Note: Analysis of Jay Levy's paper "AIDS Retrovirus (ARV-2) Clone replicates in Transfected Human and Animal Fibroblasts".
Nature 1984 312: 76-763.
DID LEVY "ISOLATE" HIV?
There is no correlation between the data presented in the paper and the title. Perhaps it would be useful to first state what conditions
should be satisfied for claiming cloning of a virus and then analyse Levy's data.
Conditions
1. Have a particle which contains, among others, proteins and nucleic acids (RNA or DNA).
2. Show that there is a direct relationship between the particle's nucleic acids and proteins i.e. the proteins are coded by the nucleic
acids (the viral genome).
3. Introduce the viral genome (RNA or cDNA) into cells and show that the DNA (cDNA) is transcribed into RNA and the RNA is
translated into proteins (transfect the cells).
4. Show that the cells produce particles and that the particle's proteins are coded by the particle's nucleic acids.
5. Show that the particle's nucleic acids and proteins are identical with those of the initial particle.
6. Because all cells contain retroviral genomes, which under the right conditions may be expressed in culture, that is both the cells in the
culture from which the original particle was obtained as well as the transfected cells may release identical retroviral particles even
when there is no transfection, when one attempts to clone a retrovirus a control culture is of pivotal significance.
There should be only one difference between the control and the cells transfected with the viral genome. Instead of the viral genes one
must use some other genes. This is essential because the act of transfection and the condition used may lead to retroviral
expression.
Levy's et al. data
- Neither in the above paper nor in any of the references they cite for "ARV Isolation" are there any electron micrographs of viral
particles.
- For transfection they used the cDNA of an approximately 9-Kb RNA which was selected from the culture medium of HVT-78 cells "infected
with ARV", without ever presenting evidence that it belongs to a particle. (This is also the case for all the other HIV genomes).
- They have no evidence that transfection actually took place not even that the cDNA actually was incorporated in the cells.
- There is no evidence that the "transfected" cells released any particles.
The only evidence which Levy and his colleagues present as proof for "ARV cloning" is:
(a) detection in cultures of RT activity;
(b) detection in "Extracts of normal PMC", "infected" with "virus recovered from transfected" cells of proteins which reacted with sera
from AIDS patients. It is not stated what they mean by infection with "virus recovered from transfected" cells. In all his other work
on HIV, Levy, as well as all other HIV researchers, by infection means cultivation of cells with either supernatant from "infected"
cultures, or the material from these cultures which in sucrose density gradients bands at 1.16 g/ml (without proof of the existence
of particles).
However:
(1) RT activity is not specific to HIV or even to retroviruses. In fact given the culture conditions they have used, it would be surprising
not to detect RT activity irrespective of "infection with HIV".
(2) The following proteins from the cellular "Extracts" were found to react with the sera from AIDS patients. p16, p25, gp41, gp120 and
gp160.
Neither in the above paper nor in any of his previous publications on HIV, Levy (or any other HIV researchers) presented unambiguous
evidence that the above proteins are indeed coded by the 9-kD RNA he used for transfection.
However, there is evidence that the proteins are non specific.
A few examples:
gp41
Although Levy did not find a gp41 in the "Extracts" from the normal "non infected" cells, Montagnier did. (Barr=82-Sinousi et al Science
1983;220:868-871). The difference may be in the fact that apparently Montagnier stimulated the non-infected cells but Levy did not. According
to Gallo the molecular weight of the glycosylated protein corresponding to the "HIV gene" which is supposed to code for gp41, "should be about 52.000 to 54.000 daltons", not 41.000 daltons. (Medrow et al J Virol 1987; 61:570-578).
gp120 and 160
There is evidence that the proteins of molecular weight 120-160KD which in the WB react with sera from AIDS patients "represent oligomers of gp41", that is they are not coded by a specific HIV genetic sequence. (Pinter et al J. Vir. Meth. 1989; 63:2674-2679; Zallo-Pozner et al NEJM
1989; 320:1280-1281).
p16-18
Monoclonal antibodies to p18 react with dendritic cells in lymphatic tissues of a variety of patients with a number of non-AIDS related
diseases. (Parravilini et al AIDS 1988; 2:171-177).
The protein is not detected in normal non stimulated cells, but it is detected in the stimulated "non HIV infected" normal cells. (Stricker et
al Nature 1987 327:710-717).
p24/25
The reaction of AIDS sera and of monoclonal antibodes to p24 which from "infected" cultures bands at 1.16g/ml, is obiquitous. The whole blood
cultures of 49/60 (82%) of "presumably uninfected but serologically indeterminate" individual and 5/5 serogenative blood donors were found
positive for p24 by Schupbach et al (AIDS 1992 6:1545-1546). [Schupbach is the principle author of the third paper (the first paper ever on HIV WB),
in the series of 4 published by Gallo et al in 1984 in Science on HTLV-III isolation].
94% of sera from gay men with lymphodenopathy or AIDS react "with a 25KD membrane antigen found in platelets from healthy donors and AIDS patients, as well as a 25KD antigen found in green-monkey kidney cells, human skin fibroblast, and herpes simplex cultured in monkey kidney cells". (Styker et al NEJM 1985; 313:1375-1380).
Conclusion
In the above paper by Levy et al there is no evidence for viral cloning or even transfection of any genes, viral or non viral. *
-- Eleni Papadopulos et al.