The inevitable union: XMRV and Vpu!

Two things I didnt intend on writing about on ERV more than a few times, but turned into repeat guests: XMRV and Vpu.

Theyve finally come together.

Susceptibility of xenotropic murine leukemia virus-related virus (XMRV) to retroviral restriction factors

Know how I was talking about intrinsic immunity yesterday?

Well, some scientists wondered how the heck XMRV was replicating in humans (especially human PBMCs), when other murine gammaretroviruses are restricted in humans because of our intrinsic immunity: Tetherin, APOBEC, Trim5. So they set out to investigate what effect (if any) these human antiretroviral proteins have on XMRV.

RECEPTOR: First things first. If a cell doesnt have the receptor XMRV needs, then it cant infect a cell. The cell doesnt have the right lock for XMRVs key. But what if you give XMRV the key to locks on every cell (VSV-G)? Can XMRV infect anything, then?

Nope.

XMRVs can infect some cells a little better with the VSV-key, but it doesnt make a huge difference. This means that there is something else restricting infection and cell tropism. A greater pressure than something as simple as receptor, and present in every cell… Intrinsic immunity!

APOBEC3G: This protein does not prevent the production of babby viruses. But, the babby viruses are mutated beyond repair, and are thus noninfectious. But not every APOBEC works on any retrovirus, and some retroviruses have figured out ways to interfere with APOBEC (HIV-1 Vif).

Well, human APOBEC3G does not like MLV (XMRVs ancestor). It shuts that mo-fo down. Weirdly, though, APOBEC3G is found in high levels in human PBMCs… but infectious XMRV was found in human PBMCs… The resulting viruses should have been duds.

So, they tested whether human APOBEC3G can mess up XMRV. Um… it could. Human APOBEC3G knocked XMRV infectious offspring levels down to MLVs crappy levels. Hmm.

TETHERIN: These experiments are so cool! Okay, first of all, XMRV is restricted by tetherin. That is, you get really really shitty viral production off of infected cells that express human tetherin (or tetherin from a couple non-human primates). Now, HIV-1 has Vpu to counteract tetherin, right? So they added Vpu to XMRV infected cells, and voila! Tons of XMRV.

Furthermore, XMRV does not appear to have any obvious way of countering this host restriction. Yes, HIV-1 has Vpu. But viruses related to HIV-1 (HIV-2, SIVs) counter tetherin a different way– with the envelope protein. Well, you dont need a complex retrovirus for Env, maybe XMRV counters tetherin that way?

Nope. HIV-1 with Vpu deleted replicates really shitty in human cells. If you add in XMRV envelope… HIV-1 with Vpu deleted still replicates shitty. Hmmm.

TRIM5alpha: Human TRIM5alpha messes up the structural proteins of regular MLV so it doesnt uncoat properly. So, they wanted to see if human (and twelve non-human primate) TRIM5alpha can screw with XMRV too…

It cant!

This is the one innate immunity hurdle that XMRV can clear, while MLV cannot. Its odd, because XMRV-MLV structural amino acid sequences are super similar (95%), and TRIM5alpha has to interact with specific amino acids to work, but, eh, XMRV can do it.

But it still doesnt entirely make sense why the Reno group could find infectious XMRV in PBMCs. Evolutionarily, it shouldnt be there. Well, rather than screaming about how Judy Mikovits is a liar and a fraud, Harriet et al reacted in another normal, scientifically acceptable way– by proposing some putative explanations, and excitedly waiting for future results. Their entire discussion was a pleasure to read.

In fact, the last author on this paper is Dr. Kate N. Bishop, also the last author on the second XMRV paper from the UK. Ignored the WPIs frantic wailing and claims everyone is incompetent but them, and kept doing science.

*tips hat to Dr. Bishop*

Neat science in this paper, Ma’am.

Comments

  1. #1 gf1
    March 5, 2010

    Understanding this is all very provisional, and no-one’s going to really know, I’ve got a couple of Qs (you said I should ask):

    Does this make it unlikely that XMRV is related to prostate cancer, as well as CFS? I’ve read a few things which seem to now assume XMRV can infect humans, even if we don’t know what this might lead to – would this new paper make you think that this is probably not the case? (Assuming it’s right). Would cancerous cells have these defenses too?, ie: could the link between XMRV and cancer just be that cancerous cells lack the natural defenses to the virus (I don’t know how the virus would get to these cells though?)

