Re: Re: Re: Two questions to Brian Foley 4 May 2004
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Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos, NM 87545

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Re: Re: Re: Re: Two questions to Brian Foley

The Perth group wrote:

4 May 2004
Is it true that the ultimate origin of all the “HIV” molecular clones (“HIV” genome) is a poly(A)-RNA present in the 1.16gm/ml band? Yes or No?

The answer is certainly No. Not all molecular clones of HIV, whether they are infectious molecular clones or non-infectious, have an origin in a density gradient ultracentifugation band. Some of them do, but many of them do not have this origin. Of those that did have an origin in a density gradient band, I have not seen any solid evidence that the viral genomic RNA was polyadenylatyed. I could very well be wrong, but I am under the impression that the genome of lentiviruses is not polyadenylated prior to packaging into virion particles.

Now that I dig deeper, reading the “RETROVIRUSES” textbook (John M. Coffin, Stephen H. Hughes and Harold E. Varmus, editors; Cold Spring Harbor Laboratory Press; copyright 1997), I get the impression that the packaged full-length genomic RNA is indeed polyadenylated. It has figures on pages 206 and 239, both of which indicate that it is known that polyadenylation takes place soon after transcription, before the Rev protein binds to export the unspliced full-length genomic RNA from the nucleus, to the cytoplasm where the Gag polyprotein binds to the Psi elements to package the viral genomic RNA into virions. [26-30].

Thus, the subset of clones of lentiviral RNA which are made from virus particles are indeed made from polyadenylated RNA. Many clones have been made using other methods, such as cloning the integrated proviral DNA.

The Perth group wrote:

28 April 2004
"What “infectious molecular clone of HIV” means?"

"...Would please Christopher Noble tell us what he understands “infectious
molecular clone” of “HIV” to be? "

28 April 2004
Would please Brian Foley tell us what does he mean by “infectious molecular clone” of “HIV”?

4 May 2004
Would please Brian Foley tell us what does he mean by “infectious molecular clone” of “HIV”?

Answer:

An infectious molecular clone of HIV, is a complete nondefective genome, including complete Long Terminal Repeats (LTRs), of any one of the lentiviruses known to infect humans. To date, the only lentiviruses known to infect humans are the HIV-1 M, N and O groups, each of which apparently entered humans via distinct chimpanzee (Pan troglodytes troglodytes subspecies) to human transmission events; and the HIV-2 groups A through G, each of which apparently entered humans via distinct Sooty mangabey (Cercocebus atys) to human transmission events. Infectious molecular clones of FIVs, SIVs, SHIVs, EIAV, visna and other lentiviruses have also been made, but they are not infectious molecular clones of HUMAN Immunodeficiency Viruses (HIVs).

Many complete genomes of HIV (or SIV, BIV, visna, FIV or any other lentivirus) are defective, due the high error rate of the retroviral reverse transcription process. Almost all defects in the gag, pol and env genes render the genome non-infectious. Retroviruses can tolerate such a high error rate because they produce more progeny than are necessary to replace the current generation. If each virion produces ten or ten thousand progeny then the viral population can survive even if 90% or more of the progeny virions are noninfectious. Defective genomes can often be corrected in vitro, via site-directed mutagenesis, PCR or other methods, to generate infectious molecular clones [1].

Defects, or mutations in genes other than the gag, pol and env genes, often render the resulting virus infectious but less pathogenic than the average virus. For one example, the Sidney blood bank cohort of individuals who were all infected from a single unit of blood which contained an HIV-1 M group subtype B virus with a major deletion in the nef gene, all lived much longer than the average person infected with non-defective HIV-1 M group subtype B [2-5]. For some period of time, this type of finding gave hope that a live attenuated clone of HIV-1 might be safe and effective as a vaccine [6-7]. Such hopes have not been completely eradicated, but they have been severely diminished, by the finding that many attenuated lentiviruses regain virulence over time [8-10].

Infectious molecular clones of HIVs, SIVs and other lentiviruses are not something new or unique to the field of lentiviral research. Quite to the contrary, the entire field of molecular genetics was largely based upon the ability of genetic researchers working with E. coli and various phages (bacterial viruses) to work with clonal isolates of both the bacterial host strains and the phages [11-13]. Nowhere in the history of the study of phages and viruses has it been required that 100% “pure” phage or viral particles be viewed under electron microscopes and shown to be free of all contaminating materials. This is a fiction invented by the Perth group purely for the benefit of an attempt at discrediting research on HIV-1. While some phages can be conveniently “plaque purified” on lawns of E. coli, most other phages and viruses cannot, so many other means of ensuring that isolates are clonally derived have been invented. Anyone who has studied molecular genetics and/or virology in the last 20 years is familiar with many of these methods.

Infectious molecular clones of HIVs must be proven to produce infectious virions which use normal cellular receptors and coreceptors (CD4 and CCR5 for example). Any DNA fragment can be transfected into mammalian cells, but only complete nondefective viral genomes will make infectious viral particles [14-17].

Transfection of cells with HIV clones (infectious or not) and/or infection of cells with virions produced from infectious molecular clones, results in production of viral proteins which are of course reactive with antibodies from HIV-infected people (or with antibodies from rabbits or other animals injected with HIV proteins, etc). The point is that the molecular genetics matches up exactly with the serology. If you transfect or infect cells with HTLV-I or some other non-lentiviral retrovirus, infectious virus particles are produced but those particles do not match up serologically with HIV-infected patients or animals injected with HIV proteins. There is some cross-reactivity between RT of HIV and RT of other retroviruses, and Gag of HIV and Gag of other retroviruses, but no cross-reactivity with Env (gp120, gp41), Nef, Rev, Vif, etc...[18-25].

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Competing interests: None declared