VIRUSMYTH HOMEPAGE

Continuum
Vol.4 No.3 Sept./Oct. 1996

THE ISOLATION OF HIV -- HAS IT REALLY BEEN ACHIEVED?
THE CASE AGAINST

Eleni Papadopulos-Eleopulos (1) Valendar F.Turner (2) John M. Papadimitriou (3) David Causer (1)

(1) Department of Medical Physics, (2) Department of Emergency Medicine, Royal Perth Hospital, Perth, Western Australia; (3) Department of Pathology, University of Western Australia.

"Listening to both sides of a story will convince you that there is more to a story than both sides"
- Frank Tyger

The definite existence of any virus, including a retrovirus, can be proven only by isolating it. For nearly half a century retroviruses have been isolated by banding in density gradients. It is accepted that the procedures incorporated into this method, which is by no means perfect, have not been followed by the researchers who claim isolation of the human immunodeficiency virus, HIV-1. Nonetheless, it is said that at present, there is ample evidence that HIV has been isolated and shown to be a unique exogenous retrovirus.(1) In this critique we have analysed the relevant data that purport to prove that HIV has been isolated. To simplfy the presentation for readers of this article, the major arguments (1) for HIV isolation are used as the headings in the discussion. Since the topic is both complex and controversial it is necessary to present substantial original data and sometimes to repeat it in order to critically assess the basis for the view that HIV has been isolated.

(Please note that some Greek characters may not print. Lamda may appear as the control character (^) and alpha, beta and gamma be g, b or a.)

1. "In 1983 Montagnier et al isolated a retrovirus".

In the 1983 Montagnier et al study there is no proof of virus isolation by "the most rigorous method available to date". Nor did they follow the "traditional...Pasteur rules". How then did they isolate a retrovirus? Even if Montagnier and his colleagues or others had followed the "Pasteur rules", since "viral and cellular proteins, and cellular contaminants...copurify with virus purified by conventional density gradients",(1) there is no reason to accept any claim of HIV isolation by any research group who did not use "the most rigorous method available to date, i.e. molecular cloning of infectious HIV DNA". However, to prove that HIV "has been isolated" by "the most rigorous method available to date", virus cloning, one must start with HIV RNA (DNA). Since the propriety of naming an RNA "HIV RNA" is contingent upon prior isolation of a particle proven to be a retrovirus, on this basis alone, "the most rigorous method available to date, i.e. molecular cloning of infectious HIV DNA", cannot prove HIV isolation.

2. "reverse transcriptase associated with such particles".

There is not one single study which proves that the enzyme present in the "growth medium" or even in the material which in sucrose density gradients bands at 1.16 gm/ml, (the density which defines retroviral particles), and which catalyses the transcription of RNA into DNA, is a constituent of particles of any kind, much less of retroviral-like particles or a unique retrovirus. The only association between "particles" and "reverse transcriptase" (RT) arises from experiments which show that some cultures/cocultures with tissues from AIDS patients exhibit both particles, many of which are not even retroviral-like, and transcription of the synthetic RNA template-primer A(n).dT15. However, this does not constitute proof of the existence of RT or RT as a constituent of a retroviral particle. Furthermore, since: (a) the presence of reverse transcriptase (RT) is proven indirectly, that is, by demonstrating transcription of the RNA template-primer A(n).dT15; (b) the template-primer A(n).dT15 can be transcribed not only by RT but by other cellular DNA polymerases. All the cellular DNA polymerases, à, á and y, can copy A(n).dT15 (2). In fact, in 1975, an International Conference on Eukaryotic DNA polymerases, which included Baltimore and Gallo (3) defined DNA polymerase y, "a component of normal cells" (4), "found to be widespread in occurrence" (2), whose activity can be increased by many factors including PHA stimulation (5), as the enzyme which "copies A(n).dT15 with high efficiency but does not copy DNA well";(3) it is impossible to say that the polymerase in the "growth medium" or in the material banding at 1.16 gm/ml which catalyses reverse transcription of A(n).dT15 is RT or one of a number of other cellular DNA polymerases.

3. "...indeed, each of these criteria could reflect another retrovirus, and some of these criteria, eg, particles and proteins, could reflect non-viral material altogether".

Although the HIV/AIDS experts, including Montagnier, Gallo and Barr-Sinoussi claim that RT is "unique to retroviruses" and "the hallmark of a retrovirus",(6-8) this is not the case, a fact accepted by some of the best known scientists.(9) "Reverse transcriptase (RT) was first discovered as an essential catalyst in the biological cycle of retroviruses. However, in the past years, evidence has accumulated showing that RTs are involved in a surprisingly large number of RNA-mediated transcriptional events that include both viral and nonviral genetic entities...the possibility that reverse transcription first took place in the early Archean" is supported by a number of facts and "the hypothesis that RNA preceded DNA as cellular genetic material". (10) According to Varmus: "Reverse transcription was assigned a central role in the replication of other viruses [hepatitis B and cauliflower mosaic viruses] and in the transposition and generation of other kinds of eukaryotic DNA". (11) "The hepatitis B viruses (HBVs) are small DNA viruses that produce persistent hepatic infections in a variety of animal hosts and replicate their DNA genomes via reverse transcription of an RNA intermediate. All members of this family contain an open reading frame (ORF), "P" (for pol), which is homologous to retroviral pol genes" (pol=polymerase).(12) "Hepatitis B virus (HBV) resembles retroviruses, including HIV, in several respects. In particular, both viruses contain reverse transcriptase, and replicate through an RNA intermediate". Because of this, it has been suggested that hepatitis B infection should be treated with the same antiretroviral agents as HIV infection.(13). At present, evidence exists which shows that although the major target organ for hepatitis B virus is the liver, cells other than hepatocytes "including peripheral blood lymphocytes and monocytes, may become infected with HBV" (14). Lymphocyte stimulation in general and PHA stimulation in particular is associated with production of hepatitis B virus from peripheral blood lymphocytes in patients infected with HBV including "viral replication in chronic hepatitis B infection of childhood".(15,16) According to Doolittle et al, "...there are many reverse transcriptase-bearing entities other than retroviruses, including mobile elements found in a wide variety of eukaryocytes, some plant and animal DNA viruses, and even some introns" (17). In one of his most recent publications, one of the best known retrovirologists, Robin Weiss from the Institute of Cancer Research, London, UK, wrote, "Now we know that a broader group of genetic elements than retroviruses utilise reverse transcription at some stage of replication; these include hepadnaviruses (including hepatitis B virus), cauliflower mosaic virus and retrotransposons of eukaryotes and prokaryotes. Indeed lamivudine may find a place in the treatment of hepatitis B infections as well as HIV".(18) In other words, RT does not seem to be more specific to retroviruses than ATPase, an enzyme now known to be ubiquitous but which, before the discovery of RT, was used to both detect and quantify retroviruses.(19) Since in all the HIV literature, by HIV isolation is meant nothing more than the detection of "HIV particles", proteins and RT (and frequently only one of them), and since any or all of these phenomena "could reflect non-viral material altogether", does it not therefore follow that HIV could reflect non-viral material altogether?

4. "HIV antigens or proteins associated with such particles".

To date nobody has presented evidence that the "HIV antigens or proteins" are constituents of retrovirus particle or even a retrovirus-like particle let alone a unique retrovirus, HIV.

5. "Antibodies against Montagnier's HIV strain-the global standard of all "HIV tests"".

5.1 In the 1983 paper entitled "Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS)",(20) where Montagnier and his colleagues reported the "isolation" of their "HIV" strain, cells obtained from a lymph node biopsy of a gay man with lymphadenopathy (lymphadenopathy syndrome [LAS]) were put in culture with PHA, IL-2 and antiserum to human interferon. (The latter had previously been shown in mice to lead to "increased retrovirus production by a factor of 10 to 50"). After 15 days RT activity was detected using the synthetic primer- template A(n).dT15. The reverse transcription of A(n)dT15 was considered proof that a retrovirus was present in the lymph node cells. The finding of the same activity in the supernatant of a co- culture of the same cells with lymphocytes from a healthy individual was considered proof that the retrovirus could be transmitted. In another experiment, polybrene and supernatant from the co-cultures were added to two, three day old umbilical cord lymphocytes cultures. After seven days "a relatively high titer" of A(n.)dT15 transcription was detected. This was considered proof not only of transmission but isolation as well. "That this new isolate was a retrovirus was further indicated by its density in a sucrose density gradient, which was 1.16, and by its labelling with [3H] uridine (fig. 1)". In figure 1 evidence was presented that A(n)dT12- 18 could be transcribed by the material from the supernatant of the umbilical cell cultures which, in sucrose density gradients, banded at 1.16 gm/ml. The "infected" umbilical cord lymphocytes as well as "HTLV- producing" cells were lysed. The proteins from a "cell extract" obtained from the lysates were reacted with the sera from the patient with lymphadenopathy, another patient with "multiple adenopathies", a healthy individual, a normal goat and goat antiserum "to HTLV-I p24". Many proteins from both cell types but especially from the "infected" umbilical cords, reacted with ALL sera. However, the "infected" umbilical cord cells did not react with the antiserum to "HTLV-I p24". The proteins from the culture supernatant which banded at 1.16 gm/ml were also reacted with the sera but instead of the goat anti-p25 antiserum they used sera from another healthy donor. In the published strips it is difficult if not impossible to distinguish any reactive bands with any serum. In the text it is stated "three major proteins could be seen: the p25 protein and proteins with molecular weights of 80.000 and 45.000" in the strip with the serum from the patient with LAS. Montagnier et al also reported that "Electron microscopy of the infected umbilical cord lymphocytes showed characteristic immature particles with dense crescent (C-type) budding at the plasma membrane". They gave no electron microscopic (EM) data on the material banding at 1.16 gm/ml but concluded "A retrovirus belonging to the family of recently discovered human T-cell leukemia viruses (HTLV) but clearly distinct from each previous isolate, has been isolated from a Caucasian patient with signs and symptoms that often precede the acquired immune deficiency syndrome (AIDS). This virus is a typical type-C RNA tumor virus, buds from the cell membrane, prefers magnesium for reverse transcriptase activity, and has an internal antigen (p25) similar to HTLV p24" (20), (When it was realised that individuals who have antibodies which react with this "virus strain" did not rapidly progress to AIDS, without proof, the taxonomically distinct "typical type-C " retrovirus became a taxonomically distinct, typical Lentivirus).

5.2 THE WORD "ISOLATION" IS DERIVED FROM THE LATIN "INSULATUS" MEANING "MADE INTO AN ISLAND". IT REFERS TO THE ACT OF SEPARATING AN OBJECT FROM ALL EXTRANEOUS MATTER THAT IS NOT THAT OBJECT. The object of interest is not a protein, nor a fragment of RNA (DNA) but a unique exogenous retrovirus, HIV. Nothing more and nothing less. No such evidence was presented by Montagnier et al. Obviously, at the very best, the finding of phenomena such as virus-like particles in cell cultures, antibody/antigen reactions and evidence for reverse transcription of A(n).dT15 can be considered proof only for detection of a retrovirus, and then if and only if each are shown to be specific to the retrovirus. This cannot be done unless the retrovirus is first isolated. Thus it comes as no surprise that Popovic, Gallo and their colleagues did not consider Montagnier et al's data as proof of "true isolation".(21) [In their 1984 papers Gallo and his colleagues defined isolation as detection of "more than one of the following:", "repeated detection of a Mg2+ -dependent reverse transcriptase activity in supernatant fluids; virus observed by electron microscopy (EM); intracellular expression of virus-related antigens detected with antibodies from seropositive donors or with rabbit antiserum to HTLV-III; or transmission of particles". (By transmission of particles was meant detection of RT or particles in cultures of human umbilical cord blood, bone marrow or peripheral blood T lymphocytes, cultured with supernatants from the "infected" cultures). Since this is no different from the experiments that Montagnier and his colleagues performed, it follows that Gallo and his colleagues did not prove "true isolation" either. In fact, Gallo et al's definition of isolation raises additional questions including: How was it possible to obtain rabbit antiserum "to HTLV-III" before the virus was isolated and how was it possible, before the virus was isolated, to ascertain that both the rabbit antiserum and the patient sera used to test material from the cultures interacted specifically with the virus? According to their definition, one can isolate HIV even if no RT is detected. How is this possible since RT is the "hallmark" of HIV?(22).] It is also significant that in his and his colleagues' 1986 patent application "Improvements relating to viral isolates and their use", Robin Weiss referred to Montagnier's "HIV strain" as "the material". "A so-called Aids virus isolate was first reported in 1983 by Montagnier and his colleagues in France who named the material "Lymphadenopathy Associated Virus One"".(23) Furthermore, isolation of a retrovirus from the umbilical cord cultures is not proof that the retrovirus was introduced from the outside, that is, that it originated from the patient with lymphadenopathy. All cells contain endogenous retroviruses (see 6.3.2). In fact sperm, ova, placenta, foetal and embryonic tissues, and to a lesser extent, umbilical cord lymphocytes, were extensively studied because retroviruses were said to be transmitted vertically (in the germ cell line) and because they were thought to play a significant role in differentiation. By the beginning of the AIDS era one or more of the following phenomena were reported from experiments with such cells: retrovirus-like particles, reverse transcriptase activity and retroviral antigens.(24-26) Thus such findings cannot be proof for the existence of HIV.

