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Abstracts from 11th Symposium Antiviral Drug Resistance

Discussion in 'XMRV Research and Replication Studies' started by Rita, Nov 16, 2010.

  1. Rita

    Rita Senior Member

    XMRV abstracts from the
    11th Symposium on Antiviral Drug Resistance
    Hershey, PA, Nov. 7-10- 2010.

    Oral Presentations


    Chawaree Chaipan1, Kari A. Dilley2, Tobias Paprotka1, Krista A.
    Delviks-Frankenberry1, Narasimhan J. Venkatachari1, Wei-Shau Hu2, and
    Vinay K. Pathak1*

    1Viral Mutation Section and 2 Viral Recombination Section, HIV Drug
    Resistance Program, National Cancer Institute at Frederick, Frederick,
    MD 21702, USA

    Xenotropic murine leukemia virus-related virus (XMRV) is a
    gammaretrovirus recently isolated from human prostate cancer and
    peripheral blood mononuclear cells (PBMCs) of patients with chronic
    fatigue syndrome (CFS). Previously, we showed that host restriction
    factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in
    human PBMCs, potently inhibit XMRV replication. The recovery of
    infectious XMRV from human PBMCs in CFS patients suggested that XMRV
    can replicate in these cells despite the expression of APOBEC3
    proteins. To determine the extent of XMRV replication and spread, we
    infected phytohemaglutinin-activated human PBMCs and A3G/A3F-positive
    and negative cell lines (CEM and CEMSS, respectively) with different
    amounts of XMRV and monitored virus production using quantitative
    realtime PCR. We found that XMRV efficiently replicated in CEM-SS
    cells but not in CEM cells and activated PBMCs. However, infectious
    XMRV could be recovered from the infected PBMCs by co-cultivation with
    canine indicator cells, and we observed low-level hypermutation of
    XMRV genomes in PBMCs. Overall, these results indicate that A3G/A3F
    expression in human PBMCs constitutes a potent block to replication.


    Amanda L. Aloia1, Karen S. Sfanos2, Jessica L. Hicks2,6, William B.
    Isaacs3-6, Qizhi Zheng2,6, Kenneth J. Pienta7, Frank Maldarelli1,
    Angelo M. De Marzo2-6, and Alan Rein1

    1HIV Drug Resistance Program, National Cancer Institute, Frederick, MD
    21702; Department of 2Pathology, 3Urology, and 4Oncology, 5The Brady
    Urological Research Institute and the 6Sidney Kimmel Comprehensive
    Cancer Center at Johns Hopkins, Baltimore, MD 21231; 7The University
    of Michigan Comprehensive Cancer Center, Ann Arbor, MI 48109

    Several recent papers have reported the presence of a gammaretrovirus,
    termed XMRV (xenotropic murine leukemia virus-related virus) in
    prostate cancers (PCa). If confirmed, this could have enormous
    implications for the detection, prevention, and treatment of PCa.
    However, other papers report failure to detect XMRV in PCa. We tested
    nearly 800 PCa samples, using a combination of real-time PCR and
    immunohistochemistry (IHC). The PCR reactions were simultaneously
    monitored for amplification of a single-copy human gene, in order to
    confirm the quality of the sample DNA and its suitability for PCR.
    Controls demonstrated that the PCR assay could detect the XMRV in a
    single infected cell, even in the presence of a 10,000-fold excess of
    uninfected human cells. The IHC used two rabbit polyclonal antisera,
    each prepared against a purified MLV protein. Both antisera always
    stained XMRV-infected or -transfected cells, but never stained control
    cells. No evidence for XMRV in PCa was obtained in these experiments.
    It is possible that XMRV is not actually circulating in the human
    population; even if it is, the data do not seem to support a causal
    role for this virus in PCa.


