Re: A further plea for references on HIV purification 10 July 2003
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Brian T Foley,
HIV Researcher
Los Alamos National Lab Los ALamos NM 87545 USA

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Re: Re: A further plea for references on HIV purification

The Perth group states:
“In fact, molecular cloning of a viral genome cannot be achieved unless the virus is first purified. “

This is indeed NOT a “fact”. A viral genome can indeed by cloned, sequenced and studied independent of any 100% pure preparation of viral particles. Thousands of viruses, including hundreds of retroviruses have in fact been cloned, sequenced and analyzed. No virus has ever been “purified” and analyzed by the exact methods that the Perth group claims are required. When asked to name one virus that has ever met their conditions, they listed two (the Bryan high titer strain of Rous sarcoma virus, and the Moloney Murine Sarcoma Virus) which are mixtures of two or mor viruses and thus have not been “purified and characterized” by the Perth group’s criteria.

If it is indeed a “fact” that a virus must be purified before it can be cloned, then the dozens of infectious molecular clones of HIV-1 that have been cloned, sequenced and studied would be de facto evidence that HIV has indeed been purified.

The human immunodeficiency virus is a retrovirus of the lentivirus family or group of retroviruses. All retroviruses have two major life stages of the life cycle, the DNA provirus integrated into host cell DNA, and the viral particle containing two copies of an RNA genome. Most of the early infectious molecular clones of HIV-1 were created from the proviral DNA stage of the viral life cycle, and thus infected cells and not free viral particles were the source of the material. When Dolly the lamb was cloned, her genome was never "isolated" it was always mixed together with chromosomal proteins nuclear membrane proteins, and many other macromolecules as well as nucleotides and water and hundreds of other smaller molecules. The "proof" of her cloning cannot be made by any claims of 100% pure sheep DNA or anything like that. Anyone can make such claims. The proof needs to come from examining the DNA of the alledgely cloned sheep and showing that it is identical (or very very nearly identical) to the alledged parental clone. The Raelians have recently made news by claiming to have cloned a human. Are we to take their word for it that they "purified" a human genome or "isolated" a human nucleus conatining that genome? Or do we need evidence that the clone is genetically identical to the parent?

Isolation of 100% pure viral particles by centrifugation or any other method is NOT a necessary step in cloning a retrovirus or any other virus.

All lentiviruses look alike by the resolution of currently available electron microscopic methods. If someone were to obtain a 100% pure batch of viral particles we could not tell by looking at them if they were HIV or SIV or BIV, let alone exactly which subtype of the HIV-1 M group of viruses they belonged to. As with the case of the Perth group's examples of the Byan high titer strain of RSV and the Moloney murine sarcoma virus, the preparation of identical-looking particles could even be a mixture of several different viruses.

A molecular clone of a virus, whether it is a complete genome or only a fraction of one, can be sequenced to tell us exactly what virus the clone is derived from. An infectious molecular clone tells us not only what virus it is derived from, but also that the clone is viable and can produce progeny virions. In the case of human viruses it is not considered ethical to inject the progeny virions nor the cloned infectious DNA into a group of humans to prove that the clone produces disease in these medical "volunteers", so we can only test them in cell cultures to show that they produce similar pathologies in the cells (some strains of HIV-1 M group viruses induce syncytia in T-cell cultures, others do not, for example) and that the progeny virions have the properties of the parent (for example antibodies harvested from the infected patient from which the virus was derived, and from other patients infected with similar strains of HIV-1 M group virus bind to the progeny virus).

The sequences of the genomes (or fractions therof) of any organism from viruses to bacteria to mammals, can tell us not only about the organism the DNA was directly derived from, but also about the relationship of that parent to other organisms. If we look at a gorilla, a human and a chimapnzee for example we cannot tell if the human is more closely related to the chimpanzee or the gorilla. However if we sequence almost any small region (say 10 kilobases) of the genomes of the 3 organisms we can see that the chimpanzee and human DNA are a bit more similar to each other than the gorilla is to either the chimp or the human.

Likewise with viral DNA or cDNA sequences, we can tell the relationships of the viruses of the HIV-1 M group to each other, to the viruses of the HIV-1 O and N groups, and to the various SIVs that have been sequenced to date. These primate lentiviruses in turn can be compared to non-primate lentiviruses such as FIVs, BIVs, EIAVs, Visna and Jembrana disease virus. At some distance (which varies from gene to gene) it becomes impossible to dtermine the exact relationship between the organisms. For example, it is not exactly clear whether fungi are more closely related to vertebrates or to plants. Likewise it is not clear whether the primate lentiviruses are more closely related to one or another of the non-primate lentiviruses. It is not clear whether the common ancestor of lentiviruses is more likely to have lived in a primate or a nonprimate host.

If the Perth group thinks that there is some problem with the theory that HIV-1 and HIV-2 cause immune deficiency in humans, perhaps they should try to explain why the detection and sequencing of HIV-1 M group genomes in humans so very well correlates with the detection of immune deficiency diseases in only infected and not non-infected people. Not only does the pattern of epidemic spread of these viral genomes precisely coincide with the pattern of immune deficiency on a global scale (for example sequences of HIV-1 M group CRF01_AE virus appeared in Thailand coincident with the AIDS epidemic in Thailand) but it also coincides on local scales (for example the people infected with nef gene defective virus from transfusion in Australia survived longer than most people infected with viruses that have all genes functional). To just claim that they think that the chicken has to come before the egg instead of visa versa, does not explain why the whole barnyard is full of chickens and eggs with more eggs hatching into chickens every day.

Competing interests:   None declared