Re: Where is the proof for HIV purification by any method? 26 June 2003
Previous Rapid Response Next Rapid Response Top
Brian T Foley,
HIV Researcher
Los Alamos National Lab, Los Alamos NM 87545

Send response to journal:
Re: Re: Where is the proof for HIV purification by any method?

The Perth group says "The method of banding in density gradients is not our method." and I realize that, but it is the Perth group who placed further requirements on this method in order to claim that HIV-1 has never been purified by this method. Of course thousands of HIV researchers have used sucrose and other density gradient preparations of HIV-1, HIV-2 and various SIVs. It is after all a tried and true method of seperating materials based on their densities. HIV-1, HIV-2 and many SIVs have been "enriched" or seperated from the vast majority of other material obtained from cell cultures using this method, as have other viruses such as Rous Sarcoma Virus (also known as Avian Leukosis Virus). 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.

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 by Crawford and Crawford to "purify" a sample of that virus to the Perth group's satisfaction in 1961.

Crawford LV, Crawford EM. (1961)
The Properties of Rous Sarcoma Virus Purified by Density Gradient Centrifugation.
Virology 13:227-232.

Likewise, there are hundreds of murine retroviruses that have been studied both before and after the 1973 publication by Francois Sinoussi (now Francois Barre-Sinoussi).

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.

The M. MSV in this title refers to the Moloney strain of murine sarcoma virus, which is replication defective due to loss of viral genome regions which were replaced by the mos oncogene (a serine/threonine kinase family member). Most Moloney MSV strains thus require a helper virus to replicate. I would therefor suspect that the "pure virus" in the 1973 paper might be a mixture of both the helper and Moloney MSV viruses.

In the 1950-1980 time period, density gradient centrifugation would have been one of the best methods of seperating viral particles from most other constituents of a virus-infected cell culture. However, since 1980 many new methods have been developed which increase our ability to study viruses, particularly advances in monoclonal antibody productition for serological characterizations and molecular genetic techniques for even more detailed analyses. With centrifugation one can look at the strains of Moloney Murine Sarcoma virus that produce angiosarcomas and the strains of Moloney Murine Sarcoma Virus that produce rhabdomyosarcomas, and see that they look identical by electron microscopy. With serological techniques it might be possible to identify subtle differences between these strains. With infectious molecular clones, it becomes possible to disect the exact regions of the genome that contribute to the differences in tumor production of various strains, by making hybrids between the two strains of by introducing single genetic differences from one strain into the other.

It is useful to look at electron micrographs of viruses, and thousands of electron micrographs of HIV-1, HIV-2 and other lentiviruses have been produced in the last 20 years. However, for most research it is not necessary to have 100% "pure" virus. When the research does require "pure" virus, cloning of a complete viral genome into an infectious molecular clone is by far more accurate and informative than centifugation of whole virus from cell cultures.

In summary, viruses including retroviruses have been studied long before they were "purified" by gradient centrifugation, and they continue to be studied by many other methods besides gradient centrifugation. Centrifugation is useful, but it never was and is not now, a "requirement" for the study of any virus.

Competing interests:   None declared