Neither is the presence of antibodies in the AIDS patients, but not in the healthy controls, which react with the proteins which band at 1.16 gm/ml, proof that such individuals are infected with an exogenous retrovirus, HIV. For example, in a study published this year, one of the best known retrovirologists, Reinhard Kurth, from the Paul-Ehrlich Institute in Germany, and his colleagues, reported that 70% of "HIV-positive patients", compared to only 3% of blood donors, had antibodies which reacted with the retrovirus HTDV/HERV- K. However, HTDV/HERV-K is not a retrovirus which is present only in AIDS patients, that is, an exogenous retrovirus as HIV is said to be, but HTDV/HERV-K is an endogenous retrovirus or, as Kurth put it, a retrovirus present "in all of us". How is it possible then to say, based just on an antibody test, that "Montagnier's strain", if one assumes Montagnier did isolate such a virus, is not another endogenous retrovirus generated by the conditions present in these patients? (see 6.3.2).

5.3 Apparently Montagnier's group found reactions between patient sera and three proteins, p25 (p24), p45 (p41) and p80 in banded material but only p24 was considered to be HIV protein. However, in 1984, Gallo's group reported that "No antigen from the uninfected clones reacted with the sera, with the exception of a protein with a molecular weight of 80.000 in H17 which bound antibodies from all of the human serum samples tested [including normal serum] but not from rabbit or goat serum". Because of this the p80 protein was considered to be non-specific. "Antigens newly expressed [reactive with sera in the cell extracts] after viral infection and recognized by the human serum used for this analysis included p65, p55, p41, p39, p32 and p24. A large protein with a molecular weight of approximately 130,000 and a protein of 48,000 were also detected". Unlike Montagnier, Gallo's group also reported that, "With normal human serum, none of the antigens was detected (not shown)", and concluded, "These results show clearly that the antigens detected after virus infection are either virus-coded proteins or cellular antigens specifically induced by the infection".(27) Gallo and his colleagues also reported that of the proteins from the supernatant of the "infected" cultures which in sucrose density gradients banded at 1.16 gm/ml, only two proteins, p41 and p24, reacted with patient sera and concluded that "these molecules are the major components of the virus preparation. p24 and p41 may therefore be considered the viral structural proteins". In the two years following their discovery of HIV, although Montagnier's group apparently made repeated attempts, unlike Gallo's group, they could not detect "high molecular weight" protein which reacted with different sera but which "was not present in the supernatant of uninfected control cells". In experiments reported in 1985, instead of using umbilical cord lymphocytes, they used "infected" H9 and CEM cells, two leukaemic cell lines, and cultured (labelled) them with radioactive cysteine, 35S cysteine, (an essential amino acid constituent of human proteins). They reported that in the supernatant "a protein of approximately 110-120K could be specifically immunoprecipitated by sera from pre-AIDS or AIDS patients, in addition to core proteins, and not by sera from normal, healthy blood donors or of laboratory workers. The protein was absent in supernatants of uninfected T lymphocytes, T- or B- cell lines" . They also showed that the 110K protein was a glycoprotein (gp110). For reasons not stated, they thought that the 110K protein had a cellular precursor. To demonstrate this, instead of using the CEM or the H9 cell lines, they formed "A cellular hybrid, between normal T4 lymphocytes and the MOLT-4 cell line, which was then "infected" with LAV and cultured with radioactive cysteine. The resulting syncytia were lysed and the proteins were reacted "with LAV-positive serum". "After 3 hr labelling, a band of 150K was detected, Upon longer labelling, (12 hr) another band of 135K appeared". Curiously, this was interpreted as "suggesting that it [135] was derived from the 150K precursor" and that "either in the cytoplasm or at the cell membrane, the gp150 is converted into the gp135 form...During virus morphogenesis, the gp135 is converted into gp110-120 by partial enzymatic removal of carbohydrates, without proteolytic cleavage. The virus-associated [Not one single piece of their data was derived even from a viral-like particle or material which banded at 1.16 gm/ml. All was either from "infected" cells or culture supernatant] gp110 may itself be further processed during virus aging...besides the main 110-120K band seen after labelling of the virus, three other thin bands of 70K, 40K and 34K respectively, could be specially immunoprecipitated by patients' sera. Since some of these sera did not precipitate any gag protein, it may be assumed that these proteins are antigenically related to gp110 and are cleavage products of the latter".(28) This conclusion can be questioned on several grounds. Suffice it to mention only two: (a) The culture supernatant and the cells cannot be considered synonymous with a retrovirus. (b) Although Montagnier et al did not comment, their data shows that many proteins, including a p40 found in the supernatant of both "non-infected" CEM and H9 cells react with sera from the patients with lymphadenopathy. Somehow, without proof that they are coded by "HIV DNA", or they belong to a retrovirus-like particle, the following proteins, gp160/150, gp 120, gp45/40, p34/32, p24, p18/17 found either in cells, supernatants, or banding at 1.16 gm/ml in sucrose density gradients became known as the HIV proteins. In other words, contrary to all scientific reasoning, it was postulated that AIDS sera contain specific HIV antibodies and the proteins with which these antibodies react were defined HIV specific proteins.

5.4 The "HIV glycoproteins", gp160, gp120 and gp41.

(a) In 1983,(20) and again in 1984 Montagnier and his colleagues (29) claimed that although p45/41 reacted with patient sera, this protein was not viral but the ubiquitous cellular protein, actin. It is interesting that even this year, the criteria used by Montagnier to define a positive HIV Western blot is: "the presence of antibodies against products of the env gene (gp160, gp120) and reactivity at least with one gag gene product (WHO criteria)"(30). However to date, no other criteria, not even the WHO criteria, exclude p41. The WHO criteria is "2 env bands (precursor, external gp, or transmembrane gp" with or without any other bands (transmembrane=gp41)(31) Unlike Montagnier, Gallo considers gp41 the most specific HIV protein.

In 1985, Gallo and his colleagues, comparing the fourth open reading frame (ORF) of the "HIV DNA" which they called env-lor, with the env genes of other retroviruses, reported that, "The predicted product of the fourth reading frame env-lor shares many features in common with the envelope gene precursors of other retroviruses, the most striking of which is a hydrophobic region near the middle of the protein...The amino-terminal domain of the translation product of the fourth open reading frame also resembles the env protein precursors of other retroviruses...we believe that the fourth open reading frame encodes an env precursor...In its mature form it is probably cleaved into a large heavily glycosylated exterior membrane protein about 481 amino acids long and a transmembrane protein, 345 amino acids long which may be glycosylated. The size of these predicted products agrees with the detection of a large glycosylated protein of Mr 120-160K in HTLV- III-infected cells which is probably the glycosylated env gene precursor and a smaller, virion-associated gp41 which is probably the membrane protein".(32) However, in a study published in 1987 by Gallo and his colleagues, where they performed a "Computer- assisted analysis" of "the amino acid sequences of the envelope protein complexes derived from the nucleotide sequences of seven AIDS virus isolates", it was reported that, "Although the overall sizes and structures of the seven surface proteins are rather similar, the deduced amino acid sequences differ substantially. On the average, only 66% of the amino acids are conserved in the exterior part of the protein...gp41, the transmembrane part of the envelope protein complex, shows more than 80% conserved amino acids", but "gp41 should be about 52.000 to 54.000 daltons by calculation".(33) Even if the molecular weight of the glycoprotein predicted from the length of the "HIV" fourth ORF was found to be identical to that of the protein present in the Western blot (41,000), the claim by Gallo that the interaction of gp41 with antibodies found in AIDS patient sera is proof that gp41 is coded by the "HIV genome", and that both gp41 and the antibodies are specific to a retrovirus, is at odds with what Gallo was saying in 1981. In the mid 1970s, Gallo and his colleagues reported the isolation of the first human retrovirus, HL23V. In fact, the evidence for the "isolation" of HL23V surpassed that of HTLV-I and HIV in at least two aspects. Unlike HIV, Gallo's group: (a) reported the detection of reverse transcriptase activity in fresh, uncultured leucocytes;(34) (b) published an electron micrograph of virus-like particles banding at a sucrose density of 1.16 gm/ml. (35) Following the discovery of HL23V, some researchers attempted to determine its prevalence utilising antibody tests (36) while others were interested to determine the specificity of the antibody reactions. The former included two of the best known HIV experts Reinhard Kurth and Robin Weiss, and their colleagues who, for this purpose used "the simian sarcoma-associated helper virus (SSAV) and the M7 strain of baboon endogenous virus (BEV) to survey human sera for specific antibodies. Also included is a virus (HL23V-1) originally isolated from cultured peripheral blood leukocytes of a patient with acute myelogenous leukemia. HL23V-1 was shown to comprise a mixture of two viruses, one closely related to SSAV, the other to BEV" and found that "A survey of human sera from healthy individuals revealed the presence of naturally occurring antibodies that react in radioimmunoprecipitation assays with proteins of mammalian type- C viruses" including the internal (gag) and envelope (env) proteins of HL23V, SSAV and BEV and concluded, "The serological studies presented here and by others provide indirect evidence that the infectious mode of transmission remains a real possibility in humans, and suggests that infection with an oncornavirus [retrovirus] may be extremely widespread".(37) Three years later, in 1980, two research groups, (38,39) one from the Laboratory of Cellular and Molecular Biology, National Cancer Institute and the other from the Laboratory of Viral Oncology, Memorial Sloan-Kettering Cancer Center, using the "viral glycoproteins", found that the antibodies present in human sera which reacted with these proteins were "directed against carbohydrate structures" and concluded that "The results are consistent with the idea that the antibodies in question are elicited as a result of exposure to many natural substances possessing widely cross-reacting antigens and are not a result of widespread infection of man with replication competent oncoviruses". In 1981 Gallo accepted the evidence that the antibodies which reacted with proteins of HL23V were directed not against the proteins "but against the carbohydrate moieties on the molecule that are introduced by the host cell as a post-transcriptional event, and which are therefore cell-specific and not virus-specific".(40) This discovery was of such significance that today nobody, not even Gallo, considers HL23V as being the first human retrovirus, or even a retrovirus. In fact, in 1981 when Gallo and his colleagues reported the presence in humans of antibodies to what he now calls the first human retrovirus, HTLV-I, (according to Weiss, "The first 'human' retrovirus to be isolated in 1971 was human foamy virus (HFV) from a nasopharyngeal carcinoma line",(18)) the title of the paper was, "Antibodies in human sera reactive against an internal structural protein of human T-cell lymphoma virus".(40) In this paper Gallo and his colleagues described the finding of antibodies to a "major internal structural protein (p24) of HTLVCR" and claimed that such antibodies were "specifically directed at HTLVCR proteins and not at cell-specific determinants-in other words, the immunological reactions are not those reported in human sera against animal virus glycoproteins which, lacking virus specificity, are directed against the carbohydrate residues of the glycoprotein".

