From: sbharris@ix.netcom.com(Steven B. Harris) Subject: Re: Viral Isolation: Pasteur Institute Rules Need Revision (fwd) Date: Sat, 11 Oct 1997 Newsgroups: misc.health.aids,misc.health.alternative In <Pine.BSF.3.95.971010092504.26439C-100000@agora.rdrop.com> Greg Nigh <gnigh@agora.rdrop.com> writes: >I would like to pose an open question: > >How were the proteins that are considered HIV proteins derived? IOW, how >was it determined that the HIV proteins *are* HIV proteins? From what >material were those proteins derived? How was it *confirmed* that they >were in fact produced by the retrovirus? The retrovirus has been cloned. You take RNA from the sucrose gradient and turn it into DNA with reverse transcriptase. You get a roughly 10 kB piece of DNA. That DNA has pretty much the same sequence whenever you do the experiment, so it's not some random piece of RNA. It's a special piece. It has the genes of a retrovirus, with common sequences found in other lentiviruses. It codes for a number of proteins. You can clone the DNA into bacteria and they MAKE the proteins. SDS-PAGE and various immune blot methods tell you that these are the same proteins that infected cells secrete, and which are in the sucrose gradient along with the RNA. Thus, they must be viral proteins. You can raise antibodies to them, label these with gold, and see with EM what they stick to. Yep, they stick to those particles that look like retroviruses from the sucrose gradient. Also they stick to the particles which look like retrovirus which bud from cells WHEN you infect with the cloned set of DNA genes. What the heck more do you need? Steve Harris, M.D.Search for Google's copy of this article
From: sbharris@ix.netcom.com(Steven B. Harris) Subject: Re: How Know You Have a Virus in Culture (was Re: Chemo (Ultrastructure of HIV)) Date: Sat, 23 Aug 1997 Newsgroups: misc.health.aids,misc.health.alternative,sci.med,sci.med.nutrition,sci.med.pharmacy In <Pine.BSF.3.95.970821191742.11757A-100000@agora.rdrop.com> Greg Nigh <gnigh@agora.rdrop.com> writes: >This is not accurate. It is accurate to say that particles have been >pictured which bud from cells and even that may (or may not) have the >characteristic features of retroviruses. However, particles budding >does not necessarily mean that they are what we think they are. Comment: Let me try again. There is this family of proteins which are known as "HIV proteins," but which we can call "X-proteins" for he sake of discussion, here. There isn't any question of their identificat- ion or their structure or their "isolation." We know their sequences exactly, and we produce them routinely by genetic engineering. For example, in the 3rd generation ELISA tests for >HIV-antibodies, the proteins for use in the tests are actually made by recombinant techniques in microbes, using genes for the "X-proteins." We know what these proteins, are, okay? We have given these "X proteins" names like HIVp24, HIVp17, and HIVgp160. This is not misidentification, because these proteins are called these names by definition. Ultimately the name does not matter, so long as we can agree as to the nature of these proteins, and the fact that they aren't a part of normal cells, and must come from elsewhere. Do you have any problem with this? Now, these X proteins are viral proteins, and they are not endogenous viral proteins. We know this because the genes that code for the proteins are NOT present in normal cells as those of an endogenous virus would of course be. There isn't any where they can hide from a cell DNA digest and Southern blot. Or from PCR techniques targetted to find them. However, these genes can be transferred to normal cells in a filtered, cell-free extract, and this transfer causes cultured cells to suddenly start actively making all these X proteins, which can then be collected, sequenced, and/or identified by various means (SDS-PAGE, for instance). So the ability of cells to make these proteins is infectious, and can be transferred from culture to culture. Moreover, such cultures secrete the genes to make these proteins, contained in lipid particles of a characteristic density, just like those secreted particles that contain the proteins themselves. Addition of a cell-free extract containing these particles to cells in culture, causes the cells to incorporate the genes, and to make more of the particles. A virus, by definition, is a package of genes and proteins secreted by cells, which causes other cells to make more of the same such packages. Our X-proteins and the genes that code for them fit this definition, and X-proteins are thus viral proteins, and the genes that code for them are viral genes. Moreover, limiting dilution assay shows that only one virus type is involved with this protein family, since dilution of cell-free extracts of X-proteins and their genes reaches the point where the extract either transforms a culture into making the whole family of X-proteins and genes, or else does nothing. If more than one virus was involved, dilution would eventually reach the point that cultures might be induced to only make some of the viral X proteins in question, and not others. But all these X-proteins we're talking about go together, always. They are the proteins of one virus species, clearly. That's really all that is necessary. We are talking about a new virus species, defined by the family of viral proteins it causes cells to make in culture. Science has given this virus the name "HIV." That's the end of that story. That's what HIV is. You'll notice that I haven't talked about antibodies or electron microscopes. They are not needed to tell if you have captured a particular virus in culture. However, we have used such things to see what "HIV" looks like. The HIV proteins can be used to make antibodies that react with HIV proteins, but not other proteins in cells. These antibodies can be stuck to heavy metal atoms so that they can be seen on electron micrographs. If they are added to cell in cultures where HIV proteins are not made, the antibodies don't stick to anything, and we see nothing special about the cells. However, in cultures where virus has been added, the cells are budding small particles into the culture media fluid, and HIV-protein antibodies are seen to stick heavily to these structures. It is thus eminently reasonable (and to decide otherwise would be perverse) that these structures are where the HIV-proteins are, and are thus the HIV virus. It helps that they look like virus particles. Now, remember, we have shown that have a virus (self-propagating collection of proteins and genes) in HIV cultures, which we call HIV. It's always possible that what we see on electron microscopy isn't the virus which we know we're growing, and is some other virus--- but it's rather unlikely. If it's some other virus, how come it has such an affinity for antibodies which we carefully made to HIV-proteins, which are the viral proteins we're interested in? You tell me. Steve Harris, M.D.Search for Google's copy of this article