Re: Where is the "HIV-1 infectious molecular clone"? 14 July 2004
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Nicholas Bennett,
Infectious Disease Postdoc/Clinician
Department of Pediatrics, University Hospital, Syracuse NY

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Re: Re: Where is the "HIV-1 infectious molecular clone"?

In their correspondance with regard to Brian Foley, the Perth Group ask for an "infectious molecular clone", and state that they "do not know of any 'HIV-1 infectious molecular clone'".

Might I interject with what, firstly, an infectious molecular clone is, according to my understanding. It is, simply, a full-length genome clone (usually DNA, for stability and ease of malipulation) which when put into a culture system produces virus which itself can them self-propogate. Therefore, the cloning of a virus genome is exactly all that is required to get an "infectious molecular clone". The two terms are practically synonymous, with the caveat that the virus is demonstrably infectious in its own right (2nd passage of culture supernatant). I don't believe the Perth Group are correct in their persistence in denying this.

As such, there are a plethora of infectious molecular clones of HIV-1 in circulation: the genome of the strain HXB2 (accession number k03455 in Genbank) is in common use as the infectious molecular clone pSVC21. HXB2 is referred to as the "Human immunodeficiency virus type 1 (HXB2), complete genome; HIV1/HTLV-III/LAV reference genome".

I use this as an example because of its nature as the reference genome, and because I can personally attest to its ability to produce virus from the DNA plasmid which then self-propogated.

As the existance of this DNA construct cannot be in debate, the only issue must therefore be its identity: is it "HIV" in terms of "the infectious cause of AIDS" or merely "HIV" because that nucleotide sequence has been labelled as HIV, and we might as well have called it Fred?

Therefore we must look to additional evidence: firstly, and most obviously, the genes in HXB2 encode a collection of 9 proteins (16 proteins after cleavage) which are all found in infected/transfected cells and most of which are also virion proteins [1]. These proteins are clearly those of a retrovirus (Gag Pol Pro Env), and indeed Reverse Transcriptase is produced and can be used as a measure of viral replication [2]. The sequences are not endogenous, and therefore must be exogenous [3]. Together therefore, the evidence above clearly shows the existence of an exogenous complex retrovirus (simple retroviruses like RSV encode no accessory genes - HIV has 6, vif vpr vpu tat rev nef). That cannot be in debate - the genetic code exists and is operational, therefore HIV exists.

Having proven the existence of an infectious molecular clone of a virus, it remains to associate it with a disease. Early in the investigative story, patients with persistent generalised lymphadenopathy (PGL) were found to have antibodies that reacted with the proteins produced by a virus (this virus) that was cultured from them [2, 4]. These days we also know that these antibodies react with the proteins produced by molecular clones of the virus (the basis of many of the ELISA and WB diagnostic tests). PCR detects the virus in the blood stream and lymph nodes of seropositive people. Virus culture can also demonstrate active infection in those who are seropositive [5-7].

The rate of progression to AIDS is clearly linked to the level of virus replication as judged by viral load [8-9]. Low viral load is associated with a more active specific anti-HIV immune response [10]. Indeed, cellular immunity to HIV can be induced and is protective against infection [11-13]. Genetic polymorphisms that affect aspects of the viral life cycle are protective against infection [14]. Viral mutations are protective against progression [15].

Infection with the virus induces changes in lymphocyte kinetics that are reversible with specific antiretroviral treatment [16]. Such changes are consistent with the immune suppression seen in late-stage infection, which is also reversible upon treatment [17-19].

As for how the molecular clones were derived, it appears as if HXB2 at least was cloned from patient tissues and/or cultured virus obtained from primary samples (standard virological procedure) [3]. In my mind, I actually have to side with Duesberg on the concept that the purist form of any virus is the molecular clone, and sequencing of viral RNA or proviral DNA (with subsequent analysis) is a perfectly acceptible method of proving existence. Given the option of trying to get extremely pure viral particles before even attempting characterization, and using partially purified particles to get a molecular foot on the ladder, I'd take the molecular approach every time. This is exactly what happened in the history of HIV. As I have said before, in many respects the proof of HIV's existence and causation of AIDS has been done retrospectively by "backfill" of knowledge after having made entirely reasonable assumptions, but this supports rather than detracts from those original assumptions.

Given the wealth of evidence presented above (merely the tip of the iceberg), one has to ask how any other explanation can possibly fit so well? Oxidative stress, drug use, malnutrition, even antiviral therapies themselves have all been promoted as possible causes of AIDS. In the face of the facts above, only one cause remains plausible - that of infection with HIV.

If the Perth Group persist in questioning the authenticity of the HIV molecular clones, they must explain why people produce specific antibodies to proteins encoded by the clone, why DNA/RNA sequences from the clone can be found in seropositive people, why viral proteins can be produced in culture systems seeded from seropositive people, why the virus can be seen under EM of seropositive lymph node samples, why immune responses to the viral proteins are protective and slow progression, why defective virus results in slower progression and (finally) why treatment with specific drugs results in reversal of the disease processes. Additionally, they must explain why responses taken by society to control the spread of HIV have been successful in doing so.

