Department of Medical Physics, Royal Perth Hospital, Western Australia,
Valendar F Turner, John Papdimitriou, Barry Page, David Causer, Helman Alfonso
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A plea for the references on HIV purification
According to the Oxford dictionary, to “purify” means to “clear of foreign elements”. There are several reasons why it is necessary to purify a retrovirus. These reasons include the need to characterise the viral proteins and viral RNA. As Montagnier pointed out: “It is necessary to do that” (1). Unless this is done, it is not possible to claim the existence of viral proteins and a viral genome and talk of “cloning of a complete viral genome into an infectious molecular clone” as Brian Foley wrote in his rapid response entitled “Re: Where is the proof for HIV purification by any method?” (26 June 2003).
Brian Foley wrote “With the Perth group’s further requirements, no virus with any morphological variation can be purified because they require that “all particles be IDENTICAL”, and they place further restrictions on what has to be done to the virus before and after the centrifugation.”
Let us be very clear on this matter. These “requirements” and “restrictions” which are really a common sense approach were formulated by virologists such as Barre-Sinoussi, Chermann, (2) Crawford, (3) Toplin (4) and are consequences of the very nature of retroviruses. Since retrovirus-like particles are ubiquitous in nature it follows you must prove that the particles in question: (i) are indeed viral particles, that is, that they are infectious; (ii) have reverse transcriptase and RNA but not DNA; (iii) they represent a unique retrovirus, that is, they must have unique RNA and proteins.
Brian Foley wrote: “Of course thousands of HIV researchers have used sucrose and other density gradient preparations of HIV-1…It is after all a tried and true method of separating materials based on their densities. HIV-1… have been “enriched” or separated from the vast majority of other material obtained from cell cultures using this method…” This is just what we have been after for more than the past 15 years!!! Could Brian Foley or anyone else please give us a few of the “thousands” of references where HIV-1 has been separated “from the vast majority of other material”, in other words, purified?
Brian Foley wrote: “Peyton Rous began working with the group of avian retroviruses that became known as Rous Sarcoma Virus in 1909 and stopped in 1915, long before sucrose density gradient centrifugation was used…” In a subsequent rapid response “Re: Re: Where is the proof for HIV purification by any method?” (27 June 2003), Brian Foley wrote: “…a virus [the Rous Sarcoma Virus] characterized by Peyton Rous in the 1908 to 1915 time period…” It was in 1911 when Peyton Rous induced malignancy in chickens by injections of cell-free filtrates obtained from a muscle tumour. Rous did not characterize a virus and in fact, Rous only suggested that the causative agent was a virus. 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 tumour in another host and bring about in consequence a further production of the same stimulant.” (5) Decades after 1911, Rous was reported to have said “that he had never said it was a virus, carefully using the term ‘tumour agent’”.(6)
Brian Foley wrote: “…since 1980 many new methods have been developed which increase our ability to study viruses, particularly advances in monoclonal antibody production for serological characterizations and molecular genetic techniques for even more detailed analyses… When research does require “pure” virus, cloning of a complete viral genome into an infectious molecular clone is by far more accurate and informative than centrifugation of whole virus from cell cultures.”
We agree that genetic techniques and monoclonal antibodies (not forgetting that like all other antibodies, monoclonal antibodies may cross -react) can be used for more detailed analyses of retroviruses. However, this cannot be done unless one first obtains the viral proteins (antigens) and the viral genome and these can only be obtained by purifying the virus. It appears Brian Foley either has not read or has forgotten the comments we made regarding the cloning of HIV or the HIV genome in our rapid response entitled “A simple request from the Perth Group” (5 June 2003). So we will repeat them.
To obtain a molecular clone means to produce an identical copy of a DNA fragment. At present this can be achieved for any such molecule without undue difficulty. However, to clone an object such as a virus, bacterium or an animal is a different matter. It requires three main steps. For example, to clone Dolly the sheep, it was absolutely necessary for the researchers to obtain the genome from a sheep’s cell, introduce it into the ova and lastly prove the birth of a sheep. Similarly, to claim proof of HIV cloning it is absolutely necessary to obtain the HIV RNA from HIV particles, introduce it (or its cDNA) into a suitable cell and ultimately prove the appearance of similar HIV particles. In our view at present, the only way to obtain the HIV genome is first to obtain material which consists of purified HIV particles or at least a material which does not contain any impurities with nucleic acids.
In 1997 some of the best known HIV researchers wrote: “Virus to be used for biochemical and serological analyses or as an immunogen is frequently prepared by centrifugation through sucrose gradients. The fractions containing viral antigen and/or infectivity are considered to contain a population of relatively pure virus particle.” This is preceded by: “However, in none of the studies…has the purity of the virus preparation been verified.” (7) In other words, at least up to 1997, the HIV genome was obtained from a “pure” HIV whose contents were unknown (except Montagnier’s “purified” virus which did not even have particles with “the morphology typical of retroviruses”.(1)) This means that at least up to 1997, nobody could claim proof of either HIV cloning or molecular HIV cloning. Perhaps Brian Foley has information to the contrary. If so, we implore him to provide a few references that prove: (i) the molecules used in “cloning of a complete viral [HIV] genome” originated from HIV particles; (ii) cloning of HIV; (iii) the HIV “Genetic Sequences” in his databases originated from HIV particles.
