Department of Medical Physics, Royal Perth Hospital, Western Australia, 6001,
Valendar F Turner, John Papadimitriou, Barry Page, David Causer, Helman Alfonso, Sam Mhlongo, Todd Miller, Christian Fiala
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Nicholas Bennett and the "HIV-1 infectious molecular clone"
In his rapid response: "Re: "HIV", HHV-8 and KS", 21 August, Nicholas Bennett wrote: "In my post entitled "Re: Where is the infectious molecular clone" of the 14th July I clearly spelled out for the benefit of the Perth Group and other lay readers what a molecular clone was, how it was obtained for HIV and how we know it belongs to an exogenous retrovirus".
In his rapid response: "Re: Where is the "HIV-1 infectious molecular clone?", 14 July, Nicholas Bennett wrote: "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 manipulation) which when put into a culture system produces virus which itself can then self-propagate. 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."
Molecular cloning is not "practically synonymous" with virus cloning. In "The Isolation of HIV – has it really been achieved? The case against",  we defined a number of terms including molecular cloning and 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, translation of RNA into proteins. The genetic material does not have to be of viral origin and transfection can be achieved by various methods. As far back as 1969 it was known that these methods may include "infection of cells with bacteria and viruses, formation of hybrids of two cell types of fusion, transplantation of isolated single nuclei in eggs and embryos, microinjection of nuclei and mitochondria fractoins, and pinocytic uptake of purified DNA". In that year Margit Nass from the University of Pennsylvania, taking advantage "of the phagocytic properties of mouse fibroblasts (L cells) grown in suspension culture" demonstrated that, "Mouse fibroblasts (L cells) in suspension culture incorporated isolated chloroplasts of spinach and African violets and isolated mitochondria of chicken liver…Green cells divided like normal cells. Green chloroplasts were followed for five cell generations or 5 days, at which time hybrid cells were greatly outnumbered by nongreen progeny cells". (214) By 1989 it was realised that the delivery of DNA into cells could be facilitated by polycationic reagents such as poly-DEAE dextron and polyornithine. "An aliquot of the aqueous reagent is simply added to the tissue culture experiment together with the DNA or RNA of interest". (215) (It is of interest that cultures/cocultures derived from tissues of HIV positive and AIDS patients are treated with the polycation polybrene and/or oxidising agents which may lead to the formation of cations). In 1990, researchers from the University of Wisconsin showed "that injection of pure RNA or DNA directly into mouse skeletal muscle results in significant expression of reporter genes within muscle cells…RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and á-galactosidase were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was comparable to that obtained from fibroblasts transfected in vitro under optimal conditions". (216) One year later another group of researchers from the USA showed that after direct injection into animal hears "of the firefly luciferose gene coupled to the myosin heavy chain…the heart can be transfected in vivo with greater efficiency than the skeletal muscle. (217)
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 a particle(s) separated from everything else (isolated) and show that the particle contains, among other molecules, proteins and nucleic acids (RNA), and that the particle(s) is indeed an infectious particle (see (6.1);
(b) Show that there is a direct relationship between the particles' nucleic acids and proteins, that is, the proteins are coded by the nucleic acids (the viral genome);
(c) Introduce the viral genome (RNA or DNA) 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 that the particles' proteins are coded by the particles' nucleic acids;
(e) Show that the particles' nucleic acids and proteins are identical with those of the ancestral particle and that they too are viral particles;
(f) Because 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, 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 genes 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". (Reference numbers as per the paper).
Obviously molecular cloning or even transfection is not synonymous with viral cloning. If molecular cloning or even transfection are synonymous with virus cloning, then given that: (a) any fragment of human DNA can be taken up by a cell following after which cloning and transfection may ensue; (b) any fragment of human RNA can be introduced into a cell cloned, reverse transcribed (the reverse transcriptases and reverse transcription are as much retroviral as cellular characteristics), the cDNA may be transcribed and translated;
then the human genome would be viral genome and we would be nothing more than viruses.
In his rapid response: "Re: Re-phrasing our two questions to Brian Foley", 11 May, Brian Foley defined infectious molecular clone as follows: "The clone must produce virus particles that are identical by serology, morphology, protein sequences, RFLP, Southern blotting, etc., to the parental virus, and the particles must also be infectious. If a cloned viral genome does not meet these criteria, it is not an INFECTIOUS molecular clone of the virus, be it HIV-1 or any other virus" (his emphasis)".
It appears that there is a difference between our definition of an infectious "HIV-1" infectious molecular clone and that of Brian Foley's. In our definition the molecular clone (RNA, cDNA) must originate from an HIV-1 particle. In Brian Foley's definition the origin of the molecular clone is not specified. If there is no proof that the molecular clone originated from a given retrovirus particle it is difficult to compare the produced "virus particles" with "the parental virus". If one goes to a blood bank which may or may not have sheep blood, select some DNA, clone the DNA and by sheer luck obtain a sheep, the evidence may be interpreted as proof for cloning a sheep, any sheep, not Dolly the sheep. Would Nicholas Bennett give us a reference and a few confirmatory studies with evidence which satisfies at least Brian Foley's if not our definition of an "HIV-1 infectious clone".
Competing interests: None declared