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Hypermutation: my view of its importance

Discussion in 'XMRV Research and Replication Studies' started by anciendaze, Mar 15, 2011.

  1. anciendaze

    anciendaze Senior Member

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    When the Virology blog gets around to directly addressing the recombination/contamination argument, I want to be prepared, and I want others prepared. To this end, I'm putting together some thoughts on the huge hole in all denier arguments I have seen. I am not addressing contamination by mouse cells, as in Huber's work. Only indirectly does the question of contamination from infected patients, which I have discussed in a posted comment on the Virology blog, enter this argument. (I think two integration sites do show evidence of laboratory contamination -- from infected humans.) Critics have frequently conflated different meanings of contamination.

    N.B I am not claiming originality myself. Nor am I acting as a mouthpiece for someone invisible. I am simply restating arguments made in public which do not seem to have been noticed or connected.

    The original observation, as Mikovits pointed out, goes back to a paper by Paprotka et al. which found XMRV sequences "extensively hypermutated". Other parts of the argument can be checked independently. We need not treat Judy Mikovits as an authority to make these arguments. Anyone with sequence data can check that many codons altered by hypermutation will produce the same amino acid. This is not a perfect defense, but it is more than enough to yield a significant survival advantage.

    The central point is that this virus has evolved to exploit mechanisms human genes use to escape damage by hypermutation, a natural defense against retrovirus. The virus uses codons which hypermutation converts to synonyms for the same amino acid. This allows enough sequence variation to escape detection of latent virus, either by immune system or PCR, while still producing replication-competent virions when replication is stimulated by hormones matching receptor elements in the initial long tandem repeat (LTR). Even in cases where APOBEC enzymes are not active, this also gives the latent proviral sequence some ability to survive point mutations. It is very likely we do not currently know all such mechanisms active against retroviral infection. There is abundant evidence retroviruses have been around for a long time.

    Hypermutation is a powerful evolutionary force on retroviruses. Most random sequences will not have this resilience. The process by which a cell line is created is highly selective, so initial virus in a new cell line may be expected to initially show a narrow range of variability. Cell lines free from selection by hypermutation will diverge quickly thereafter. Together these two processes give the appearance of de novo creation of a novel pathogen, if you ignore data from patients. One clue to this selection/divergence is an original sequence with resilience to hypermutation.

    Arguments being marshaled to suggest contamination use probability, but their primary resonance is with prejudices common among virologists. PCR has been such a powerful tool that any suggestion it can be defeated by viruses makes life much harder for them. Also, working on things which even might be contaminated is not generally a wise career move. A virus cares nothing for the convenience of the host, let alone the convenience or careers of virologists. The brilliance of this 'strategy' for evading defenses argues for a long evolution, not recent origin in a one-off laboratory event.

    Finding this strategy itself is far more important than either XMRV or ME/CFS. Nothing prevents other retroviruses from operating the same way, even if their sequences are substantially different. The strategy need not be confined to hypermutation by APOBEC3G either. Once you have identified a pattern of hypermutation, you can use a computer to check proposed retroviral sequences for resilience to such changes. Pathogens which take a slow strategy, (as opposed to the fast strategy of a disease like cholera,) will need this resilience to survive long latent periods. Provirus which can survive as long as the host need not replicate rapidly to persist. Diseases where such a retrovirus would be likely include breast cancer, ALS, MS, RA and Lupus.

    Statistical arguments can reach astonishing heights of sophistication, but most advanced techniques I once learned have fallen by the wayside as far as applicability. The shocking truth is that data going into statistical reasoning is seldom free from selection effects which destroy the validity of those arguments. Hue's argument on the common source of these sequences is a prime example. The problem is not that she used powerful (Bayesian) techniques, but that her input data came mostly from cell lines.

    If all data were drawn from studies of cell lines, there is no question a phylogenetic tree constructed from that data would be rooted there. If you have a large amount of data from cell lines, and only a tiny amount from virus in the wild, the probability is that algorithms for constructing phylogenetic trees will construct a similar tree.

    Aside: Switzer's paper on extensive recombination in XMRV actually undermines the idea of trusting phylogenetic tree models. Recombination makes inheritance into a directed acyclic graph. Viable virus emerging from extensive recombination also argues for a long evolution. The recombination-contamination argument shows great similarity to a famous cartoon by Sidney Harris. Even with the assumed minor miracle of recombination producing a potent pathogen, we have a missing wild-type ancestor for those pre-XMRV sequences some 99% homologous to XMRV. Who is looking for this?