In his rapid response “Re: Re: Where is the proof for HIV purification by any method?” (27 June 2003), referring to the Barre- Sinoussi 1973 paper (2), Brian Foley wrote: “In fact, this paper states on 240 in Results: “the particles isolated under conditions of rate sedimentation considered [sic] mainly of virus particles and very little amounts of contaminating material.” This quote does not refer to the electron micrographs obtained from the density gradients bands for which the authors wrote: “From the electron- photo micrographs…these fractions contained mainly typical spherical C- type particles. No apparent differences in physical appearance could be discerned among the viral particles in these regions.” (2)
Brian Foley wrote: “The point I want to make is that Sinoussi et al. did get viral particles that looked identical to each other by electron microscopy, but without molecular clones, sequences, or serological profiles they could only take the word of the people they obtained their 78A rat fibroblasts from, that these particles were derived from the Moloney strain or MSV and not the Kirsten strain or some endogenous mouse or rat retrovirus that had become activated. All of these retroviruses look alike.”
We are glad that Foley agrees Sinoussi et al. produced an electron micrograph which showed only “viral particles that looked identical to each other”. Their scope was not to characterize the MSV. It is only when you want to prove the existence of a unique new retrovirus that you must proceed with all the other steps, namely, “molecular clones, sequences, or serological profiles” which are the requirements to characterize the viral particles as being unique, steps which can only follow and not precede purification.
Referring to the Crawford et al. 1961 paper (3), Brian Foley wrote: “Again, in this paper, the authors do not state that the virus preparations are 100% pure. In fact they state on page 229 “The third preparation was RSV from a tumour which has been purified by density gradient centrifugation. Such preparations contain much nonviral material, and counts of physical particles are therefore less accurate.”
Since the third preparation was taken from a tumour, we can expect nonviral particles to be present. However, regarding the first and second preparations which were obtained from culture, the authors wrote: “Figures 1 and 2 are electron micrographs of a preparation of purified RSV after fixation and sectioning. It will be seen that the preparation is homogeneous and little nonviral material is evident.”(3)
Brian Foley wrote: “The Rous Sarcoma Virus paper also states “DNA was barely detectable in the preparation; it would be equivalent to 2 x 10^5 molecular weights units per particle if it were actually present in the virus.”
This was an initial observation by the authors who then went on further and stated: “Table 3 gives the results obtained for the base composition of RSV…In the initial separation by paper chromatography any DNA present would remain at the origin. No ultraviolet-absorbing material was detected in this position, confirming the virtual absence of DNA in the RSV preparation.”(3)
So perhaps Brian Foley may reconsider his conclusion: “Again showing that this was not 100% “pure” virus and nothing else.”
Brian Foley wrote: “I fail to see why these two papers which describe centrifugation of retroviruses are accepted by the Perth group as meeting all of their criteria for isolation and purification of a virus, when similar papers describing the same procedures carried out with HIV-1 or HIV-2 are dismissed. It is especially irritating to me that they like the Moloney Murine Sarcoma Virus paper even when the authors state that the prepared virus is a mixture of at least two different viruses; the Moloney MSV and its helper MLV virus.”
Our criteria cover not only isolation/purification but also characterization of the virus. We would be grateful if Brian Foley could provide us with references showing HIV-1 preparations in which “No apparent differences in physical appearance could be discerned among the viral particles in these regions” as Sinoussi et al. did and with “a high degree of homogeneity” and “virtual absence of DNA”as Crawford et al did. Then and only then can we proceed and characterize the viral particles.
In fact Brian Foley gave his rapid responses the titles: “Re: Where is the proof for HIV purification by any method?” and “Re: Re: Where is the proof for HIV purification by any method?” but has failed to give us any published proof. So we repeat “Where is the proof for HIV purification by any method?” That is, give us the published references.
(1) Tahi D. Did Luc Montagnier discover HIV? Text of video interview with Professor Luc Montagnier at the Pasteur Institute July 18th 1997. Continuum 1998 5:30-34
(2) Sinoussi F, Mendiola L, Chermann JC, Jasmin C, Raynaud M. (1973) Purification and Partial Differentiation of the Particles of Murine Sarcoma Virus (M. MSV) According to their Sedimentation Rates in Sucrose Density Gradients. Spectra 4:237-243.
(3) Crawford LV, Crawford EM. (1961) The Properties of Rous Sarcoma Virus Purified by Density Gradient Centrifugation. Virology 13:227-232.
(4) Toplin I, Sottong P. (1972) Large-Volume Purification of Tumor Viruses by Use of Zonal Centrifuges. Applied Microbiology 23 (5): 1010-1014.
(5) Rous PA. (1911) Sarcoma of the Fowl transmissible by an agent separable from the tumor cells. J Exp Med 13: 397-411
(6) Broxmeyer L. (2003) Medical Hypotheses 60 (5):671-688
(7) Gluschankof P, Mondor I, Gelderblom HR, Sattentau QJ. (1997) Cell Membrane Vesicles Are a Major Contaminant of Gradient-Enriched Human Immunodeficiency Virus Type-1 Preparations. Virology 230: 125-133
Competing interests: None declared