(b) By 1989, researchers from New York showed that in Western blot analyses, "the components visualized in the 120-160 kDa region do not correspond to gp120 or its precursor but rather represent oligomers of gp41". It was also shown that the WB pattern obtained is dependent on many factors including temperature and the concentration of sodium dodecyl sulphate used to disrupt the "pure virus". "Confusion over the identification of these bands has resulted in incorrect conclusions in experimental studies. Similarly, some clinical specimens may have been identified erroneously as seropositive, on the assumption that these bands reflected specific reactivity against two distinct viral components and fulfilled a criterion for true or probable positivity. The correct identification of these bands will affect the standards to be established for Western Blot positivity: it may necessitate the reinterpretation of published results".(41,42) (Little if any notice was taken of this report!). Indeed, if, as it is claimed, HIV Western blots are prepared from lysates of purified HIV virions, then it would be impossible for p160 and p120 to be found in WB strips since: (i) All HIV researchers agree with Montagnier and Gallo that gp160 is a precursor to gp120 and gp41 and unlike the latter two proteins, is only found in infected cells and not in mature particles; (ii) Although many EM have been published of virus-like particles in non-banded material nobody,(43,44) not even the CDC, (45) or Hans Gelderblom and his colleagues who have most thoroughly studied these particles, has proven the existence in the cultures of cell-free particles possessing knobs (spikes). In one of his latest publications Gelderblom and his colleagues have estimated that immediately after being released, "HIV particles" possess an average of 0.5 knobs per particle but also pointed out that "it was possible that structures resembling knobs might be observed even when there was no gp120 present, i.e., false positives".(46) It is accepted that gp120 is present only in the knobs (spikes). Since there is no proof for the presence of knobs in the cell-free particles, even immediately after release from the cell, it is not possible for the gp120 to be present in the Western blot.

5.5 The "HIV pol protein", p31/34.

In 1987 Henderson isolated the p30-32 and p34-36 of "HIV purified by double banding" in sucrose density gradients. By comparing the amino-acid sequences of these proteins with Class II histocompatability DR proteins, they concluded that "the DR alpha and beta chains appeared to be identical to the p34-36 and p30-32 proteins respectively";(47)

5.6 The "HIV gag protein", p24

As far as Montagnier is concerned, p24 is THE HIV protein, and for at least three years after the introduction of the "HIV" antibody test, a p24 band found in the WB was considered by most laboratories, including the CDC, as proof for HIV infection. At present there is ample evidence that antibodies which react with p24 are ubiquitous in both human and animal sera, which can only be interpreted that either p24, the antibodies, or both, are non-HIV- specific or a significant proportion of both humans and animals are infected with HIV. For example, if the p24 band in the WB is considered proof of HIV infection then about 30% of individuals who are transfused with HIV negative blood become infected as a result.(48) Since, according to the AID vaccine Clinical Trials Group, (49) "The presence of p24 band was common among low-risk, uninfected volunteers and complicated the interpretation of the Western blot test results", HIV infection should be common among healthy at no risk individuals. In fact, because of such evidence, since 1987, with perhaps only two exceptions, Montagnier and researchers conducting the Multicenter AIDS Cohort Study in the United States, no laboratory anywhere in the world considers a reaction between the p24 in the WB and antibodies present in sera, as proof of HIV infection. Yet, when the same reaction takes place between an antibody to the p24 of the WB and a patient serum, it is considered proof of viraemia, and when between an antibody to p24 and material present in a cell culture, the same reaction is considered proof of HIV isolation!

Obviously, the detection of a protein, even if known to be virus specific, in sera or even culture, does not constitute proof for isolation or viraemia. That such a finding is non-specific can be best illustrated by a few examples. In 1992, Jorg Shupbach, the principle author of one of the first four 1984 papers published by Gallo's group on HIV isolation, reported that the whole blood cultures of 49/60 (82%) of "presumably uninfected but serologically indeterminate individuals and 5/5 seronegative blood donors were found positive for p24".(50) If p24 is an HIV protein then it must be present in all AIDS patients if not all seropositive patients and not in persons not at risk of developing AIDS. In 1989, David Ho and his colleagues used p24 measurements in serum and in cultures of non-infected cells cultured with plasma from "infected" patients, to estimate active virus, "infectious HIV-1", viraemia, viral load. The serum from 14/53 patients whose plasma cultures were positive, was negative for p24. They concluded, "Thus, plasma culture was more sensitive than serum p24 antigen measurement in detecting the presence of cell-free HIV-1 in blood". They also reported that treatment with AZT for four weeks induced "a 94 percent reduction in the load of cell-free virus".(51) Even Jackson et al who claim an overall 98.3% "HIV isolation" rate, can detect p24 in serum of 42% of AIDS patients, 37% of ARC patients and 17% of asymptomatic seropositive individuals (52) which is a much lower rate than in non-HIV infected organ transplant recipients. "In one kidney recipient (the donor was negative for p24 antigen) who, 3 days following transplantation developed fever, weakness, myalgias, cough and diarrhoea, all "Bacteriological, parasitological and virological samples remained negative [including HIV PCR]. The only positive result was antigenaemia p24, positive with Abbot antigen kits in very high titers of 1000pg/ml for polyclonal and 41pg/ml for monoclonal assays. This antigenaemia was totally neutalizable with Abbot antiserum anti-p24...2 months after transplantation, all assays for p24-antigen became negative, without appearance of antibodies against HIV. Five months after transplantation our patient remains asymptomatic, renal function is excellent, p24 antigenaemia still negative and HIV antibodies still negative".(53) Using two kits, the Abbot and Diagnostic Pasteur, in one study, p24 was detected transiently in 12/14 kidney recipients. Peak titres ranged from 850 to 200 000 pg/ml 7-27 days post- transplantation. Two heart and 5/7 bone marrow recipients were also positive, although the titres were lower and ranged from 140-750 pg/ml. Disappearance of p24 took longer in kidney (approximately 6 months) than in bone-marrow (approximately 4-6 weeks) recipients. According to the authors: "This may be related to differences in immunosuppression therapy". Discussing their findings they wrote: "The observation of a 25-30kD protein binding to polyclonal anti- HIV human sera after immunoblots with reactive sera raises several questions. This protein could be related to a host immune response to grafts or transplants...Its early detection after transplantation might indicate the implications of immunosuppression therapy...The 25-30kD protein could therefore be compared with the p28 antigen recently described with human T-cell- related virus lymphotropic-endogenous sequence...The characterization of this 25-30kD protein may represent an important contribution to the detection of HIV-1-related endogenous retroviruses".(54) The disagreement between Montagnier and Gallo about which proteins were actually "HIV" proteins was not limited to gp41 but included p24. Montagnier always mentioned that "no cross-reactivity existed between HIV p24 and other antibodies including antibodies to HTLV- I, II". Until 1985 he also maintained that there was "a very close homology between LAV and HTLV-III but an absence of homology with HTLV-I and -II".(28) However, in 1985 he wrote, "We have also compared the deduced amino-acid sequences of LAV proteins with those of HTLV-I and other retroviruses and find no significant homology, except for domains pol and gag which are generally conserved among retroviruses".(55)

Gallo always maintained that homology exists between the HTLV-I, II and HIV gag genes (56) and the many features shared by all "human retroviruses" include "a small (p24/p25) major capsid protein; p24 cross-reactive antigenic determinant detected with either heterologous (rabbit) antisera or human monoclonal antibodies".(57) Indeed, gag stands for group specific antigens. As far back as 1974 Gelderblom and his colleagues wrote, "While the virus envelope antigens are primarily virus-strain specific, the bulk of internal proteins of the virion with molecular weight (mw) between 10,000 d and 30,000 d are group-specific (gs) for viruses originating in a given animal species (gs-spec. antigens). The major protein constituent of mammalian C-type oncornaviruses [retroviruses] with a molecular weight in the range of 30,000 d was found to possess, besides gs spec. antigen, an antigenic determinant that is shared by C-type viruses of many mammalian species including monkeys and was thus termed gs interspecies (gs-interspec.) antigen".(58) In 1989 William Blattner, a well known HIV/AIDS expert stated: "It may be feasible to use viral antigen probes to look for cross-reactive antibodies, since certain viral proteins, particularly the polymerase and gag proteins may be highly conserved between subtypes of virus".(59) Thus, even if p24 were to be specific to retroviruses, it cannot be HIV specific. If p24 detected in culture supernatants is a component of similar particles, viral or non-viral, then in density gradients all the p24 should be found at least in one band (fraction), even if not at a density of 1.16 gm/ml. That this is not the case has been demonstrated by Montagnier himself. In one experiment Montagnier and his colleagues divided the density gradient into sixteen fractions. The RT peak was found in fraction five and six, while the p24 and gp110 were present in all but three (1, 2, 3) fractions. (28)

5.7 The role of actin and myosin in particle budding.

There is no scientific reason to define a protein present in a cell, culture supernatant, or even in material banding at 1.16 gm/ml in sucrose density gradients as being retroviral on the basis that it reacts with antibodies in AIDS patient sera, as Montagnier and Gallo's groups did. According to Gelderblom, AIDS patient sera are "polyspecific"(60,61) and at present there is ample evidence that these sera react with a plethora of self and non-self antigens including proteins of "non-infected" lymphocytes. Why then should they not also react with the "HIV proteins", even if such proteins are cellular proteins, or with a variety of recombinant or synthetic antigens? If the proteins in the cultures/co-cultures of tissues derived from AIDS patients and which react with AIDS patient sera are indeed retroviral, then what are the proteins in the "non-infected" cells and supernatants which Montagnier repeatedly reported to also react with AIDS patient sera? On the basis of reactivity with AIDS patient sera, only 20% of the proteins which band at 1.16 gm/ml can be considered "HIV proteins" and, as the HIV/AIDS experts claim, without proof, are coded by "HIV DNA".(47,62) Even if there was proof that pure (isolated) "HIV" particles are present at 1.16 gm/ml, then all the proteins banding at 1.16 gm/ml should be embodied in such particles. However, since only 20% of these proteins are "HIV" proteins, the question then arises, what is the origin and role of the remaining 80% of the proteins in such particles and by what genes are they coded? Why are only 20% of the proteins viral and non-cellular? Why not all of them and vice versa? If the gp41 protein present in the Western blot band and which reacts with AIDS patient sera could be the ubiquitous protein actin, then why should not one consider the p24 protein as being one of the light chains of myosin, another equally ubiquitous protein especially given that: (a) Matsiota, Montagnier and their colleagues at the Pasteur Institute have shown that AIDS patients and those at risk have high levels of antibodies to this protein;(63) (b) at present there is ample evidence that the plethora of cellular proteins (á2 microglobulin, the à and á chains of human lymphocyte antigen (HLA) DR, CD71, CD63, CD43, CD8, "the major leukocyte adhesion receptors LFA-1 (CD11A/CD18) and CD44) which are present in the "HIV particles", include actin and myosin.(64-68) Indeed, in the last few years researchers from a number of institutions expressed the view that actin polymerisation (or actin/myosin interaction) "mediates HIV budding" and release. Researchers from New York and Philadelphia found that colchicine treatment of "MOLT4/HIV-1IIIB" cells, "induced lymphocyte polarization, redistribution of F-actin into a pseudopod, and secretion of HIV from the pseudopod", and that the particles were "observed exclusively on the tip of the pseudopod". 65 Two of the studies which examined the role of actin and myosin in "HIV particle" budding and release are by researchers from Japan. In one publication the authors concluded, "Since F-actin is essential for maintaining cell-shape and cellular function, polarization of F- actin might change the cell membrane configuration or cell fragility, which may be essential for HIV release".(67) In the other study, the authors "demonstrated that myosin and actin are colocalised at the budding site of viral particles. In particular, myosin was concentrated on the same area of the plasma membrane as the dense spots of the viral particles. In contrast, actin was widely distributed on the plasma membrane and was always found in areas where viral particles were present". They concluded, "actin might participate with myosin in an active process leading to the release of viral particles from the membrane". Because these researchers, like most others, are of the opinion that "the initiation of a myosin-actin interaction requires an increase in free intracellular calcium", they have performed a preliminary experiment using two calcium chelators, one, BAPTA which they consider chelates only intracellular free calcium and the other, EGTA, which in their view chelates only the free calcium on the outer side of the cell. They found that "HIV-1 release was suppressed most pronouncably when both" the inner and the outer free calcium was chelated, and that inhibition was stronger with the outer chelater than the inner. "From these results, we suggest that [Ca2+]o might enter the cell by the stimulation of viral budding itself at the budding site...it may be difficult to detect an increase of [Ca2+]i...because the budding mechanism is going on continuously and slowly in a very narrow region without any synchronization".(64)

At present evidence also exists that: (a) there is an association between the redistribution of polymerised actin, myosin and other cellular proteins (glycoproteins) and many cellular processes including budding unrelated to HIV release;(69-73) (b) polymerisation of actin, actin-myosin interaction and cross- linking of polymers in general is regulated by the redox state, oxidation leading to interaction;(74-76) (c) both AIDS patients and cultures derived from AIDS patients are subjected to oxidising agents. In fact, for the detection of "HIV", proteins and particles the cell cultures must be stimulated (treated with oxidising agents).(77)) Ten years ago Montagnier wrote, "Indeed, LAV infection of resting T4 cells does not lead to viral replication or to expression of viral antigen on the cell surface, while stimulation by lectins or antigens of the same cells results in the production of viral particles, antigenic expression and the cytopathic effect".(78) (d) in the presence of antioxidants no "HIV" phenomena can be observed.(77,79,80) In a study presented at this year's International AIDS Conference, researchers from Rome reported, "The results obtained using 3-ABA, NAC [antioxidants] and a combined treatment 3-ABA/NAC given together seem to confirm the role of intracellular redox balance in the modulation of the HIV expression. In fact, a significant reduction in the number of viral particles was observed in cultures which have received the combined treatment with NAC/ABA".(81)

Given the above data, may one be tempted to speculate that the "HIV" particles and proteins are nothing more than "non-viral material altogether", induced by the agents to which the AIDS patients and cultures are exposed?

