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| A Brief History of Retroviruses Eleni Papadopulos-Eleopulos 1 Valendar F. Turner 2 John M. Papadimitriou 3 Barry A. Page 1 David Causer 1 Note: The information on this website is presented
for educational purposes and This document was provided by 1 Department of Medical Physics 2 Department of Emergency Medicine, Royal Perth Hospital,Perth, Western Australia 3 Department of Pathology, University of Western Australia. Eleni Papadopulos-Eleopulos is a biophysicist and leader of a group of HIV/AIDS scientists from Perth in Western Australia. Over the past decade and more she and her colleagues have published many scientific papers questioning the HIV/AIDS hypothesis including the widely referenced critical study Is A Positive Western Blot Proof of HIV Infection in Bio/technology ( now Nature/Bio/technology) in March 1993 and a comprehensive critique The Isolation of HIV: Has it really been achieved? The Case Against in Continuum, October 1996. We would like to thank Continuum for asking us to comment on Professor Luc Montagnier’s answers given in his interview with Djamel Tahi. We think it is useful to consider a brief review of the methods used to prove the existence of retroviruses and Montagnier et al’s 1983 evidence for the existence of "HIV".It is generally accepted that Peyton Rous discovered retroviruses in 1911 when he induced malignancy in chickens by injections of cell-free filtrates obtained from a muscle tumor. Similar experiments were repeated by many researchers and the tumor inducing filtrates became known as filterable agents, filterable viruses, Rous agents, Rous virus. However, Rous himself expressed doubts that the agents which caused tumors were infectious in nature. Indeed, Rous warned, "The first tendency will be to regard the self-perpetuating agent active in this sarcoma of the fowl as a minute parasitic organism. Analogy with several infectious diseases of man and the lower animals, caused by ultramicroscopic organisms, gives support to this view of the findings, and at present work is being directed to its experimental verification. But an agency of another sort is not out of the question. It is conceivable that a chemical stimulant, elaborated by the neoplastic cells, might cause the tumor in another host and bring about in consequence a further production of the same stimulant".1 In 1928, AE Boycott, the President of the Royal Society of Medicine, Section of Pathology, in his Presidential Address entitled "The Transition from Live to Dead: the Nature of Filtrable Viruses", said: "Another analogous phenomenon takes us, I think, a step further. The products of autolysis of dead cells in the body, in suitable concentration, stimulate tissue growth. It is a beautiful self-regulating mechanism in which the amount of stimulus is proportionate to the amount of cell destruction, and therefore to the amount of cell growth required, and it is obviously of the highest importance for survival - a far more potent factor in selection and evolution than any disease has ever been. As it normally operates in healing our cut fingers, the final result is simply the restoration of the cells which were destroyed. But if the normal restraint exercised by neighboring tissues is evaded and use made of tissue cultures, the products of autolysis or metabolism (in the form of extracts of tissues, tumors, or embryos) stimulate growth indefinitely and a much larger quantity of tissue may be obtained than we started with. From the autolysis of this a larger amount of stimulating substance may be obtained, and there seems no reason why this process of multiplication should have any limit: normal tissues in the physical isolation of tissue cultures are as immortal as malignant tissues in their physiological isolation from the rest of the body...These products of autolysis...have not received nearly as much attention as they deserve, but they are probably of relatively simple and discoverable constitutions. Yet applied to cells they cause growth, and in so doing potentially increase their own quantity; this is very much what the Rous agent does...As to its origin, all the evidence seems to concur in indicating that the Rous virus arises de novo i n each tumor. There is no epidemiological evidence that cancer comes into the body from outside; everything we know supports the classical view that it is a local autochthonous disease. Experimental sarcomas produced by embryo extract and indol, arsenic or tar have been transmitted by filtrates. Epitheliomas are easily produced in mice by tar and in men by chronic irritation; and if we believe that all malignant tumors contain more or less of a carcinogenic agent akin to the Rous virus, it follows that we can with a considerable degree of certainty stimulate normal tissues to produce virus".2Twenty years later in an article entitled The Plasmagene Theory of the Origin of Cancer, Darlington, discussing the induction of cancer by the Rous