    John M. Coffin1, Oya Cingz1, Ann Wiegand2, Jon Spindler2, and Mary Kearney2

    1Department of Molecular Biology and Microbiology and Program in
    Genetics, Tufts University, Boston MA 02111; 2HIV Drug Resistance
    Program, National Cancer Institute, Frederick MD 21702

    Xenotropic MLV-related virus (XMRV) was first described in a few cases
    of prostate cancer about 5 years ago. XMRV is closely relate to, but
    distinct in sequence from, known endogenous xenotropic MLV (Xmv)
    proviruses. It has since been associated with larger numbers of
    prostate cancers, as well as a significant fraction of patients with
    chronic fatigue syndrome (CFS). In these cases, it has been possible
    to isolate infectious virus and study its properties. Some more recent
    studies have identified other types of endogenous MLV-related
    sequences (polytropic-Pmv- and modified polytropic, Mpmv) in a cohort
    of CFS cases, but not in random blood donors. In the latter case,
    sequences have been identified only by PCR amplification of a fragment
    of the genome. Neither complete genome sequences nor infectious virus
    has been obtained. In the meantime, a number of other investigators
    have reported nearly complete failure to replicate these results by
    finding virus or antiviral antibodies in other cohorts of CFS and
    prostate cancer. The very close similarity of XMRV and the large
    numbers (around 60 in inbred mice) of endogenous MLVs raise
    unclarified issues regarding the provenance of virus and virus-like
    sequences identified in clinical samples. Our groups have been
    investigating the relationship between endogenous MLVs and XMRV
    with the goal of understanding the origin of the virus and the
    potential for confusion due to inadvertent contamination of assays
    with mouse DNA. Using a highly specific PCR assay for XMRV, based on
    characteristic conserved indels in the LTR and gag, we have been
    looking for closely related sequences in wild and inbred mouse DNA, so
    far without success. In another study, the DRP group has developed
    highly specific and sensitive PCR assays (X- single copy assay (SCA)
    and X- single genome sequencing
    (SGS)) to quantitate and distinguish XMRV and endogenous MLV
    sequences. In reconstruction experiments, these assays could detect
    single copies of XMRV RNA in plasma and very low numbers of infected
    cells in whole blood. They are also very sensitive detectors of mouse
    DNA contamination, allowing detection and genetic analysis of the
    proviral DNA im less than one-tenth of a cell.

    To distinguish mouse DAN contamination, we have developed a PCR assay
    based on the LTR sequences of the intracisternal A particle (IAP)
    retroelement, of which there are more than a thousand copies in the
    mouse genome, but none in human DNA. This assay provides a clear and
    unambiguous means of detecting much less than 1 cells worth of mouse

    Poster Presentations

    POSTER 5


    Tanya Ndognwe1, Karen Kirby1, Bruno Marchand1, Adeyemi Adedeji1,
    Eleftherios Michailidis1, Yee-Tsuei Ong1, Atsuko Hachiya1, Emily
    Ryan1, Shun-Lu Liu1, Angela Whatley1, Donald H. Burke1, Sanath Kumar1,
    Marc Johnson1, Ei-Ichi Kodama2, Krista A. Delviks-Frankenberry3, Vinay
    K. Pathak3, Hiroaki Mitsuya4, Michael A. Parniak5, Kamal Singh1, and
    Stefan G. Sarafianos1

    1Department of Molecular Microbiology & Immunology, University of
    Missouri School of Medicine, Columbia, MO; 2Division of Emerging
    Infectious Diseases, Tohoku University School of Medicine, Sendai,
    Japan; 3HIV Drug Resistance Program, National Cancer Institute,
    Frederick, MD; 4Department of Internal Medicine, Kumamoto University
    School of Medicine, Kumamoto, Japan & Experimental Retrovirology
    Section, HIV/AIDS Malignancy Branch, NIH, Bethesda, MD; 5Department of
    Molecular Genetics & Biochemistry, University of Pittsburgh School of
    Medicine, Pittsburgh, PA

