroviruses or had some particles more closely resembling the morphology of retroviruses among a mass of microvesicles.  The microvesicles contained both proteins and poly(A)-RNA.(4)

 

Q8.      Would Brian Foley please tell us what is the scientific basis for his claim that Montagnier’s and Gallo’s Southern, northern and western blots were valid?

 

 

Brian Foley wrote:

"The Perth Group wrote:

"…

Q6.      Is it true the, that the origin of IHXB-2 "one of the best known infectious molecular clones" of "HIV" is a poly(A)-RNA found in Gallo's "purified" HIV.

…"

 

no.

 

We have been over this at least twice before.  The Lambda-HXB-2 infectious molecular clone of HIV-1 M group subtype B did not originate in poly(A) RNA.  It was cloned from one of the several proviral genomes integrated into an H9 cell line".

 

 

We are astonished by Brian Foley’ response.

Let us repeat how the “proviral genomes” and thus lHXB-2 were obtained.

In their paper entitled:  "Complete nucleotide sequence of the AIDS virus, HTLV-III" ("HIV"), Ratner, Gallo and their colleagues wrote:  "Clone HXB2 was derived from a recombinant phage library or Xbal-digested DNA from HTLV-III-infected H9 cell cloned in lJ1 (ref. 53)"(4).

In ref. 53 by Shaw, Gallo et al, the authors wrote:  that the "H9/HTLV-III DNA" [the DNA of the "infected" H9 cells] was screened with an HTLV-III cDNA probe (8) to obtain molecular clones of full-length integrated provirus [emphasis ours] with flanking cellular sequences…Figure 1 illustrates the restriction maps of these two clones, designated lHXB-2 and lHXB-3" (5).  Ref. 8 is a paper published by Arya, Gallo and their colleagues.  There one reads:  "The virus particles were purified from supernatant fluids of HT cells, clone 9 (H9) infected with HTLV-III (HTLV-IIIB) by centrifugation through a sucrose density gradient at equilibrium…The particles were lysed with sodium dodecyl sulfate (SDS), digested with proteinase K, and directly chromatographed on an oligo(dT) cellulose column.  The resulting polyadenylate [poly(A)]-containing RNA was used as template to synthesise 32P-labelled complementary DNA (cDNA) ["HIV" genome] in the presence of oligo (dT) primers" (6) [emphasis ours].

That is, lHXB-2 was obtained using the cDNA of the poly(A)-RNA as its probe which in turn was obtained from the 1.16g/ml band, the “purified HIV”.

 

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.

            Yes or No?

 

 

Brian Foley wrote:

"The Perth Group wrote:

"…

Q7.      If this is the case, is it not true that to claim the poly(A)-RNA in the 1.16g/ml band (the "purified" virus) is the "HIV" genome, evidence must exist which proves beyond reasonable doubt that the "purified" virus contained nothing else but particles with the morphology of retroviruses.  (If impurities existed, they must not contain RNA).

…"

 

No.”

 

 

We consider Brian Foley’s response incredulous.   Our question 7 was prefaced by:

“In his rapid response, "Re: A question and a request to Brian Foley", 13th May, Brian Foley agrees that poly(A)-RNA is not specific to retroviruses.

In his rapid response, "Re: Re-phrasing our two questions to Brian Foley", 11th May, Brian Foley agrees that the poly(A)-RNA which Montagnier and Gallo claimed to be the "HIV" genome originated from their 1.16g/ml band, which they claimed was "purified" virus.”

 

Q10.   Would Brian Foley please tell us what is the scientific basis for his response?

 

 

Brian Foley wrote: “The Perth Group wrote:
“…
Q8. Doesn’t it follow that we must start with Montagnier’s and Gallo’s groups 1983-1985 “raw data”.
…”

No.

The laboratory groups lead by Robert Gallo and Luc Montagnier were the first to publish identification of a virus, referred to at the time as HTLV-III by Gallo and LAV by Montagnier, which later became known as HIV-1 M group subtype B as the probable cause of AIDS. These two labs were not the only two labs in the world to independently reach the same conclusion. One can examine the raw data of other labs, such as the lab that Jay Levy worked in, and ignore the work done by the Gallo and Montagnier labs, and still reach the conclusion that HIV-1 and HIV-2 the causes of AIDS in humans.

1: 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

2: Cooper DA, Gold J, May W, Kaminsky LS, Penny R, Levy JA. .
Contact tracing in the acquired immune deficiency syndrome (AIDS). .
Evidence for transmission of virus and disease by an asymptomatic .
carrier. Med J Aust. 1984 Oct 27;141(9):579-82. .
PMID: 6092883

3: 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”

 

 

We are amazed by Brian Foley’s response that we need not start with Montagnier’s and Gallo’s evidence.  Whenever researchers claim to have made a scientific discovery the duty of scientists is to critically analyse their evidence.

 

The Cooper et al paper is not relevant to our debate.   It declared three individuals who had many sexually transmitted agents as being infected with “HIV” on the basis of a reaction between antibodies in their sera and cells “infected” with “HIV”.  All one has to do to realise that this antibody test is totally non specific is to read Montagnier's 1983 paper and Gallo's 1984 papers.  In this regard, it is of interest also to note that patients also tested positive for Hep B, EBV, CMV;  one of the contacts for Hep B, EBV, CMV and TPHA and the other for Hep A, Hep B, EBV and CMV.

 

Levy simply repeated what Montagnier and Gallo did.   Like them, he reported the finding of reverse transcriptase activity and reaction between AIDS patients’ sera and “HIV” “infected” cells.   However, unlike Montagnier and Gallo who reported finding “typical type-C” particles, in the Levy et al study "Particles with characteristic type D retrovirus morphology were detected by electron microscopy".