agent, the filterable viruses and the "self-propagating" particles transmitted by heredity but lying outside the nucleus found in plants and "known as plasmagenes", wrote: "These infections, it will be seen, are artificial, or at least unnatural. Now the distinction between natural and artificial infection has long been known, although little regarded, in the discussion of plant viruses. A number of aberrant conditions can be transmitted from stock to scion, and some even have arisen in a scion after it has been grafted on a healthy stock. These are artificial diseases; they are not transmitted in nature, but only by grafting. Some may have arisen by the mutation of self-propagating proteins in the cells of plants propagated over long periods by vegetative means (as tumors can be). Others have certainly arisen by the migration or transplantation of proteins from one organism to another. In either case they have a property of infection which they can reveal only in artificial circumstances...We make a great mistake therefore in calling them viruses; they are proviruses... One more question is worth answering: What form would the mutant protein be likely to take in the tumor cell? On account of its rapid multiplication it might well show a higher degree of aggregation than its progenitor. It would then appear as an alien particle in the mutant cell. This is borne out by the electron microscope observations on two chicken tumor agents of provirus type by Claude, Porter and Pickels (1947)".3 The electron microscope observation by Claude et al is the first report of virus-like particles in a tumor, the first electron micrographs of the "Rous virus". Soon after many other researchers reported this type of particles in many tumors, and as Boycott predicted in "stimulated normal tissues". As far as Darlington's prediction that these particles may be due to "a higher degree of aggregation" of the cytoplasm it may be interesting to note that: (a) for proteins, nucleic acids or protein/nucleic acid aggregation (condensation, contraction) to take place, oxidation is necessary;4 (b) tumor tissues are oxidised;4 (c) all the agents used to "stimulate normal tissues" to induce retroviruses are oxidizing agents.5-7 In the 1940s, following the development of the electron microscope (EM) and the technique of ultracentrifugation in density gradients, the particles observed in malignant tissues could be isolated and thus purified, that is, separated from everything else. Because these particles were seen in malignant tissues "it has been judged that the particles constitute the aetiological agent of the disease" and by the 1950's Rous’s filtrable agents became known as oncoviruses (onkos=tumor). The principal morphological characteristic of these particles is a restricted range of diameters and the main physical characteristic their density. 8 When the ultrastructure of these particles was determined they were defined as particles with a diameter of 100-120nM containing "condensed inner bodies (cores)" and surfaces "studded with projections (spikes, knobs)".9 By the 1950’s well-known retrovirologists such as JW Beard, recognized that cells including uninfected cells, under various conditions, were responsible for the generation of a heterogeneous array of particles, some of which may look like oncoviruses. This "particle problem" led to the opinion that to prove the existence of a retrovirus "the scheme of approach, as well illustrated by that devised and rigorously tested in investigations of viral agents, is relatively simple. This consists in (1) isolation of the particles of interest; (2) recovery (purification) of the particles in a given preparation that are homogeneous with respect to particle kind; (3) identification of the particles, and (4) analysis and characterization of the particles for the physical, chemical, or biological properties desired". Beard also stressed that "identification, characterization, and analysis are subject to well-known disciplines established by intensive investigations, and the possibilities have by no means been exhausted. Strangely enough, it is in this field that the most frequent shortcomings are seen. These are related at times to evasion of disciplines or to their application to unsuitable materials. As was foreseen, much of the interest in the more tedious aspects of particle isolation and analysis has been diverted by the simpler and undoubtedly informative processes of electron microscopy. While much can be learned quickly with the instrument, it is nevertheless clear that the results obtained with it can never replace, and all too often may obscure, the need for the critical fundamental analyses that are dependent on access to homogeneous materials"10 (italics ours). Retrovirologists also agreed that "Virions of RTV (retro-viruses ) have a characteristic buoyant density, and centrifugation to equilibrium in density gradients is the preferred technique for purification of RTV".11 At a European meeting on the use of centrifugation in density gradients held at the Pasteur Institute in 1972 with Jean-Claude Chermann as its secretary, it was stressed that once the culture fluids (supernatants) are banded, the density band at which retroviruses are trapped (this varies slightly with the substance used to manufacture the gradients), must be thoroughly assayed. The assays consist of the following: "Assays for RNA Tumor Viruses *With specific reagents for enveloped and internal antigens gs and env ". 