CONCLUSION

The statement "antibodies against Montagnier's HIV strain-the global standard of all "HIV tests"", presumes proof of: (a) the existence of more than one "HIV strain", including one of Montagnier's. Such evidence can be obtained only by isolating the retrovirus. However, Montagnier's evidence does not prove the isolation of a retrovirus; (b) the existence of "HIV" specific immunogenic proteins. Again, such proof can be obtained only by isolating the retrovirus; (c) antibodies specifically induced by HIV infection. It is true that for detection of such antibodies one does not need to use HIV or the HIV immunogenic proteins. For example, serological tests for both infectious mononucleosis and syphilis employ antigens derived from horse red blood cells and ox heart respectively but nonetheless predict infection with Epstein-Barr virus and Treponema pallidum. However, the only way to prove that "HIV antibodies" are directed against "HIV", that is, the only way to use the antibody test to prove HIV infection, is to present evidence which proves that the antibodies are HIV specific. Such proof can be obtained only by using HIV isolation as a gold standard. Since this has not been done it is not possible to say that "the global standard of all "HIV tests"" proves HIV infection.

6. "HIV DNA"

In debating the proof for the existence of a unique, exogenous retroviral agent one cannot adopt as an initial premise ("Full- length HIV-1 and HIV-2 DNAs...") that is contingent upon proof of the arugment ("ergo...HIV exists and has been isolated"). The a priori designation of a particular fragment of DNA as "HIV DNA" merely begs the question under consideration.

6.1 MINIMUM EVIDENCE REQUIRED TO PROVE THE EXISTENCE OF HIV DNA

If "HIV DNA" is the genome of a unique retroviral particle then the most basic requirement is proof for the existence of a unique molecular entity "HIV DNA", that is, unique fragments of DNA identical in both composition and length in all infected individuals. The claim that a stretch of RNA (cDNA) is a unique molecular entity which constitutes the genome of a unique retrovirus can be accepted if and only if it is shown that the RNA belongs to a particle with the morphological, physical and replicative characteristics of a retroviral particle. Proof of these properties can only by obtained by isolating the putative viral particles, that is, by obtaining them separate from everything else, extracting the nucleic acids and demonstrating that such particles are identical (their constituents including their nucleic acids are identical) and infectious. The correct procedures, now having been used for over half a century to achieve this proof, require demonstration that: 1. In "infected" cell cultures (cocultures) there are particles with a diameter of 100-120nM containing "condensed inner bodies (cores)" and surfaces "studded with projections (spikes, knobs)"; (82) 2. In sucrose density gradients the particles band at a density of 1.16 gm/ml; 3. At the density of 1.16 gm/ml these is nothing else but particles with the morphological characteristics of retroviral particles; 4. The particles contain only RNA and not DNA and that the RNA consistently has the same length (number of bases) and composition no matter how many times the experiment is repeated; 5. When the particles are introduced into secondary cultures, but mindful of the critical caveat discussed below: (a) the particles are taken up by the cells; (b) the entire RNA is reverse transcribed into cDNA; (c) the entire cDNA is inserted into the cellular DNA; (d) the DNA is transcribed into RNA which is translated into proteins; 6. As a result of 5 the cells in the secondary cultures release particles into the culture medium; 7. The particles released in the secondary cultures have exactly the same characteristics as the original particles, that is, they must have identical morphology, band at 1.16 gm/ml and contain the same RNA and proteins.

The caveat is that while the introduction of the majority of infectious particles into cell cultures and subsequent release of similar particles is proof that such particles are indeed infectious, this is not the sufficient case for retroviruses. The basis of this exception is the fact that "one of the most striking features that distinguishes retroviruses from all other animal viruses is the presence in the chromosomes of normal uninfected cells, of genomes with those of infectious viruses".(83) In fact, a cell may contain the genome of many retroviruses. As far back as 1976 retrovirologists recognised that "the failure to isolate endogenous viruses from certain species may reflect the limitations of in vitro cocultivation techniques".(84) In other words, the finding of a retrovirus in both the primary and secondary "infected" cultures/cocultures is not proof that the cells have been infected with an exogenous retrovirus.

One way which will suggest but will not prove that the cells acquired virus from the outside (exogenously acquired retrovirus, infectious retrovirus) and have not assembled a retrovirus from information already existing in normal cells (endogenous retrovirus) is to conduct experiments that use controls, that is, to run in parallel with test cultures/cocultures control cultures/cocultures. The only difference between the test and control cultures should be the introduction of particles into the test cultures. In other words, apart from the introduction of particles, in every other respect control cultures must be dealt with identically. For example: (a) because detection of RT and retroviral genetic sequences and release of retroviral particles depends on the metabolic state of the cells, the physiological state of the cells used in the control cultures should be as close as possible to those of AIDS patients; (b) because the mere act of co-cultivation alone may lead to release of endogenous retroviral particles, if test cells are cocultured, so should the cells used in control experiments; (85) (c) extracts, even from normal unstimulated cells, when added to the cultures may increase endogenous retroviral expression. (86) Because of this, when cells are cultured with "HIV" (supernatant or material which bands at 1.16 gm/ml), the controls must be cultured with similar material from cell cultures originating from sick individuals with illnesses similar to AIDS, that is, matched individuals who are immunosuppressed; (d) the appearance of endogenous retrovirus can be accelerated and the yield increased a million fold by stimulating the cultures with mitogens,(87) mutagens, chemical carcinogens and radiation.(88,89) If test cultures are exposed to or employ such agents so should the controls; (e) since AIDS patients and those at risk of developing the syndrome are exposed to strong oxidising agents,(79,90) the control cells should also originate from such patients; (g) to avoid observer bias and in the best interests of science, blind examination of test and control cultures/cocultures should be performed.

6.2 EVIDENCE FOR THE EXISTENCE OF "HIV DNA"

6.2.1 In 1984, in the first of two papers, Montagnier and his colleagues described the following experiment: "Because LAV can induce T-cell fusion and because EBV [Epstein Barr virus] is known to have fusion activity in B cells, we performed co-infection experiments of unfractionated lymphocytes (B and T) with both viruses. It was hoped that stable hybrids of LAV-infected T cells and of EBV-transformed B cells would be formed and that such hybrids would be able to continuously produce LAV. Several regimens were tried. The one that gave rise to continuous productive infection of LAV was the following. Whole lymphocytes of F. R. were first stimulated for 24 hours with Protein A and then infected with and EBV strain, M81, derived from a nasopharyngeal carcinoma. Five days later, half of this culture was infected with LAV as described (1) and then divided in two subcultures: one was cultured in medium lacking T-cell growth factor (TCGF: interleukin-2), the other in medium containing TCGF. As expected, the TCGF-fed culture produced LAV as detected by a peak of RT activity appearing between day 12 (day 6 after LAV infection) and day 21 in the supernatant. In contrast, the cells cultured in the absence of TCGF did not yield any detectable RT...On day 19, at the time of decline of LAV production, a subculture of the TCGF-fed cells received fresh T cells from the same donor: these T cells had been activated for 3 days with phytohemagglutinin (PHA)...Six days later (day 25), a new peak of RT appeared, but contrary to the first infection, it was not transient...At the time of the second LAV infection, large round cells transformed by EBV could be readily seen in this culture, as well as in the control culture not infected with LAV, indicating that immortalization of the B cells by EBV had already occurred. The immortalized B-cell line was termed RF8".(29) [Reference 1 to which Montagnier refers is the 1983 paper in which Montagnier et al described the first "isolation" of HIV (see 5)]. In the second study, 200 ml of supernatant from the "HIV infected" FR8 cells were banded in sucrose gradients, "Virus containing fractions were pooled" and centrifuged. (It is not stated how they determined the existence of "virus", in which band(s) (fraction(s)) "virus" was found, how many bands if any were found to have particles, or why there were more bands than one (1.16 gm/ml) containing the "virus"). The pellet was incubated with several substances, dATP, dGTP, dTPP, dCTP including 32dCTP and an oligo(dT) primer. From the cDNAs thus obtained, three clones "pLAV13, 75 and 82, carrying inserts of 2.5, 0.6 and 0.8 kilobases (kb), respectively, were characterized further. All three inserts have a common restriction pattern at one end, indicative of a common priming site. "The 50-base pair (bp) common HindIII-PstI fragment was sequenced and shown to contain an oligo(dA) stretch preceding the cloning dC tail. The clones are thus copies of the 3' end of a poly(A) RNA. The specificity of pLAV13 was determined in a series of filter hydbridization experiments using nick-translated pLAV13 insert as a probe". Firstly, "using an adapted spot-blot technique" they tested the pellet obtained from the supernatant of "LAV infected" normal lymphocytes and CEM cells as well as non-infected lymphocytes. The "infected" pellets were positive and the non- infected negative. "Second, the probe detected DNA in the Southern blots of LAV-infected T lymphocytes and CEM cells. No hybridization was detected in DNA from uninfected lymphocytes or from normal liver". No details are given regarding the method used to produce "infection", but it would appear that the normal cells and the CEM cells were cultured with supernatant from the FR8 cells, that is, the same supernatant they used to obtain the probe! They concluded: "Together, these data show that LAV pLAV13 DNA is exogenous to the human genome and detects both RNA and integrated DNA forms, derived from LAV-infected cells. Thus, pLAV13 is LAV specific".(91)