    Does this mean that, if XMRV can infect people, our natural defenses would be far more likely to be able to fight off an infection, rahter than it necessarily being life-long?

    Would any of this have any implications for the varying results we’ve seen with CFS and prostate cancer? (Implying either that it would be unusually difficult to detect in patients, or that the positive results were likely to have been mistaken).

    Ta.

  2. #2 qetzal
    March 5, 2010

    Adding to gf1’s questions:

    – Are there normal (non-cancerous) human cell types that are known to lack significant APOBEC3G & Tetherin activity?

    – Would the activation procedures that WPI used when culturing PBMCs be likely to alleviate the restrictions imposed by APOBEC3G & Tetherin?

    If so, perhaps the XMRV that WPI claims to find in CFS PBMCs is actually coming from other cells and merely ‘contaminates’ their PBMC preps. If other labs’ PBMC preps were cleaner, that could help explain some of the discrepancies.

  3. #3 gf1
    March 5, 2010

    I just came upon this patient write up of an XMRV presentation, which seemed relevent. It mentions that XMRV does seem to be surprisingly difficult to detect by PCR, and also that contamination of cell lines seems common too.

    “Dr. Yasuhiro Ikeda Assistant Professor, Dept. of Biochemistry & Molecular Biology, Mayo Clinic
    Characterization of a novel human retrovirus Characterization of a novel human retrovirus XMRV: Prevalence, antiviral drug sensitivity, and small animal model development
    UCDMC Cancer Center auditorium, 4501 “X” Street, Sacramento Thursday, March 4, 2010, 9:00 a.m. – 10:00 a.m.

    Disclaimer, the information Dr. Ikeda presented is not formally published. After his talk, I asked him to e-mail me what he would of the research information available from his lecture or otherwise. He seemed agreeable. I sat in the first row but my audio recording turned out very poor, and he has an accent. I have to get a different cable to download the audio file to my computer. I have a science background, but it’s not in virology. This is only my interpretation of what he presented from notes, recollection and my poor recording.

    Dr. Ikeda was impressive, friendly, and seemed meticulous. He presented research involving XMRV detection by PCR and antibody, cancer, antivirals, and finding a mouse model to grow the XMRV virus. The lecture was an hour long so he moved fast.

    He started with an overview of prostate and ME/CFS XMRV research, positive and negative associations. He presented the XMRV association with ME/CFS studies matter of factly as 1 for and 3 against with DeRisi skeptical.

    There was a slide about an indirect association of XMRV with H pylori, and Hepatitis C. He didn’t go into detail.

    The cell receptor for XMRV is XPR1.

    After testing many types of mice they infected Mus pahari (wild-derived) mice with XMRV and thought them suitable (but they like to play dead and bite). They discovered one California strain of mouse that harbored an endogenous retrovirus closely related to XMRV sequences. The XMRV they sequenced had very few mutations. They found the virus in their infected mice most often in the spleen, blood and brain, not prostate. They were curious about the leukemia connection because they found very few infected leukocytes.

    A number of antiviral drugs were tested at different concentrations with XMRV infected cells. They thought AZT seemed to be the only one that worked well without excessive toxicity to the cells (with about 100X reduction in viral activity). He stated that after stopping AZT treatment of the cells the virus started to replicate at rates comparable to before treatment. They had a slide that showed a sequence where HIV and XMRV were similar, and that they thought the drug was interrupting the replication at those sequences.

    They did a human prostate cancer study. 159 samples of prostate cancer tissue (grade 5-7), and 201 controls were tested for XMRV. 4.5% were XMRV positive in PC tissue. 2.5% were XMRV positive in prostate tissue controls .

    There was some confusing data on XMRV detection. He reported PCR seemed to be much less sensitive and often didn’t detect XMRV (and they used multiple PCR testing with some samples to find XMRV) than with other methods. They found real time PCR was more sensitive than nested PCR. They didn’t find XMRV in some of their first pass testing using western blot. They said that the positive tests had weak signals.

    They are looking at XMRV surface glycoproteins for targeting. I’m not sure if he was talking vaccines or other treatments.

    I found this very interesting. They found some standard cell lines of LnCap and 22Rv1 (biosafety level 2 lines) were already infected with XMRV before they came to their lab. Not all, just from some of their sources so they think that labs should be screening received cell lines by real time PCR.