12 (Reverse transcriptase is an enzyme first discovered in oncoviruses in 1970 13 hence their present name retroviruses, and 60-70S RNA, the viral RNA. Retroviruses are sometimes called RNA tumor viruses because their genome consists of RNA and not DNA). Thus the method specified at the Pasteur Institute in 1972 is no different from that discussed by JW Beard two decades earlier. Indeed, the method is basic logic applied to the definition of a virus. It is impossible to claim that a protein or an RNA are retroviral unless it is first proven these are constituents of a particle and that the particle is infectious. As can be seen, the first step is electron microscopic examination to prove that the band contains particles with the morphological characteristics of retroviruses and, as Francoise Barré-Sinoussi and Jean-Claude Chermann pointed out at the Pasteur meeting, that the band is pure, that is, it contains nothing else but particles with "no apparent differences in physical appearances".14 The second step in assaying the 1.16g/ml material is to prove that the particles are able to reverse transcribe RNA into DNA. However, as Gallo himself warned the finding of particles, even those containing reverse transcriptase, is insufficient evidence to prove a particle is a retrovirus. The complete proof depends on experiments to: (a) obtain particles from a culture that are separate from everything else (isolated) and show that the particles contain proteins and RNA but not DNA and the proteins are coded by the RNA (the viral genome); (b) show that when the particles are introduced into a culture of uninfected cells, the particles enter the cells, the particles’ RNA is reversed transcribed into DNA which is incorporated into the cellular DNA; (c) show that these cells in their turn produce retroviral-like particles; (d) show that the particles produced by these cells contain proteins and RNA which are identical with those of the original particles introduced into the cells; (e) show that cell cultures identical to those in which the retro-viral-like particles were introduced do not produce such particles when they are cultured in exactly the same conditions but instead of the retroviral particles one introduces some other culture material such as cellular microvesicles. This is because, unlike for any other infectious agent, all cells contain retroviral genomes which under appropriate conditions may be expressed in culture. That is, may lead to the appearance of retroviruses known as endogenous retroviruses. It follows that both the cells in the culture from which the original particles were obtained as well as the culture into which they were introduced may release identical retroviral particles even if the particles that were introduced were not infectious. Therefore it is absolutely imperative to have suitable controls. Thus, to prove the existence of a retrovirus, one must isolate and analyze the retroviral-like particles twice. The first time to obtain and analyze the particle constituents released in the first culture. The second time to prove that the particles released, if any, by the cell in the second culture, are identical to the ancestral particles. The crucial caveat in this procedure is the use of experimental techniques to control for the effects of cocultivation, chemical agents and the many other factors which themselves may induce retroviral phenomena independent of exogenous retroviral infection.15-17 In conclusion, by the early 1980’s, retrovirologists agreed that to prove the existence of retroviruses one must first isolate (purify) candidate particles and the method to achieve this was by banding in a density gradient. SUMMARY OF MONTAGNIER AND COLLEAGUES 1983 SCIENCE PAPER In 1983 Luc Montagnier and his colleagues from the Pasteur Institute and other French researchers published a paper which is considered the first study in which the existence of "HIV" was proven. The paper is entitled "Isolation of a T-Lymphotropic Retrovirus from a patient at risk for Acquired Immune Deficiency Syndrome (AIDS)"18 with Francoise Barré-Sinoussi as principal and Jean-Claude Chermann as second author. The authors' claim to have isolated a retrovirus and thus proven its existence was based on the following experiments: 1 Lymphocytes from the lymph nodes of two patients with lymphadenopathies as well as peripheral blood mononuclear cells from these patients "were put in culture medium with phyto-hemagglutinin (PHA), T-cell growth factor (TCGF), and