6.2.2 In May 1984, Gallo and his colleagues published four papers. To "isolate" HIV they used a leukaemic cell line which they called HT. It is impossible to known with what tissues from AIDS patients this cell line was cultured. Reading the May 1984 papers one gets the impression that the HT cell line was cultured with concentrated (supernatant) fluids originating from individual, AIDS patient, stimulated T-cell cultures. Subsequently, the Gallo investigation found the HT cell line was cultured with concentrated fluids pooled initially from individual cultures of three patients and ultimately from the individual cultures of ten patients.(92) The Gallo investigation found this procedure to be "of dubious scientific rigor". One scientist described the procedure as "really crazy".(93) In 1985, Gallo and his colleagues wrote, "The H9/HTLV-IIIB cell line was derived from the human T-cell line HT, following co- culture with T lymphocytes obtained from several AIDS patients, and contains many different HTLV-III forms".(94) The detection of reverse transcription of A(n).dT15 in the supernatant, was considered proof the HT cells were infected with a retrovirus, HIV, which originated from the patients' tissues. A clone, H9 of the HT cell line was obtained "using irradiated blood of a healthy donor as a feeder".(21) The H9 cells were cultured with supernatant from the "HIV" infected HT cells. The H9 supernatant was banded in sucrose density gradients and the material which banded at 1.16 gm/ml which, without proof, Gallo and his colleagues considered to be synonymous with retroviral particles, was "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 synthesize 32P-labelled complementary DNA (cDNA) in the presence of oligo(dT) primers. The size of the resultant cDNA ranged from 0.1 to 10 kb. When these labelled cDNAs were hybridised to poly(A)-containing RNA purified from infected [that is, cells cultured with the same supernatants as those from which the probe was obtained] and uninfected H9 cells as well as other uninfected human cell lines, only the infected H9 cells contained homologous RNA sequences as evidenced by discrete RNA bands after Northern hybridisation. Figure 1 shows that cDNA preparations from HTLV-IIIB and HTLV-IIIZ gave identical patterns, detecting species of about 9.0, 4.2, and 2.0 kb...These bands are similar in size to those corresponding to genomic size messenger RNA (mRNA) and spliced mRNAs of env and pX sequences previously observed in cells infected with HTLV-I, consistent with the anticipated relatedness of these viruses. Furthermore, viral mRNA bands of HTLV-II-infected cells were detected with an HTLV-III cDNA probe and again the sizes of the mRNA were like those with HTLV-I"!(56)

In another study by Gallo and colleagues, extrachromosomal DNA of "infected" H9 cells was extracted and "assayed for its content of unintegrated viral DNA" using the 32P-labelled cDNA as a viral probe. "Unintegrated linear viral DNA was first detected after 10 hr [of "infection"] and was also present at the subsequent time points. Figure 1 shows a Southern blot of the 15-hr sampling. A band of ~10 kilobases (kb) in the undigested DNA represents the linear form of unintegrated HTLV-III".(95) In yet another study Gallo and his colleagues reported that, "Since the HTLV-III provirus was found to lack Xba I restriction sites, a genomic library was constructed by using Xba I-digested H9/HTLV-III DNA, and this was screened with an HTLV-III cDNA probe to obtain molecular clones of full length integrated provirus with flanking cellular sequences. Fourteen such clones were obtained from an enriched library of 106 recombinant phage, and two of these were plaque-purified and characterized. Figure 1 illustrates the restriction maps of these two clones, designated ^HXB-2 and ^HXB-3. The overall length of the HTLV-III provirus is approximately 10 kilobases...To determine whether the HTLV-III genome contains sequences homologous to normal human DNA, the viral insert of ^XB-2...was isolated, nick translated and used to probe HTLV-III-infected and uninfected cellular DNA. Under standard condition of hybridization...this probe hybridized to DNA from H9/HTLV-III cells as well as other HTLV-III-infected cells, but not to DNA from uninfected H9 cells, uninfected HT cells (the parent line from which H9 was cloned), or normal human tissues (data not shown). This finding is in agreement with the results of other experiments in which the unintegrated (replicative intermediate) form of HTLV-III was used as a probe and demonstrates that HTLV-III, is an exogenous retrovirus lacking nucleic acid sequences derived from human DNA".(96)

6.2.3 In 1984, Levy and his colleagues cultured PBMC from patient suffering from Kaposis'sarcoma with IL-2, polybrene and PHA. The supernatant was tested for RT, the cells for reaction with serum from the Pasteur Institute patient BRU and "some cultures were examined for virus by electron microscopy". The finding of a positive result with "any of these tests" was considered proof of virus isolation. The supernatant from one of these cultures was "inoculated into fresh human PMC stimulated 3 days before with phytohemagglutinin". Within 6 days the supernatant of this culture had high RT activity and this was said to represent "the virus isolate ARV-2".(97) The HUT78 cell line was cultured with "ARV-2". In the HUT78 "Virus production was monitored by measuring reverse transcriptase activity". When there was maximum RT activity, the supernatant was centrifuged and the resuspended pellet, after treatment with DNAase, was centrifuged in sucrose gradients. The nucleic acid from each fraction was electrophoresed on agarose gel. The region in the gel containing an "~9kb RNA species was cut out" and used to obtain "a radioactive cDNA probe". The DNA from the HUT78 cell line cultured with "ARV-2" was digested with restriction enzymes, electrophoresed in agarose gel and Southern blotted using the "radioactive cDNA probe". "No specific bands were detected in several digests of DNA from uninfected cells...whereas bands were seen in infected cells...undigested DNA from infected cells contained a species at 5.5 kb, a faint species at 6kb and a broad band at the exclusion limit of the gel (>15kb). We suggest that the DNA species 5.5kb and 6 kb represent unintegrated viral DNA in a circular configuration containing respectively one and two long terminal repeats (LTRs); the upper broad band (>15kb) represents provirus integrated into the host cell DNA". In an additional experiment "whole-cell DNA from cells infected with ARV-2 was partially digested with ECORI; 9-15 kb cell DNA was cloned into an EMBL-4 bacteriophage ^ vector and recombinant phage were identified with the virus-specific cDNA probe". Among the recombinant phage obtained were ^-9B and ^-7A, each of which was 9.5 kb.(98)

6.2.4 SUMMARY AND DISCUSSION

It is obvious that although Montagnier, Gallo and Levy and their respective colleagues refer to virion or virus particles purification or isolation, none of these groups have presented evidence for the isolation of retrovirus particles or even the isolation of virus-like particles, the first and absolutely necessary step in proving the existence of a retroviral genome. (At the time of writing, neither has any other group of HIV/AIDS researchers). Finding some RNA which bands at 1.16 gm/ml, selecting from it a poly(A) rich fraction, or a fragment of a given length, even if always found to be the same length and sequence, and referring to it as HTLV-III, LAV, ARV does not constitute such proof. It must be stressed that even if the RNA is incorporated in a particle which in sucrose density gradients bands at 1.16 gm/ml, this is still not proof that it is retroviral RNA. According to John Coffin, one of the best known experts on the retroviral genome, there are particles "with a full complement of viral proteins, but the particles contain a collection of cellular RNAs and only about 1% genomic RNA...assembly of particles does not require the genome...in its absence other RNA molecules may be substituted".(83) It is important to note that although all groups, Montagnier's, Gallo's and Levy's refer to the material from the culture supernatants which in sucrose density gradients bands at 1.16 gm/ml as viral particles, virions, and to the RNA and proteins at that density as "particle-associated" RNA or proteins, not one of the groups presented evidence for the existence at this density of any particles, retroviral-like or otherwise, pure (isolated) or otherwise. Instead these researchers cultured lymphocytes from AIDS patients and stimulated (activated) them with a wide variety of agents. Reverse transcription of A(n).dT15 in the culture supernatant was considered proof for infection with a retrovirus or even proof of isolation. Supernatants from these cultures were introduced into cultures of leukaemic or transformed cell lines. With the supernatants from these cultures they performed two types of experiments: (a) The supernatants were banded in sucrose density gradients. At the 1.16 gm/ml band (and sometimes at other band(s), at least in Montagnier's group experiments, this is not made clear), they found fragments of RNA of certain lengths (although no two had the same length) or were rich in adenine, (poly(A) rich fragments), and called these "HIV RNA", the "HIV genome". Using a (dT) primer the "HIV RNA" was transcribed into a complementary DNA (cDNA); (b) The supernatants were introduced into another set of the transformed and leukaemic cell lines as well as into stimulated cultures of normal T-cells. The DNA from these cells, as well as the DNA from the cultures to which no supernatant was added, were hybridised using probes from the cDNA. Positive results were obtained only with the DNA from the cells to which the supernatants were added. This evidence was interpreted as proving that the "HIV DNA", the retrovirus, originated from the AIDS patients and in fact that these patient acquired it from the outside, that is, the retrovirus was exogenous.

There are many problems associated with these experiments and their interpretation. Among the many questions their conclusion raises the most obvious are: 1. HIV is said to be a retrovirus and retroviruses are particles which contain among other things, RNA. How then is it possible to claim that the RNA which banded at 1.16 gm/ml, "HIV RNA", is the genome of a retrovirus without proof that it is a constituent of a particle, viral or non-viral which bands at this density? 2. RT is not specific to retrovirus and in fact A(n).dT15 can be reverse transcribed by all cellular DNA polymerases à, á and y. Is it possible then to consider reverse transcription of A(n).dT15 as proof for HIV isolation or even detection of a retrovirus? Even if the process of reverse transcription is specific to retroviruses, can the detection of a process ever be considered proof for the isolation of an object, in this case, retroviral particles? 3. cell culture supernatants will contain both DNA and RNA including some enclosed in cellular debris (fragments) especially if cellular viability is not one hundred percent as is the case in cultures used by the three groups. The RNAs may include messenger RNA (which is adenine rich), as well as high molecular weight heterogenous nucleic RNA. These RNAs, in addition to having high molecular weight and heterogeneity in size, also have poly(A), with the poly(A) attached at the 3' end of the molecule, and may be RNAase resistant. Actinomycin, inhibits its synthesis and also interferes with its proper processing and breakdown.(99) From animal virology it is also known that non-retroviral RNA and DNA also bands at 1.16 gm/ml.(100) How is it then possible to claim that just because an RNA bands at 1.16 gm/ml and is adenine rich or has a certain length, it is "HIV RNA"? If this RNA is "HIV RNA", then what is the other RNA and the DNA which also bands at this particular density? If the latter are cellular why not the poly(A)RNA as well? 4. By definition, retroviruses are infectious particles which contain only RNA. When they enter a cell the RNA is reverse transcribed into DNA, which is then integrated into cellular DNA as a provirus, which means that "HIV DNA" will be present only in the cell and nowhere else. Yet many HIV experts including Gallo have shown that both the supernatants of "infected" cell cultures and the "HIV particles", that is, the material which bands at 1.16 gm/ml, contains "HIV DNA" which "may integrate directly into the host chromosomal DNA".(101-103) The question then arises, is the "HIV DNA" the result of "HIV RNA" reverse transcription or is it vice versa? 5. It is accepted that the HIV RNA is localised in a condensed core surrounded by a "lipid-bilayered envelope derived from the cellular membrane of the host cell, studded with virally encoded gp120 and myristylated protein, p17. The so-called core-envelope link (CEL) attaches the core to the envelope".(103) One of the best know facts in biology is that condensed cores (chromatin) is transcriptionally inactive. This is one of the reasons why viruses, including retroviruses, to multiply, must first enter cells where their chromatin is decondensed. However, in a paper published in 1993 by Hui Zhang and colleagues including Poiesz, from Suny Health Science Center at Syracuse, New York, wrote: "We have shown that in the absence of detergent, large amounts of DNAase-resistant viral DNA can be synthesized within intact HIV-1 virions, indicating that this phenomenon is not dependent on perturbation of the viral envelope. [Not to mention decondensation of chromatin]. Nascent viral DNA synthesis also occurred in purified virions incubated at 37ø in cell-free human physiological fluids including seminal plasma, breast milk, and fecal fluids"(103) This means that either (i) the "intact HIV-1 virions" perform a function that no other biological system with very condensed and protected chromatin can perform or (ii) the "HIV RNA" found in the supernatants or in the "purified virions" is present in an unembodied form or (iii) the "HIV RNAs" are de novo synthesised in the cell cultures (see 6.3.5); 6. At present there is ample evidence that any RNA or DNA present in the supernatant, irrespective of its origin, especially when cells are stimulated by polycations and oxidising agents, will be taken up by the cells (see 7.1). How is it then possible to claim that a positive hybridisation signal in cells cultured with the same "HIV DNA" containing supernatant as the supernatant from which the "HIV DNA" probe originated but not in other cells is proof that the "HIV DNA" is the genome of an exogenous retrovirus? 7. The first, absolutely necessary step in proving that the "HIV DNA" originated from the lymphocyte cells of AIDS patients and those at risk, is to perform hybridisation experiments using the DNA of their fresh, uncultured lymphocytes and the "HIV DNA" as a probe. It is hard to understand why neither Montagnier's nor Levy's group reported such experiments. Gallo's group did and the results were negative (see 6.4.4). How is it then possible to claim that "HIV DNA" is the genome of an exogenous retrovirus which originated from AIDS patients and those at risk? 8. Reading the seminal paper on HIV isolation entitled "Detection, Isolation and Continuous Production of Cytopathic Retroviruses (HTLV-III) from patients with AIDS and Pre-AIDS", one gets the impression that the leukaemic HT cell line which Gallo, Popovic, and their colleagues used was a new cell line and one which they established. The Gallo inquiry revealed that the HT (H9) cell line is the same as that used by Levy's group, HUT78, a leukaemic cell line established in another laboratory. However, the abundant evidence for the existence of endogenous human retroviruses has largely been obtained from experiments on leukaemic and transformed cells. Evidence exists that both H9 and EBV-transformed B lymphocytes release retrovirus-like particles even when not "infected with HIV".(104) Furthermore, the HUT78 (H9) cell line was established from a patient with "malignancies of mature T4 cells", a disease which, according to Gallo, is caused by the exogenous retrovirus, HTLV-I. Indeed, as far back as 1983, he claimed to have shown that the HT (H9) cell line contained HTLV proviral sequences.(105) According to some American researchers, EBV- transformed normal human peripheral blood B lymphocytes contain HTLV-I related transcripts.(106) Since all retroviral particles by definition band at 1.16 gm/ml, assuming that all the groups had a retrovirus at this density, how is it possible to claim that the retrovirus originating from the HUT78 and EBV-transformed B- lymphocytes is a new retrovirus HIV, and not one which was already present? Can one claim that the "HIV RNA" and thus the probes and primers originating from it are the RNA and probes and primers of a unique exogenous retroviral genome? 9. The biological dogma states that DNA is synthesised on a DNA template, RNA on a DNA template, and proteins on an RNA template. In other words, the only way for a cell to acquire new nucleic acid entities is for them to be introduced from the outside, exogenously either from another cell type, an infectious agent or a synthetic nucleic acid. If the biological dogma is correct then the "HIV RNA", be it a cellular or viral molecular entity, should have originated either from the patients' lymphocytes or the transformed and leukaemic cell lines. However, when "HIV cDNA" was used a probe, not one of the groups reported positive hybridization results from any of the cells, not even from the lymphocytes of AIDS patients. The question then arises, does a unique molecular entity, "HIV DNA" exist? What does it mean and from where did it originate?