    At the end there was about a 10 minute presentation about stem cell research in his lab. He talked about adult stem cells and differentiating them into specific cell functions; heart, nerve, and so forth. He didn’t say if this was related to his XMRV research.

    In our discussion after his presentation he stated he is only consulting with Silverman. They will probably not so ME/CFS research because Mayo isn’t big into ME/CFS. The docs there think the patient population is too heterogenous. I said the Science study used the most restrictive patient criteria and others the least restrictive. My impression was that he didn’t know the details of the different definitions. “

  4. #4 ragarth
    March 6, 2010

    Dr… Bishop?

    By any chance is he working with the FBI to save us from destruction with a parallel universe that he created?

  5. #5 KevinS
    March 6, 2010

    I think these findings on restriction are exactly in line with what we are seeing in patients who are positive for the virus. Since XMRV is a gamma retrovirus it needs dividing cells (rapidly dividing is better) to infect. Cancer patients obviously have rapidly diving cells, and some CFS patients have markers of chronic immune activation so lots of lymphoproliferation.

    The study shows XMRV has the tools to use the receptor on most cells. It looks like TRIMs do not touch the virus. This means there is no stopping this virus from integrating. Since it integrated into a cell that is proliferating the virus gets copied as a pro-virus. This is especially true if the virus integrated into an area of the genome where it does not get copied well.

    Once the virus is in it has trouble getting out and infecting other cells since APOBEC and Tetherin put the breaks on other rounds of infection.

    If the virus is being shed from a mouse with an endogenous virus in its genome, there would be very little sequence diversity of the virus that is being shed. If the virus can infect cells but not get out very well, then the provirus we see has been copied by accurate polymerases. This explains the apparent lack of sequence diversity. We don’t see viral sequences that look like they encountered APOBEC because the virus is not really replicating very well. This is why it may be hard to detect it. You need to find the cell or subset of cells with the provirus among trillions of cells. We have found the virus in dividing cell types that are easy to access, cancer and blood. It would be likely that we would see it in other dividing cell types that are less accessible.

    This is all wild speculation, but in my head it makes sense.

  6. #6 sardine
    March 8, 2010

    I know this is off topic, but I read this site to try to get a handle on some biology, of which I am sorely ignorant (MechE,) but when you see some of these bacteria speciate in the lab (and other observed forms of speciation) are there changes in the DNA? And is there any basic articles I could read on the subject.
    I have spent some time searching but haven’t found anything that I could understand.

    Thanks in advance for any help you all could give me on this,

    Dale

  7. #7 GK
    March 16, 2010

    Hi, erv-Germany-is-not-part-of-Europe-because-there’s-no-XMRV-in-Europe-although-it-was-found-in-Germany-in-2008.

    See if you’re capable of being an objective scientist again when it comes to XMRV….

    THE PREVALENCE OF XENOTROPIC MURINE LEUKEMIA VIRUS-RELATED VIRUS IN
    HEALTHY BLOOD DONORS IN JAPAN

    Rika A. Furuta1, Takayuki Miyazawa2, Takeki Sugiyama3, Takafumi
    Kimura1, Fumiya Hirayama1, Yoshihiko Tani1 and Hirotoshi Shibata1

    1 Department of Research, Japanese Red Cross Osaka Blood Center, 2
    Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto
    University, 3 Department of Urology, Nishiwaki Municipal Hospital.

    To estimate the impacts of infection with xenotropic murine leukemia
    virus-related virus (XMRV) on the blood service, we investigated the
    prevalence of this virus in both prostate cancer patients and healthy
    blood donors in Japan. All specimens from the prostate cancer patients
    were collected after obtaining their written informed consent. The
    ethical committee of the Japanese Red Cross Society approved the
    examination of XMRV antibodies, but not nucleic acids, in random donor
    sera.

    All serum samples of healthy blood donors tested negative for HIV-1,
    HIV-2, HTLV-1, hepatitis B virus, hepatitis C virus and human
    parvovirus B19.

    To make a recombinant virus as test antigens for the antibody
    screening, 293T cells were transfected with an expression vector
    carrying an XMRV provirus clone, namely, VP62 (kindly gifted by Dr. R.
    H. Silverman). We used an env-defective mutant of HIV-1 derived from
    pNL4-3 (kindly gifted by Dr. A. Adachi) as a negative control.