antiserum to human interferon...In the mouse system, we had previously shown that antiserum to interferon could increase retrovirus production by a factor of 10 to 50". The supernatants were regularly assayed for reverse transcriptase activity (RT) using the synthetic template primer A(n).dT12-18. "After 15 days of culture, a reverse transcriptase activity was detected in the culture supernatant of the lymph node culture" of one of the patients, the first patient. (The level of activity is not given). "Peripheral blood lymphocytes cultured in the same way were consistently negative for reverse transcriptase activity even after 6 weeks". So were both cultures from the second patient. Apparently the detection of RT activity was considered evidence for infection with a retro-virus. 2 Lymphocytes from an adult healthy blood donor were cultured (culture conditions not given) and after three days half the culture was cocultured with lymphocytes from the patient’s culture in whose RT was detected. (Conditions not given). "Reverse transcriptase activity could be detected in the supernatant on day 15 of the cocultures", (level of activity not given) but not in the culture of the blood donor. (It is not mentioned if the conditions in the blood donor culture were the same as the coculture conditions. However, it is obvious that the blood donor cells were not cocultured with lymphocytes from lymph nodes of patients who were not at risk of AIDS but who otherwise had similar clinical and laboratory abnormalities as patient number one. Given that cocultivation leads to the appearance of endogenous retroviruses this is a significant omission from the experimental protocol). 3. Normal umbilical cord lymphocytes were cultured for three days (culture conditions not given), after which supernatants from the coculture and polybrene were added. "After a lag period of 7 days, a relatively high titer of reverse transcriptase activity was detected". (In fact the activity was relatively low, no more than 8,000 counts/min. Background activity as high as 4000 counts/min have been reported.19 ) "Identical cultures" to which supernatant has not been added remained negative. (Since no supernatant was added the cultures could not have been identical. Since supernatant from non-infected cultures added to normal non-infected cells leads to the appearance of endogenous retro-viruses this is also a significant difference). Commenting on the findings in the three experiments the authors wrote: "These two successive infections clearly show that the virus could be propagated on normal lymphocytes from either newborns or adults". The data from the three experiments apparently were also considered proof of "isolation", however, "That this new isolate was a retrovirus was further indicated by its density in a sucrose gradient, which was 1.16". 4. The evidence from the sucrose gradients consisted of two parts: ( a) the supernatant from the cord blood lymphocytes in which RT activity was detected was banded in sucrose density gradients. Maximum RT activity was reported at the 1.16g/ml band. ( b) to the cord blood lymphocyte culture in which RT activity was detected [ 35 S] methionine was added, that is radioactive methionine, an amino acid which is incorporated into growing protein chains and whose radioactivity allows detection of such proteins. Two types of experiments were performed with this culture, one with the cells and the other with the supernatant: (i ) a cell extract was lysed (broken apart) and centrifuged. To parts of the cellular supernatant various sera (containing antibodies) were added and the proteins were electrophoresed (separated using an electric field) on a polyacrylamide-SDS slab gel. Many proteins were found to react, not only with the sera from the two patients with multiple lymphadenopathies but also with sera from a healthy donor and a normal goat. (ii) the culture supernatant was banded in a sucrose density gradient. Although no mention is made of EM studies of the 1.16g/ml band, it was claimed that the band represented "purified, labeled virus from patient 1". The 1.16g/ml band was reacted with the sera of the two patients as well as two healthy blood donors and was processed in the same way as the cellular extract. Although in the published manuscripts it is virtually impossible to distinguish proteins reacting with any sera, even with the sera from the two patients, in the text it is stated that "when purified, labeled virus [the 1.16g/ml band] was analyzed [reacted with the sera] three major proteins could be seen: the p25 protein and proteins with molecular weights of 80,000 and 45,000. The 45K protein may be due to contamination of the virus by cellular actin which was present in immunoprecipitations of all cell extracts." (italics ours) EM studies of the cord blood lymphocytes culture "showed characteristic immature particles with dense crescent (C-type) budding at the plasma membrane...The virus is a typical type-C RNA tumor virus". |