6.3. SPECULATIONS ON "HIV DNA"

If one wishes to speculate on the nature and origin of RNA (cDNA) derived from the cultures containing tissues of AIDS patients and those at risk, and which bands at 1.16 gm/ml, there are many possibilities including:

6.3.1 Although to date no such evidence exists, it is possible that the stretch of RNA, presently called "HIV RNA", is the genome of an exogenous retrovirus, HIV. However, for this to be considered proven in addition to satisfying all the requirements in 6.1 one must also show that: (i) the unique stretch of RNA can be obtained only from cultures of particular individuals; (ii) when the RNA (or cDNA) is used as a probe to test fresh, uncultured lymphocytes, a positive test is obtained only from the fresh cells of individuals who also have a positive culture; (iii) that in animals or humans, the retrovirus is horizontally (animal to animal, person to person) transmitted.

6.3.2 The genome of an endogenous retrovirus, that is, a stretch of RNA with a corresponding DNA template present in the cellular DNA of uninfected animals and which is passed from generation to generation vertically (from parents to offspring via the germ cell line) and which under certain conditions can be expressed and incorporated into retroviral particles. For many decades it has been known that animal DNA contains sequences "closely related or identical with those of infectious viruses". However, the human genome was considered to be an exception and as late as 1994, both Gallo and Fauci were of the opinion that "...there are no known human endogenous retroviruses".107 In fact, in the 1970s and in the 1980s after Gallo's claim of the discovery of HL23V, HTLV-I and later HTLV-II, and especially after Montagnier's claim of the discovery of HIV, considerably greater interest was engendered in retroviruses with the result that it became "increasingly clear that the DNA of man, like that of other vertebrates, contains many integrated retroviral genomes", (25,108) and that in many cases the genes are expressed, "including mRNA transcripts related to full-length endogenous retroviral DNA" (109,110) with open reading frames for the gag, pol and env proteins.111 By 1987, many researchers reported the expression of the genome of the human endogenous retrovirus, HERV-K, homologous to the mouse mammary tumor virus (MMTV). "In several cell lines, HERV-K genome was expressed as an 8.8 kilobase poly(A)+ RNA which appears to be the full-length transcript of this genome". When the human breast cancer cell line T47D was "grown in RPMI 1640 supplemented with 10% fetal calf serum, HERV-K genome expression was slight". However, when the cells were treated with estradiol and then progesterone, they produced "retroviruslike particles and soluble protein sharing antigenic determinants with MMTV env gene product".(112) In support of their thesis "that a human endogenous RT might mediate gene movements leading to leukemia and cancer", researchers from Hahnemann University, Philadelphia, including David Gillespie, a long time collaborator of Gallo "demonstrated the presence of a reverse transcriptase-like enzyme in retroviral particles from patients with essential thrombocythemia, polycythemia vera, and chronic myelogenous leukaemia. It was subsequently shown that the human genome contains 50 copies of HERV-K. HERV-K is a human endogenous class I retroviral element that contains gag, pol and env open reading frames...as well as intact LTR regions...Expression of a 9 kb genomic HERV-K RNA transcripts were detected in human cell lines...We were able to show for the first time the expression of HERV-K pol gene in human blood leukocytes. The HERV-K pol gene was expressed in peripheral blood cells from two sets of non-leukemic individuals. The first set consisted of 7 normal donors, while the second set consisted of 3 patients with PV, all of which expressed HERV-K pol gene. Five different nucleotide sequences were obtained from the 7 normal donors. Four of the 5 normal sequences contained heterogenous open reading frames for pol as detected by both RT-PCR and RNAase protection. Unlike normal donors which randomly express HERV-K proviruses, analysis of HERV-K pol from PV patient showed selective expression of a restricted family of related proviruses".(113) By 1995, Gallo admitted that the human cell does contain retroviral genomes but he still insisted they are defective, "Retroviruses are transmitted either genetically (endogenous forms) or as infectious agents (exogenous forms). As do many other animal species, humans have both forms...The DNA of many species, including humans, harbor multiple copies of different retroviral proviruses. The human endogenous proviral sequences are virtually all defective, and comprise about one percent of the human genome".(114) The view regarding defectiveness is not shared even by Reinhard Kurth who, with his colleagues, have extensively studied the human endogenous retroviruses (115) and have shown that HERV-K sequences are transcribed and that a human teratocarcinoma cell line, GH, which contains these sequences, when examined by EM was found to produce "human teratocarcinoma-derived retrovirus (HTDV) particles". By 1993 Kurth and colleagues reported that in the GH cell line, "Four viral mRNA species could be identified, including a full-length mRNA. The other three subgenomic RNAs are generated by single or double splicing events...Sequence analysis of expressed HERV-K genomes revealed non-defective gag genes, a prerequisite for particle formation. Open reading frames were also observed in pol and env. Antisera raised against recombinant gag proteins of HERV-K stained HTDV particles in immunoelectron microscopy, linking them to the HERV-K family". Discussing their findings they wrote: "In Northern blots, expression of HERV-K could only be demonstrated in teratocarcinoma cell lines but not in other human lines. Preliminary RT PCR studies suggest, however, that HERV-K may be expressed in many if not all human cells at levels to low to be detectable in Northern blots. The basis of the significant quantitative differences in expression between teratocarcinoma cells and other cell lines is not clear. It is intriguing to speculate that a cellular factor(s) may regulate the synthesis of HERV-K mRNA depending on the cell type or the state of differentiation. In this context, it should be remembered that other retroid elements [ERV-9, RTLVL-H, LINE-1] are also preferentially expressed in human teratocarcinoma cells".(116) It is of interest to note that Montagnier and his colleagues reported their "HIV genome" from a transformed cell line, that Levy and colleagues' HUT78 cell line is a human leukaemic cell line and that Gallo and colleagues' H9 cell line is none other than HUT78, and thus must have HTLV-I as well as endogenous retrovirus. It is equally important to note that although Kurth et al found no sequence homology between HERV-K and "human T-lymphotropic virus" or HIV, many researchers reported HTLV-I sequences in the human genome including in cell lines derived from teratocarcinoma.

In a paper published in 1985 researchers from a number of institutions in the USA including the Laboratory of Tumor Immunology and Biology, National Cancer Institute, Bethesda, it was reported that "Human DNA contains multiple copies of a novel class of endogenous retroviral genomes. Analysis of a human recombinant DNA clone (HLM-2) containing one such proviral genome revealed that it is a mosaic of retroviral-related sequences with the organization and length of known endogenous retroviral genomes. The HLM-2 long terminal repeat hybridized with the long terminal repeat of the squirrel monkey virus, a type D virus. The HLM-2 gag and pol genes share extensive homology with those of the M432 retrovirus (a type A-related retrovirus), mouse mammary tumor virus (a type B retrovirus), and the avian Rous sarcoma virus (a type C retrovirus). Nucleotide sequence analysis revealed regions in the HLM-2 pol gene that were as much as 70% identical to the mouse mammary tumor virus pol gene. A portion of the putative HLM-2 env gene hybridised with the corresponding region of the M432 viral genome". The pol region of HLM-2 showed homology with HTLV-I which, according to the authors. "is not endogenous to human cells but is transmitted horizontally as an infectious tumor-inducing virus of humans".(117)

In 1987 researchers from Canada reported the finding of a "Human Endogenous Retroviruslike Genome with Type C pol sequences and gag sequences related to the Human T-cell Lymphotropic Viruses", HTLV-I and HTLV-II.(118) In 1989 researchers from the Department of Biochemistry, New York University showed that "human DNA contains a wide spectrum of retrovirus-related reverse transcriptase coding sequences, including some that are clearly related to human T-cell leukaemia virus type I and II, some that are related to the L-I family of long interspersed nucleotide sequences, and others that are related to previously described human endogenous proviral DNAs. In addition, human T-cell leukaemia virus type I-related sequences appear to be transcribed in both normal human T cells and in a cell line derived from a human teratocarcinoma".(119) In a paper published in 1989, researchers from the USA summarised their experimental findings as follows: "Human T-cell leukemia virus (HTLV) type I- related endogenous sequences (HRES) have been cloned from a human genomic library. HRES-1/1 is present in DNA of all normal donors examined. By nucleotide sequence analysis, HRES-1/1 contains two potential open reading frames capable of encoding a p25 and a p15. A 684 flanking region 5' from the first ATG codon of p25 contains a TATA-box, a poly-adenylation signal, a putative tRNA primer binding site, and inverted repeats at locations which are typical of a retroviral long terminal repeat...The HRES-1/1 genomic locus is transcriptionally active in lymphoid cells", including EBV- transformed normal human peripheral blood lymphocytes, leukemic cell lines, melanoma cells and embryonic tissues.(106) In a paper published in 1992 by researchers from Hungary and Britain entitled "Human T-cell lymphotropic virus (HTLV)-related endogenous sequences, HRES-1, encodes a 28-kDa protein: A possible autoantigen for HTLV-I gag-reactive autoantibodies", the "presence of a human T-cell lymphotropic virus (HTLV)-related endogenous sequence, HRES- 1, in the human genome was documented. The HRES-1 genomic locus is transcriptionally active and contains open reading frames...Antibodies to HRES-1-specific synthetic peptides were noted in patients with MS, progressive systemic sclerosis (PSS), SLW, Sjogren syndrome (SJS), and essential cryoglobulinemia (ECG). The data suggest that HRES-1 may serve as an autoantigen and correspond to a natural target of HTLV-I core protein-reactive autoantibodies".(120)

6.3.3 The genome of a retrovirus de novo assembled by genetic recombination and deletion of: (a) endogenous retroviral sequences; (b) retroviral and cellular sequences; (c) non-retroviral cellular genes.