    The second large selection effect is PCR itself, which is capable of incredible selectivity. Tiny mutations in latent proviral sequences which defeat PCR can still produce replication-competent virions at a later date. There don't have to be many survivor sequences to cause persistent infection. The irony here is that sequence variation is being used to suggest origin, while PCR eliminates sequences showing the largest variations. PCR can be used differently, as Lo and Alter demonstrated. Their results were dismissed as coming from an unrelated putative virus. You can't seem to win if you argue on these terms. Prejudices and arguments from authority will triumph. More sequence data outside of cell lines is the answer, but this runs into another roadblock.

    The most important effect of convenience may be that administrators and authorities who have ignored this disease, and denigrated or disparaged patients, find the existence of this virus highly inconvenient. They just might have some control over funding and publication.
  2. Wonko

    Wonko Senior Member

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    Interesting - thank you
  3. asleep

    asleep Senior Member

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    Very astute post, anciendaze! They've been digging in so many directions that they accidentally dug the entire foundation of their argument out from under themselves.

    With regards to the conflated definitions of "contamination," I think that an understanding of the contextual waffling in meaning is important in differentiating their modest science from their politics. I've touched on it in a bit more detail here.
  4. Megan

    Megan Senior Member

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    Thanks Anciendaze,

    Your post is very informative. I have difficulty following the details but see that the hypermutation argument seems really important. I would love to see you ask the professor about this!

    And thanks for highlighting that the Sweitzer paper contradicted the other one, I noticed that too. Perhaps more to support your argument (how ironic).

    With regards to suggesting that it was human contamination of the two samples, it seems this is harder to accept, I would think this might detract from the overall argument. I the latest episode in the beginning they say Silverman was 'aware of the problem', he was apparently well aware before the Towers paper, and is going back to do exactly the extra experiments they spoke about in the episode the paper was discussed.

    The other key question I had following the CROI presentation that I think needs asking follows from the fact that they said in the presentation that they made the prostate cell line androgen independent. Naturally prostate tumor growth is driven by androgen hormones such as testosterone, but can eventually evolve to grow without it if it is deprived of such hormones long enough (I know in people this takes about 2 years). I can only interpret this to mean that in the 22Rv1 cell line derivation process, in the early phases, it was deprived of androgen hormones (I assume for a long time) until it could grow just as well without them. I dont know how this is done because I dont understand how these cell lines are drived or how the xenografting process fits into this - but presumably the mice in the early xenografts would therefore have had to be adrogen deprived?

    Would this therefore not be a significant factor in the detection of XMRV since XMRV replication is also androgen driven? ie. could there have been a lack of detection in the early xenografts because the cell line was androgen deprived and then have shown up in later rounds when maybe it wasn't?
  5. Megan

    Megan Senior Member

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    Additionally, the following article on HTLV I contains the below quote. I don't know how this is relevant but perhaps there is a clue here - HTLV certainly seems to be a different beast to HIV. http://www.nature.com/onc/journal/v24/n39/full/1208968a.html

    What if XMRV was clonally expanding in humans, but actively replicating in the cell line, what implications would that have for sequence variability?
  6. anciendaze

    anciendaze Senior Member

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    One problem in most scientific fields is the dense cloud of jargon which develops while arguing details. Another is the use of common words with special meanings.

    In fact the culturing phase of tests on PBMCs is a clonal expansion by another name. Cell lines are subject to extreme selection to provide cells with uniform properties convenient for experiments. These could also be called clonal expansions, though the connection with use of this term in immunology would be lost. Unintended selection of an actively replicating virus originally latent in the cell line is highly plausible. I've been sticking to the broadest descriptive terms here to make an argument very similar to the one you are suggesting.

    Aside: One probable source of virus in prostate cancer tissue is not the cancer cells themselves, but infected immune system cells infiltrating diseased tissue. Not only would these be few in number, and hard to detect for this reason, any latent provirus in them would also be subject to hypermutation.
  7. Megan

    Megan Senior Member

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    I have followed up on my own query above about how they made the cell lines androgen independent. The following papers provide the information, one of which can be got in full for free. In any case it appears that the mice were given testosterone in the early rounds of the cell derivation with the androgen deprivation coming a bit later. As far as I can understand it this would not undermine the argument put by Vinay Pathak at the CROI conference.

    http://www.ncbi.nlm.nih.gov/pubmed/8433392
    http://www.ncbi.nlm.nih.gov/pubmed/8674060
    http://www.ncbi.nlm.nih.gov/pubmed/10462204

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