I find it singularly peculiar that the Perth Group say "However, evidence for the existence of an “infectious molecular clone” must be obtained from one experimental study which sets out to confirm all the properties that Brian Foley defines as properties of an “infectious molecular clone”. " Firstly, as has been repeatedly said to them, no one single study is likely to answer their questions. Asking for one is setting up a strawman argument. If they follow the link of Genbank accession K03455 they will find a total of 37 references that went into the confirmation of the HXB2 sequence! Brian Foley is also right in saying that the RFLP analysis, morphology, protein sequences etc are confirmatory parts of the isolation. It seems clear to me what Brain Foley meant in his letter of 11th May, even though perhaps it was not expressed as clearly as the Perth Group would have liked. In my opinion, isolating the viral particles and characterising them in their entirety is secondary to showing that the genetic material released by the culture is the same as that which went in (or, sensibly altered: e.g. RNA for a retrovirus, segmented genome for influenza etc). This really only works for viruses, because as intracellular parasites the genetic matertial is all that is needed to produce virus in a cell line (sometimes modified to DNA). I admit that several species of virus need virion-associated proteins to initiate replication (eg influenza, measles, herpes and retroviruses) but transfecting a DNA clone of the sequence certainly works in many instances.

I also repeat my request for the Perth Group to respond to my criticisms regarding their statements about Montagnier's original paper: do they admit they were wrong and can they provide an explanation why?

Refs:

1 Richieri SP et al. Vaccine. 1998 Jan-Feb;16(2-3):119-29 Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography

2. Barré-Sinoussi F, Chermann JC, Rey F, Nugeyre MT, Chamaret S, Gruest J, et al. Isolation of a T-lymphotropic retrovirus from a patient at risk for acquired immune deficiency syndrome (AIDS). Science 1983;220:868-71.

3. Shaw GM, Hahn BH, Arya SK, Groopman JE, Gallo RC, Wong-Staal F. Science. 1984 Dec 7;226(4679):1165-71. Molecular characterization of human T-cell leukemia (lymphotropic) virus type III in the acquired immune deficiency syndrome.

4. Barre-Sinoussi F, Mathur-Wagh U, Rey F, Brun-Vezinet F, Yancovitz SR, Rouzioux C, Montagnier L, Mildvan D, Chermann JC. JAMA. 1985 Mar 22- 29;253(12):1737-9. Isolation of lymphadenopathy-associated virus (LAV) and detection of LAV antibodies from US patients with AIDS.

5. Jackson et al J Clinical Mole Bio 1988 pp1416-1418 Rapid and sensitive viral culture method for human immunodeficiency virus type

6. Jackson et al J Clinical Mole Bio 1990 pp 16-19 Human immunodeficiency virus type 1 detected in all seropositive symptomatic and asymptomatic individuals.

7. Ho et al NEJM 1989 321:pp 1621-1625 Quantitation of human immunodeficiency virus type 1 in the blood of infected persons

8. Mellors et al. Ann Intern Med. 1995 Apr 15;122(8):573-9. Quantitation of HIV-1 RNA in plasma predicts outcome after seroconversion.

9. Mellors et al. Ann Intern Med. 1997 Jun 15;126(12):946-54. Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection.

10. Musey et al. N Engl J Med. 1997 Oct 30;337(18):1267-74. Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection.

11. Rowland-Jones et al. J Clin Invest. 1998 Nov 1;102(9):1758-65. Cytotoxic T cell responses to multiple conserved HIV epitopes in HIV- resistant prostitutes in Nairobi.

12. Kaul et al. J Immunol. 2000 Feb 1;164(3):1602-11. HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi.

13. Kaul et al. J Clin Invest. 2001 Feb;107(3):341-9. Late seroconversion in HIV-resistant Nairobi prostitutes despite pre-existing HIV-specific CD8+ responses.

14. Liu R et al. Cell. 1996 Aug 9;86(3):367-77. Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection.

15. Learmont J et al. Lancet 1992, 340:863-867. Long-term symptomless HIV-1 infection in recipients of blood products from a single donor.

16. Hellerstein et al. Nature Med January 1999 Volume 5 Number 1 pp 83 - 89. Directly Measured Kinetics of Circulating T Lymphocytes in Normal and HIV-1 Infected Humans.

17. Autran et al, Science. 1997 Jul 4;277(5322):112-6. Positive effects of combined antiretroviral therapy on CD4+ T cell homeostasis and function in advanced HIV disease.

18. Egger et al. BMJ. 1997 Nov 8;315(7117):1194-9. Impact of new antiretroviral combination therapies in HIV infected patients in Switzerland: prospective multicentre study. Swiss HIV Cohort Study.

19. Battegay et al. J Intern Med. 1998 Dec;244(6):479-87. Early participation in an HIV cohort study slows disease progression and improves survival. The Swiss HIV Cohort Study.

Competing interests: None declared