    Two days after transfection, the culture supernatants of the
    transfected cells were collected and concentrated 20 times by
    centrifugation. We implemented western blotting assay to screen
    antibodies against XMRV in sera because a high background was observed
    if we performed enzyme-linked immunosorbent assay. In the western
    blotting, the blot strips were incubated with the serum samples
    diluted 1:100 with 5 % skim milk in Tris-buffered saline overnight at
    4°C.

    Two of 32 serum samples collected from the prostate cancer patients
    and 5 of 300 serum samples collected from healthy blood donors tested
    positive for antibodies against XMRV Gag protein. We did not observe
    any specific signals against Env proteins in the western blotting. Of
    the 2 serum samples that were obtained from prostate cancer patient
    and tested positive for anti-XMRV antibodies, the XMRV specific
    nucleic acid sequence was detected in only one sample (patient #24) by
    using nested RT-PCR.

    In addition, we collected 7 mL of whole blood cells from the patient
    #24 and cultured the peripheral blood mononuclear cells (PBMCs) in the
    presence of recombinant interleukin 2 and concanavalin A.

    The PBMCs were harvested after 10 days of culture, the virus was
    isolated from the cells by performing a LacZ marker rescue assay and
    RNA and genomic DNA were extracted. The nested PCR performed to detect
    XMRV yielded positive results for both genomic and RT PCR, although
    the virus was successfully isolated in only 1 of 3 independent
    experiments. To examine the susceptibility of PBMCs derived from
    healthy individuals to XMRV, we inoculated activated PBMCs from 3
    healthy volunteers with the culture supernatant of the PBMCs obtained
    from patient #24. By using the nested PCR, we detected the
    XMRV-specific nucleic acid sequence in the genomic DNA of the PBMCs
    obtained from 2 of the 3 healthy volunteers.

    We conclude that XMRV infection is prevalent among both prostate
    cancer patients and healthy individuals in Japan. Although our study
    had a limited sample size, the prevalence among blood donors as
    determined by identifying XMRV-specific antibodies was found to be
    1.7%, while that among prostate cancer patients was found to be 6.3%
    (P<0.05, one-sided Mann-Whitney U-test).

    The results of genomic PCR performing on the PBMCs indicate that XMRV
    is sustained in a few fractions of blood cells and can spread through
    blood even though the virus replication rate appears to be very low.

    Source: 1 Department of Research, Japanese Red Cross Osaka Blood Center, 2
    Laboratory for Viral pathogenesis, Institute for Virus Research, Kyoto
    University, 3 Department of Urology, Nishiwaki Municipal Hospital.

  8. #8 ERV
    March 16, 2010

    Artard– OMFG THEY FOUND XMRV IN 8 PATIENTS IN GERMANY AND THEN NO ONE COULD REPLICATE THEIR FINDINGS!!! WARBLEGARBLE!

    Also, I already wrote about the Japanese test.

    Thanks for yet another insightful comment.

    Yours in Christ,
    Abbie

  9. #9 ERV
    March 16, 2010

    Artard– So just to be 100% clear, here: A grown, professional scientist accusing researchers who dont agree with her of scientific fraud, making up data, scamming to get published, and accepting bribes from Big Pharma/Big Insurance/whatever, with no evidence to support those assertions is absolutely fine with you.

    But calling this ‘professional scientist’ a cunt if she cannot provide evidence for her claims is ‘wrong’.

    You are a fucking moron.

  10. #10 GK
    March 18, 2010

    Touche!

    Stay classy, darlin’.

  11. #11 sue
    April 8, 2010

    so which cells have the TRIM5alpha thingie?

  12. #12 sue
    April 8, 2010

    it says most cells in primates have it….so i guess the thought of eradication of XMRV once established is a lost cause :-/

  13. #13 Dan
    April 10, 2010

    Let’s see Judy Mikovits PhD with 20 years of experience with the National Cancer Institute knows less…than…uh a student who doesn’t even have a graduate degree. Another ego manic vying for attention or do you work for UNUM?

  14. #14 BurntOffering
    August 27, 2010

    “I found this very interesting. They found some standard cell lines of LnCap and 22Rv1 (biosafety level 2 lines) were already infected with XMRV before they came to their lab. Not all, just from some of their sources so they think that labs should be screening received cell lines by real time PCR.”

    I hope that WPI/VIPDX has been running sufficient controls on pre-culture LnCap samples. This seems a plausible explanation for the disparate findings. If WPIs findings collapse under the weight of contamination, it will go badly for them. Their work is needed but they have surely alienated a lot of scientists.

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