In the virological literature there is ample evidence which shows that when a cell contains two proviruses, progeny may be found that possess the genome of one but the structural proteins of either or both viruses present. Conversely, the RNA may be viral but at least some of the proteins may be cellular. In other instances, the particles do not have a genome at all, or one or more genes are missing (genetically defective viruses). The genetic mixing can be between viral genomes or between viral and cellular genes.(83,121) According to distinguished retrovirologists such as Weiss and Temin, new retroviral genomes may arise by rearrangement of cellular DNA caused by many factors including pathogenic processes, a view that proposes retroviruses as an effect and not the cause of disease.(122,123) According to Varmus, "Retroviral genomes recombine at high frequency (estimates range as high as 10 to 30% for each cycle of multiplication), and heterodimeric RNAs are thought to be intermediates, with recombination taking place during reverse transcription. Recombination appears to be strongly favoured by homology, but joining also occurs occasionally between unrelated sequences, e.g., during the latter phase of genetic transduction by retroviruses. When viruses are grown in cells that contain related endogenous proviruses, packageable transcripts from those proviruses may participate in recombination reactions with the exogenous virus. This is most dramatically revealed by the repair of deletion mutations in the genome of an exogenous virus in a fashion that superficially resembles gene conversion". In some animals proviruses have been acquired "during recent breeding of the strains in the laboratory" and "in a few instances, endogenous proviruses have been established or increased in number during experimental observations"(121) (italics ours).

As far back as 1974, based on the then available evidence, Howard Temin proposed that the retroviral (ribodeoxyviruses) genomes originate from "normal cellular components. The relationships between the different ribodeoxyvirus groups reflect the relationships among the cellular components from which the viruses evolved and the convergent evolution of the viruses. In other words, there are relationships among ribodeoxyviruses because the ribodeoxyviruses evolved from cells which themselves had relationships deriving from common ancestors. A possible mechanism of this evolution is described in Fig. 5". In the legend to Fig. 5 Temin wrote. "A section of a cell genome becomes modified in successive DNA (W) to RNA (-) to DNA transfers until it becomes a ribodeoxyvirus genome. First, these sequences evolve as part of a cellular genome. After they have escaped as a virus, they evolve independently as a virus genome. The time scale may be millions of years in germ-line cells and days in somatic cells".(122) Temin reinforced his view in a more recent publication.(124)

In 1975, Gallo, Gillepsie and their colleagues wrote: "Even though RNA of class II [exogenous] retroviruses shows minimal homology to uninfected host cell DNA, hybridization of nucleic acids among class II leukemia viruses from different species gives a pattern which is the same as the phylogenetic relatedness among their natural hosts...We have proposed that these and other results favor the interpretation that all RNA tumor viruses are derived from cell genes, a proposal in agreement with the virogene theory...By analysis of the RNA of viruses infecting and replicating in a new host, evidence has also been obtained which indicates that the genome of type C viruses can be substantially changed by the host, probably by recombination with host DNA".(125) A few years later, Coffin wrote: "The close relationship of virion proteins as well as overall nucleic acid homology must mean that both exogenous and endogenous avian tumor viruses [retroviruses] derive from a common ancestor".(126)

In 1991 researchers from the New York University published a paper entitled, "Evolutionary Implications of Primate Endogenous Retrovirus". Discussing the presently available data they wrote, "A recent detailed phylogenetic analysis of exogenous and endogenous retroviruses (including retrotransposons) strongly suggests that a pool of endogenous retroviral sequences periodically contributes to the generation of exogenous viruses, and that the presence of endogenous primate retroviruses is probably more directly related to exogenous viruses that might have been thought".(127)

6.3.4 The "novel" RNA found in the cell culture supernatant and the material from it banding at 1.16 gm/ml, the "HIV RNA", may have nothing to do with a retroviral genome. It may be an RNA obtained by transposition, that is, by certain replicating DNA sequences (transposons) becoming inserted elsewhere in the genome, or by retroposition, that is, by particular RNA (retrotransposons) first being transcribed into DNA and then similarly being inserted into the genome. Retroposition can "use cellular mechanisms for passive retroposition, as well as retroelements containing reverse transcriptase". The retroelements may be retrovirus-like elements or nonviral elements.(128,129) Not only can retroposition "shape and reshape the eukaryocytic genome in many different ways"(128) but the nonviral retroelements may be similar to the retroviral elements. According to Doolittle et al from the University of California, San Diego,"...the entire group of reverse transcriptase-bearing agents, including retrotransposons and genuine retroviruses, has recently been dubbed, "retroids". Sequence comparisons by many other workers leave little doubt that the reverse transcriptases of all the "retroids" considered here are homologous, which is to say, the sequence resemblances are not the result of chance or convergences. Our own comparisons confirm that general notion, not only for reverse transcriptases, but also for the ribonucleases, endonucleases and proteases, although it should be understood that not all "retroids" contain all four enzymes...All of these elements have additional features in common with retroviruses including characteristic LTRs (long terminal repeats) and primer sites that are complementary to various tRNAs. Like retroviruses, most contain distinctive nucleic acid-binding and core particle proteins; in electron micrographs there is a remarkable likeness to retroviral capsids...About the only feature that regularly distinguishes many of these retrotransposons from genuine retroviruses is the absence of an envelope protein".(17)

6.3.5 Although half a century has passed since the Nobel laureate Barbara McClintock discovered the phenomenon of transposition which can lead to the appearance of new genotypes and phenotypes, at present it is still generally accepted that any time one finds a particular stretch of RNA in a cell, for example, in a T- lymphocyte, unless RNA or DNA has been introduced from outside, all T-cells, regardless of their physiological state or stresses to which they are subject, will contain a corresponding stretch of DNA. In other words, the DNA (genes) in a cell are invariant and all RNA molecules in the cell are subservient to a matching length of DNA. However, according to McClintock, the genome can be restructured and not only by transposition. In her Nobel lecture of 8th December 1983, she said, "rapid reorganisation of genomes may underline some species formation. Our present knowledge would suggest that these reorganizations originate from some "shock" that forced the genome to restructure itself in order to overcome a threat to its survival...Major genomic restructuring most certainly accompanied formation of new species". The "genomic shock" which leads to the origin of new species may be "either produced by accidents occurring within the cell itself, or imposed from without such as virus infections, species crosses, poisons of various sorts, or even altered surroundings such as those imposed by tissue culture. We are aware of some of the mishaps affecting DNA and also of their repair mechanisms, but many others could be difficult to recognize. Homeostatic adjustments to various accidents would be required if these accidents occur frequently. Many such mishaps and their adjustments would not be detected unless some event or observation directed attention to them...Unquestionably, we will emerge from this revolutionary period with modified views of components of cells and how they operate, but only however, to await the emergence of the next revolutionary phase that again will bring startling changes in concepts"(130) [italics ours and see this reference for examples].

In the 1980s a number of phenomena have been discovered which brought startling changes in concepts including the following: Up until the late 1970s, the prevailing concept was that a discrete, contiguous stretch of DNA is a structural gene encoding the genetic information to specify the manufacture of a single protein, and that the linear sequence of the nucleotides in this stretch of DNA corresponds directly to the linear sequences of the RNA nucleotides and to the amino acids in the protein. The first discovery which contradicted this belief was the discovery that the DNA base sequences which coded for a given protein were not in a continuous stretch of DNA but may be interspersed with other, non-coding base sequences, that is, the genes are split, "genes-in-pieces". A number of mechanisms have been postulated to account for this observation. In one such explanation it is hypothesised that the entire stretch of DNA is transcribed into a piece of RNA, then the non-coding regions (introns) are excised and the coding regions (exons) are spliced together to make the appropriate messenger RNA.(131) There are no rules setting an upper limit on the number of introns in a "gene", some genes may have up to sixteen or more introns. Nor are there any rules regarding the length of introns, although in general, introns are much longer than exons, the length of exons "peaking at about 40 or 50 amino acids...the shortest intron being 50 bases long, the longest extending out to some 50.000 bp".(132)

According to Gilbert introns represent "hot spots" for recombination and new genes can be created "through the coupling of exons by intron-mediated recombination", "introns are lost and more complicated exons are formed".(133) At present evidence exists showing that at least some introns are mobile genetic elements, transposable elements, they self-splice, they often contain reading frames capable of encoding a protein including "regions of homology to reverse transcriptase scattered over a roughly 250- amino acid stretch in the middle of each intron ORF".(134) The discovery of split genes "shows that the genetic apparatus of the cell is more complex, more dynamic than any of us had suspected".132 Another strongly held view was the belief that all cellular reactions and thus gene splicing were catalysed by a protein enzyme. In the early 1980s it was found that RNA can cut, splice and assemble itself, as well as assemble RNAs other than itself.(135-138)

6.3.6 One of the strongest held views in biology is the belief that nucleic acids have an inherent ability of instructing their own synthesis and that nucleic acids cannot be synthesised in the absence of a nucleic acid template. Manfred Eigen and his colleagues in Germany conducted extensive theoretical and experimental work on molecular self-replication.(139) In their experimental work they used the bacterial virus (phage) Qá. In addition to its genome, a simple strand RNA molecule of 4500 nucleotides, the virus has an RNA molecule of 220 nucleotides known as "Spiegelman's minivariant" which, like the genomic RNA, is reproduced in cell-free laboratory systems by an enzyme called Qá replicase. By mixing Mg2+ ions, the nucleoside triphosphates ATP, GTP, UTP, CTP, Qá replicase and template RNA, they could obtain RNA replication but a totally unsuspected finding was that even the absence of the template, RNA was still synthesised. They performed many experiments to prove this phenomenon and to exclude the possibility of the presence of an initial RNA template and concluded, "Finally we were convinced we had before us RNA molecules that had been synthesised de novo by the Qá replicase enzyme. What was most puzzling, the de novo product had a uniform composition which in each trial turned out to be similar to or even identical with Spiegelman's minivariant". When the template free mixture was then divided into several isolated compartments where optimal conditions for de novo synthesis were maintained they found that "each component had a uniform population of de novo product, the products differed from compartment to compartment. Further analysis revealed however that the different sequences were not completely unrelated...There was a definite, uniform final product for any set of experimental conditions, but here were as many different optimal products as there were different experimental conditions. One of the optimal products appeared to be Spiegelman's minivariant...Other products of optimization were adapted to conditions that would destroy RNAs, such as high concentrations of ribonuclease, an enzyme that cleaves RNA into pieces...Some variants were so well adapted to odd environments that they had a replication efficiency as much as 1000 times that of variants adapted to a normal environment...Any RNA formed by noninstructed chemistry would be reproduced by template-instructed chemistry at a rate proportional to the current RNA concentration. The result would be exponential growth. Furthermore, even if only a single template were formed initially by noninstructed synthesis, there would soon be a host of different sequences because errors (point mutations, insertions and deletions) would inevitably be made in the course of replication. Hence in each generation there would be not only a larger number of RNA strands but also a greater variety of RNA sequences. What would happen then? Some of the mutants would be copied more rapidly than others or would be less susceptible to errors in copying, and their concentration would increase more rapidly. Sooner or later these faster-growing mutants would take over...Hence the results of the self-replication competition had to be the master sequence together with a huge swarm of mutants derived from it and from which it had no way of escape...We call this entire mutant distribution a quasispecies. It is the quasispecies mutant distribution that survives the competition among self-replicating RNAs and not just one master sequence or several equivalent ones that are the fittest genes in the distribution. The essence of selection them is the stability of the quasispecies".(140) According to Eigen and his colleagues, the maximum length of an RNA master sequence is of the order of 10,000 nucleotides.(139,141)

6.3.7 A basic principle of molecular biology is that the primary sequence of RNA faithfully reflects the primary sequence of the DNA from which it is transcribed. However, in the 1980s RNA editing, "broadly defined as a process that changes the nucleotide sequences of an RNA molecule from that of the DNA template encoding it", was discovered. In the process a non-functional transcript can be retailored, producing a translatable mRNA, or modify an already functioning mRNA so that it generates a protein of altered amino acid sequences. Sometimes editing is so extensive that the majority of sequences in a mRNA are not genomically encoded but are generated post-transcriptionally producing the "paradoxical situation of a transcript that lacks sufficient complementarity to hybridize to its own gene!".(142-144) According to Nancy Maizels and Alan Weiner from the Department of Molecular Biophysics and Biochemistry at Yale University, "the central dogma has survived hard times. The discovery of reverse transcriptase amended but did not violate the central dogma of how genes make proteins; introns qualified the conclusion that genes are necessarily collinear with the proteins they encode; somatic rearrangement of lymphocyte DNA called stability of eukaryotic genomes into doubt...and catalytic RNA challenged the pre-eminence of proteins and breathed new life into the ancient RNA world". However, the discovery of RNA editing "could come close to dealing it a mortal blow".(145)

6.3.8 CONCLUSION

The finding of a novel stretch of RNA or DNA and proteins in: (a) lymphocytes of sick individuals or individuals who have been "shocked" with agents such as physical or chemical mitogens, carcinogens or oxidising agents in general as is the case with AIDS patients and those at risk;(77,79,90) (b) lymphocytes in cultures or co-cultures (which could lead to the appearance of hybrids) which have been additionally "shocked" with sometimes multiple, similar agents; is not proof that the given stretch of RNA comes from the outside, irrespective of its length, the presence of poly(A) and number of ORF ("genes").

