Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso
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A simple request from the Perth Group
In his rapid response “Re: Where are the experiments which prove HIV isolation, sexual transmission and antibody specificity” (23rd May 2003), Brian Foley has not addressed our request given in the title of his rapid response. In order to avoid any misunderstanding before we address his claim “There are several dozen infectious molecular clones of HIV-1”, it will prove helpful to define some terms including virus cloning.
DNA cloning- the production of identical copies of a DNA fragment, any DNA fragment, from an ancestral DNA fragment by splicing it into a suitable cloning vehicle, for example, a bacteriophage or plasmid;
Transfection- the introduction of exogenous DNA into cells and its ability to replicate and express itself in these cells. That is, transcription of DNA into RNA and translation of RNA into proteins. The genetic material does not have to be of viral origin and transfection can be achieved by various methods.
Virus cloning-the introduction into cells of genetic material, DNA or RNA, which has been proven beforehand to be the genome of a virus followed by the appearance in the same cells of viruses identical in every aspect to the viruses from which the genomic material originated.
Before one can claim proof of cloning of a retrovirus one must: (a) Obtain particles separated from everything else (isolated) and show that the particles contain, amongst other molecules, proteins and nucleic acids (RNA), and that the particle are indeed infectious; (b) Show a direct relationship between the particle nucleic acids and proteins, that is, the proteins are coded by the nucleic acids (the viral genome); (c) Introduce the viral genome (RNA or cDNA) into cells and show that the DNA (cDNA) is integrated into the cellular DNA and is transcribed into RNA and the RNA is translated into proteins (transfect the cells); (d) Show that the cells produce particles and the particle proteins are coded by the particle nucleic acids; (e) Show that the particle nucleic acids and proteins are identical with those of the ancestral particle and they too are viral particles.
In addition one must be aware that all cells contain retroviral genomes which, under appropriate circumstances may be expressed in culture, that is, both the cells in the culture from which the original particles were obtained as well as the transfected cells may release identical retroviral particles even if there is no cloning. Thus, when one attempts to clone a retrovirus, a control culture is of quintessential significance. The only difference between the control and the cells transfected with the viral genome should be that in the control cultures one should use some other nucleic acid for transfection. This is because, under suitable culture conditions, the very act of transfection may result in retroviral expression including the production of retroviral particles. It is obvious that retrovirus cloning is not synonymous with retrovirus isolation. In fact, for cloning one must isolate the virus twice. The first time to obtain the viral genome and the second to prove that if particles are released after introduction of the viral genome, they are identical with those from which the genome was originally obtained.
Not one of the five references Brian Foley provided contains any proof for HIV cloning. The most that can be claimed from these experiments is transfection with some nucleic acids which originated from material that either (a) predominantly contained cellular fragments and thus cellular RNA and DNA and a few particles which have some but not all of the morphology attributed to either the HIV or other retroviral particles or (b) consists of material which contain no particles having any of the morphology attributed to retroviruses. In other words, the transfection, if any, was of nucleic acids for which there is no proof they were either of HIV or any other retrovirus.
In his rapid response “Re: A modest proposal” (1st June 2003), Brian Foley states “Molecular genetics offers a whole world of research that was not available with pre-1970 techniques”. We agree. But we also agree with the ex-editor of Nature, Sir John Maddox: "Is there a danger, in molecular biology, that the accumulation of data will get so far ahead of its assimilation into a conceptual framework that the data will eventually prove an encumbrance? Part of the trouble is that excitement of the chase leaves little time for reflection. And there are grants for producing data, but hardly any for standing back in contemplation".
We do not care what method is used to isolate HIV. As far as we are concerned the only important fact is to obtain material which overwhelmingly consists of particles with the morphology attributed to HIV and that such particles are infectious. We have concentrated on this method because: (a) all the HIV experts who have claimed to have isolated/purified HIV and thus to have proven its existence have used the method of banding in density gradients; (b) all the proteins used as antigens in the antibody tests and the “HIV” genome have been obtained from such banded material.
We repeat our simple request: “Where are the experiments which prove HIV isolation, sexual transmission and antibody specificity?”
Competing interests: None declared