According to Brian Foley "HIV" is neither type-D or type-C particles.  "HIV" is a lentivirus, that is, it belongs to a different sub-family of the retroviral family.

 

In the Luciw, Levy et al paper, HUT-78 cells, [which according to Gallo are infected with HTLV-I see Wong-Staal et al, Nature 1983;302:626-628], were "infected" with “HIV”.  "To characterise the viral genome, RNA was extracted from purified virions and electrophoresed on agarose gels containing methyl mercury hydroxide…To purify virus particles, the culture medium was spun at low speed…to remove cells and further clarified by centrifugation…The supernatant was centrifugated to "pellet virus"_.  The pellet was resuspended and treated with DNase and "layered into a 10-50% sucrose gradient…and centrifugated in the SW41 rotor for 2 hrs at 34,000 r.p.m.   Fractions were collected, extracted with phenol, and nucleic acid from the aqueous phase was adjusted to 300 mM NaC1 and precipitated with cold ethanol.  A sample of each fraction was electrophoresed on 1% agarose gels (containing 5mM methyl mercury hydroxide) and stained with ethidium bromide to locate the fraction enriched for virion RNA (lane a).  Arrows show the positions of ribosomal RNA markers at 4.9 and 1.9 kb.  The remaining RNA from this gradient fraction was electrophoresed in low-melting 1% agarose gel (containing 5 nM methyl mercury hydroxide).  The region in the gel containing the ~9 kb RNA species was cut out…"

 

"The 9-kb RNA species was used as a template with random primers in a reverse transcriptase reaction to produce a virus-specific cDNA probe".

Note: the "virus-specific cDNA probe" is the cDNA of an arbitrary chosen 9-kb RNA originating from a pellet obtained by centrifugation.  Since no EMs of the pellet were published the possibility cannot be excluded no retrovirus particles were present.  Even if such particles were present, since the pellet was obtained from a HUT-78 cell line, the particles could very well be HTLV-I.

 

Using what they called "virus-specific cDNA" as probe they "examined the structure of viral DNA in infected cells by digestion with restriction enzymes, electrophoresis in agarose gels and Southern blotting.  Undigested DNA from infected cells contained a species at 5.5kb, a faint species at 6kb and a broad band at the exclusion limit of the gel (> 15kb).  We suggest that the DNA species at 5.5 and 6kb represent unintegrated viral DNA in a circular configuration containing respectively, one and two long terminal repeats (LTRs);  the upper broad band (> 15kb) represents proviruses integrated into host cell DNA".

 

Note:  According to Montagnier's group (5) the "HIV" provirus is 9.1 – 9.2 kb long and to Gallo's group "approximately 10 kilobases" (6).

 

Q11.    Since Levy repeated much of what Montagnier and Gallo did, (he did not use banding in density gradients and did not define the “HIV” genome as a poly(A)-RNA but arbitrarily chose a 9kb RNA from a centrifugation pellet) and since not Levy but Montagnier and Gallo are considered to be the discoverers of “HIV”, doesn’t it follow that we must start with Montagnier’s and Gallo’s groups 1983-1985 “raw data”.

            Yes or No?

 

 

Brian Foley wrote: “The Perth Group wrote:
“…
So we repeat our request: “Would Brian Foley please give us a study and a few confirmatory studies where the existence of an “infectious molecular clone” of “HIV” has been proven.”
…”

No.

If you can show me one virus (and not a mixture of three or more viruses, as you presented the last time you were asked) that has “purified” and/or cloned to your satisfaction, I will consider further discussion. Until then, adios.”

 

 We have never presented an example of purified virus with evidence that it consisted of “a mixture of three or more viruses”.  Most importantly, the examples we presented consisted of nothing but retrovirus particles containing RNA and not DNA.  The “purified” “HIV” (or more correctly, “purified vesicles” as Gluschankof’s group called it) consist of either no particles with the “morphology typical of retroviruses” or a mass of vesicles in which are dispersed some particles having some characteristics of retroviral particles which have been labelled “HIV” particles but the experts still cannot agree as to which sub-family of retroviruses they belong.  Moreover, the “purified” “HIV” contains both RNA and DNA.

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”.    Furthermore, Brian Foley has been stating that there can be no better proof for the existence of “HIV” than the evidence of  the “HIV” “infectious molecular clone”.  However when we requested him to give us just a few references we found to our utmost surprise he declined with an emphatic “No”.

 

Q12.    Why?  Is it because among the “thousands of papers published on HIVs”, Brian Foley cannot find one with evidence which proves the existence of "HIV-1” “infectious molecular clone” and thus of “HIV-1”?

 

 

References

1.      Sinoussi F, Mendiola L, Chermann JC. (1973). 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.

2.      Rey MA, Spire B, Dormont D, Barre-Sinoussi F, Montagnier L, Chermann JC. (1984). Characterization of the RNA dependent DNA polymerase of a new human T-lymphotropic retrovirus (lymphadenopathy associated virus). Biochemical and Biophysical Research Communications 121:126-33.

3.      Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ. (1997).   Cell Membrane Vesicles Are a Major Contaminant of Gradient-Enriched Human Immunodeficiency Virus Type-1 Preparations. Virology 230: 125-133.

4.      Bess JW, Gorelick RJ, Bosche WJ, Henderson LE, Arthur LO. (1997). Microvesicles are a source of contaminating cellular proteins found in purified HIV-1 preparations. Virology 230:134-144.

5.      Alizon M, Sonigo P, Barre-Sinoussi P, Chermann JC, Tiollais P, Montagnier L, Wain-Hobson S.  Molecular cloning of lymphadenopathy-associated virus. (1984) Nature 312: 757-760.

6.      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.

 

 

Competing interests: None declared