From Montagnier's, Gallo's and Levy's and their colleagues' evidence it is not possible to conclude that the "HIV RNAs" they found are a "new species" of RNAs induced by "shocking" the cells or by one or more of the other phenomena which have come to light in the 1980s. Nor is it possible to conclude that their RNAs are the genome of an exogenous retrovirus as they did. However, a number of predictions can be made: (a) If the "HIV DNA" is indeed the genome of an exogenous retrovirus then: (i) there must be evidence to prove the existence of a unique molecular entity "HIV RNA", and a corresponding fragment of DNA ("HIV DNA") which has a unique length and unique nucleic acid sequences; (ii) when the full length fragment of "HIV DNA" or "HIV cDNA" is used for hybridisation studies all infected people should give a positive result. (b) If the selected RNA which was found to band at 1.16 gm/ml, the "HIV RNA", is the genome of a retrovirus which exists "in all of us", endogenous retrovirus, then again evidence must prove the existence of a unique molecular entity, "HIV RNA", ("HIV DNA"). When hybridisation studies are conducted using the full length of the unique molecular entity as a probe, positive results should be found "in all of us"; (c) If the RNA found by the three groups, "HIV RNA", is the genome of a retrovirus assembled de novo from DNA already existing in the cells, as the result of in vivo or in vitro conditions, evidence must also prove the existence of a unique molecular entity. When the whole length of the unique fragment of nucleic acids is used as a hybridisation probe, a positive result should only be found in cells which are subjected to exactly the same in vivo or in vitro conditions as those from which the "HIV RNA" at 1.16 gm/ml was obtained. When only fragments of "HIV RNA" are used for hybridisation, the probability of finding a positive result will increase; (d) If the "HIV RNA" is a unique non-viral molecular species of RNA resulting from the transcription of a unique molecular species of DNA then when the whole fragment of "HIV RNA", ("HIV cDNA") is used a probe for hybridisation studies, a positive result should be found only in the cells of the same type as those from which the "HIV RNA" originated, in all individuals; (e) If the "HIV RNA" is neither the genome of a retrovirus nor a faithful transcript of a fragment of DNA present in the cells from which it has been obtained, but is the result of the "shock" to which the cells have been exposed, either in vivo or in vitro or both, or as a result of the phenomena discovered in the 1980s then: (i) since it is not possible to exactly reproduce the conditions in vivo or in vitro to which the cells are subjected, it would prove difficult if not impossible to always obtain a unique molecular entity "HIV RNA", that is, to always obtain a fragment of RNA or DNA of identical length and sequences; (ii) when the full-length fragments of "HIV RNA" or "HIV cDNA" are used as hybridisation probes there will be only a low probability of finding a positive result. However, the probability will increase if only small fragments of the "HIV RNA" or "HIV cDNA" are employed.

6.4. EVIDENCE THAT THE "HIV RNA" BELONGS TO AN EXOGENOUS RETROVIRUS

The Montagnier, Gallo and Levy groups claimed that the special RNA which they selected from the total RNA which in sucrose density gradients banded at the density of 1.16 gm/ml was novel to the lymphocytes and that in fact belonged to an exogenous retrovirus. Although they did not present evidence to prove this assertion, the possibility cannot be excluded that indeed this may have been the case. Since at present their claim is generally accepted one would have thought that by now they or other researchers should have been able to provide ample confirmatory proof. This does not seem to be the case:

6.4.1 If the RNA originates from a retrovirus either endogenous or exogenous then evidence must exist which proves that such RNA is a constituent of particles which possess at least the most basic morphological and physical features of retroviruses, that is, "a diameter of 100-120 nm budding at cellular membranes. Cell released virions contain condensed inner bodies (cores) and are studded with projections (spikes, knobs)".(82) To date not only has nobody shown that the "HIV RNA" belongs to such particles, there is no evidence that particles of any kind are present in the material from cell cultures/cocultures which bands at the retroviral density of 1.16 gm/ml and from which the "HIV RNA" is selected. Furthermore, although particles have been demonstrated in cultures, cultures contain many different types of particles but none display BOTH principal morphological characteristics, that is, "a diameter of 100-120 nm" AND surfaces which "are studded with projections (spikes, knobs)".146

6.4.2 If the "HIV RNA" is the genome of an exogenous retrovirus then, like the "exogenous animal retroviruses", one should be able to find it in infected material without the necessity to revert to the use of co-cultivation or mitogenically stimulated cultures. However, none of the phenomena which are thought to prove the existence of HIV can be detected unless one employs mitogens or co-cultures or both (and sometimes additional "shock"), a fact accepted by both Montagnier and Gallo.(78,147)

6.4.3 One cannot claim that "HIV RNA" is the genome of a unique retrovirus, HIV, unless evidence is presented to prove that 'HIV" is a unique molecular entity. By 1985 it was known that "the env genes of ARV and HTLV-III differ by more than 20 percent" and that "the Gallo group has sequenced another HTLV-III isolate and finds that it differs from the first by about as much as ARC".(114,148) By 1986, Gallo and his colleagues accepted that the "HIV genome" has a "far greater variability" as "compared to HTLV" and in fact "The rate of genetic change for the AIDS virus is more than a millionfold greater than for most DNA genomes and may even be tenfold greater than for some other RNA viruses including certain retroviruses and influenza A virus".(149) At present it is accepted that "no two isolates are identical. Each isolate contains many variants".(150) In one and the same patient the genomic data in monocytes differs from that in T-lymphocytes.(151) There are "striking differences" between the proviral DNA and cDNA in one and the same PBMC sample "which could not be explained by either an artefact of reverse transcriptase efficiency or template selection bias".(152) The genetic data obtained in vitro do not correlate with the data obtained in vivo, "to culture is to disturb".(153) According to the researchers from the Pasteur Institute "an asymptomatic patient can harbour at least 106 genetically distinct variants of HIV, and for an AIDS patient the figure is more than 108.(154,155) The "HIV genome" varies with time; in one case where clones were obtained 16 months apart all the clones detected in the second sample were distinct from the clones in the first sample.(156) It is also accepted that up to 99.9% of the "HIV genomes" may be defective.(157)

According to Levy, "The mechanism responsible for generating these varying strains of virions is puzzling. One theoretical possibility is that the unintegrated proviral copies of HIV that accumulate during acute replicative infection can undergo efficient genomic recombination leading to the evolution of infectious variants.(158) In Robin Weiss' view, "the source of variation is the infidelity of reverse transcription, which has no editing mechanism for transcriptional errors", as well as "genetic recombination" especially when cell fusion takes place.(159) By the late 1980s, researchers from the Pasteur Institute concluded, "it is increasingly clear that it will be very difficult to describe correctly the characteristics of HIV viruses using single molecular clones". "It is evident that HIV, either in vivo or in vitro, is extraordinarily complex and that a population-based approach", a quasispecies approach as defined by Eigen, must be used to describe HIV. They also added, "Even with a population-based approach, only small regions of the HIV genome can be studied...Given such complexity and the evident differences between quasispecies in vivo and in vitro, the task of defining HIV infection in molecular terms will be difficult".(153,160) The data which have been published since confirm their conclusion. Prior to the 1990s, the HIV sequences were classified as African and USA/European with sequence differences of 20-30 percent between these two groups.(161) In the 1990s, HIV researchers started to divide the "HIV genome" into subtypes A, B, C, D, E, etc. The basis for this classification system is: "(a) subtypes are approximately equidistant from one another in env (a 'star' phylogeny"); (b) the env phylogenetic tree is for the most part congruent with gag phylogenetic trees; (c) two or more samples are required to define a sequence subtype". However, "Subtype naming problems have arisen for several reasons. A small but not insignificant number of viral sequences are hybrid, clustering with one sequence subtype in gag and another sequence subtype in env, for example; or, to take another example, clustering over different stretches with two or more subtypes in env...Naming becomes problematic when highly divergent forms of a given subtype arises: such forms are sometimes designated A', B', F', etc". It is increasingly necessary to have sequence data from both gag and env coding sequences when a new form or subtype is being claimed".(162)

By the middle of this year "at least ten" (A-J) prevalent major (M) and a low prevalence, O, HIV-1 genotypes were described and new genotypes are still reported.(8,163) According to researchers from the Henry M Jackson Foundation Research Laboratory and Division of Retrovirology, Walter Reed Army Institute, USA, "The great majority of genotypic consignments for HIV-1 are based on subgenomic sequence segments, typically encompassing 2% to 30% of the genome", and not by comparisons of the whole genome. This is because, "it remains impractical to obtain full length genomic sequences of HIV- 1 isolates as a routine genotyping method, due to the low abundance of HIV-1 proviral DNA in clinical samples and virus cultures on PBMC substrate, and to the relative inefficiency of the polymerase chain reaction when amplicons become large". "The designation Human Immunodeficiency Virus Type-1 (HIV-1) encompassed an unanticipated complexity of viral forms".(163) According to researchers from the Los Alamos National Laboratory, "while a subtype designation based on a gene or gene fragment may be correct, recombination may have occurred. Therefore, care should be taken to not over interpret the subtype designation. If one is to discuss the subtype designation of viral isolates based on the data presented here, they should be refer to the designation as 'B-like over V3 loop region' rather than as 'subtype-B'".(164) One and the same person may be "infected" with more than one subtype.(165) This means that at present it is not possible to say what are the sequence differences, both qualitative and quantitative, between different HIV-1 subtypes. Nonetheless, some suggestive data does exist. In 1993 researchers from several institutions "reported that in the A-G HIV-1 genotypes the intra-genotypic gag distances averaged 7% whereas the inter-genotypic distances averaged 14%...The maximum level of variability in gag is still well below that observed for the envelope region of HIV-1".(166) Two HIV-1 strains, designated ANT70 and MVP5180 were isolated in 1987 and 1991 respectively from patients in Cameroon". They were classified as HIV-1 subtype O. By 1994 evidence was presented which "indicated that subtype O was endemic in Cameroon and Gabon".(167) "DNA sequence analysis of MVP-5180 showed that its genetic organisation was that of HIV-1, with 65% similarity to HIV-1 and 56% similarity to HIV-2 consensus sequences. The env gene of MVP- 5180 had similarities to HIV-1 and HIV-2 of 53 and of 49% respectively...Comparison of the MVP-5180 amino acid sequence with that of the Gabon chimpanzee virus showed similarities of 70, 78 and 53% in the gag, pol, and env genes, respectively; similarities of 70, 76 and 51% to the Uganda HIV-1 (U455) and of 54, 57 and 34% to the HIV-2 isolate D205 were found". The researchers from Germany and Cameroon who conducted this study expressed the view that "Even more divergent HIVs may exist. Such divergent HIVs are likely to be transmitted by the usual routes (sexual and blood contact and mother-to-infant transmission), leading to wider distribution. They will have to be taken into account in vaccine development and diagnostic test sensitivity and specificity".(168) Indeed, this seems to be the case. Last year, David Ho and his associates (169) studied an Australian patient with "primary infection". "Since seroconverters generally harbor a relatively homogenous population of viruses", they were surprised when they found that he was "co-infected", "by multiple subtype B HIV-1...The average genetic distances between group I and II, I and III, and II and III were 9.6, 16.5 and 18.4% respectively...One population of sequences was clearly distinguishable from the others on the basic of phylogenetic analysis, In addition, sequences suggesting recombination between tw