    The Xenotropic Murine Leukemia Virus-Related Virus (XMRV) was
    originally identified in biological samples from familial prostate
    cancer patients and was more recently reported in patients with
    chronic fatigue syndrome (CFS). Although other studies have failed to
    detect XMRV in CFS or prostate cancer patients, given the potential
    importance of this virus for human disease we initiated studies on its
    replication mechanism and susceptibility to inhibitors. We used
    biochemical, biophysical, virological, and structural methods to
    characterize the DNA polymerase and RNase H functions of XMRV reverse
    transcriptase (RT). We compared the properties of XMRV RT, XMRV RNase
    H, Moloney Murine Leukemia Virus (MuLV) RT, and HIV RT. Using steady
    state and pre-steady state kinetics we demonstrated that XMRV RT is
    the slowest and least efficient of the three enzymes in synthesizing
    DNA or cleaving RNA/DNA. Similarly, its ability to unblock
    chain-terminated primers using PPi or ATP are much lower compared to
    HIV RT. Its reduced polymerase and RNAse H activity is due in part to
    a lower affinity for nucleic acid, as judged by gel-shift assays and
    confirmed by surface plasmon resonance experiments, which revealed
    that the deficiency in DNA binding is due to a high dissociation rate.
    Trap experiments showed that XMRV RT has very low processivity
    compared to HIV RT. Transient kinetics of mismatch incorporations
    revealed that XMRV RT has higher fidelity than MuLV and HIV RTs.
    Nonetheless, XMRV and MuLV appear to have comparable fidelities in
    cell-based assays. The polymerase function of XMRV RT is susceptible
    to antiretrovirals from several classes, but not to NNRTIs. XMRV RT is
    inhibited efficiently by AZT-TP, PMEADP, d4T-TP, PMPA-DP, and by a
    nucleic acid aptamer. Two potent NRTIs that block XMRV RT efficiently
    by a different mechanism also have potent antiviral activity in
    pseudotype-based and replication competent virus-based assays. We have
    also identified compounds that efficiently block the RNase H activity
    of XMRV RT and of active XMRV RNase H fragments. Finally, we have
    solved the crystal structure of XMRV RNase H at high resolution (1.5
    ), which will facilitate the design of new and more potent XMRV

    POSTER 44


    KyeongEun Lee1, Francis W. Ruscetti2, Patricia Lloyd1, Alan Rein1,
    Gisela Fanning-Heidecker1, and Vineet N. KewalRamani1

    1HIV Drug Resistance Program and 2Laboratory of Experimental
    Immunology, National Cancer Institute- Frederick, Frederick, MD 21702,

    Human immunodeficiency virus (HIV) titer can be estimated using
    indicator cell lines, such as GHOST cells, within days of infection.
    HIV indicator cells rely on production of Tat to transactivate
    expression of a reporter gene under the control of HIV LTR sequences.
    Simple retroviruses typically do not encode transcriptional
    transactivators. For simple retroviruses that lack transformational or
    cytopathic activity, their titers are often measured by infection of
    cells after end point dilution and assaying for virus proliferation
    after weeks of culture. Replication-dependent vectors have been
    leveraged to assay the mobilization of retrotransposable elements and
    the replication of retroviruses. Here we describe an indicator cell
    line for the detection of infectious xenotropic murine leukemia
    virus-related virus (XMRV) that relies on the propagation of a vector,
    which leads to expression of a GFP reporter. We constructed an MLV
    vector encoding puromycin resistance and a CMV enhancer/promoter
    driven GFP reporter gene whose transcription was antisense to the
    vector mRNA. The GFP reporter sequence (iGFP) was interrupted by an
    intron placed in the sense direction relative to the vector. The
    prostate cell line, LNCaP, was stably transfected with the above
    construct, and several LNCaP-iGFP cell clones displaying sensitivity
    to XMRV infection after end point dilution were isolated and
    designated Detectors of Exogenous Retroviral Sequence Elements (DERSE)
    cells. GFP signal could be detected within three days of infection,
    with the number of GFP-positive cells increasing over subsequent days.
    GFP signal after virus inoculation was dosedependent and could be
    impaired by heat inactivation of virus stocks or the addition of AZT
    to cultures at the time of infection. In principle, DERSE cells should
    also detect other gammaretroviruses capable of
    infecting human cell lines. DERSE cells provide a facile assay to
    assess antiviral or antibody mediated neutralization of XMRV, and
    should be useful in assessing the presence of infectious XMRV in
    patient materials.
  2. August59

    August59 Daughters High School Graduation

    Upstate SC, USA
    Thanks Rita - There was some good stuff in here. Seems as if the have defintely compiled an assay that will screen out ALL contamination. Looks as if there is some more definitive studies as to what ARV work against XMRV, plus some that are in the works as well?
  3. eric_s

    eric_s Senior Member

    Switzerland/Spain (Valencia)
    Thanks, it's interesting. The first study seems to say that they could not find XMRV in PBMC unless they used culturing, so one more piece of evidence that a PCR study that looks for XMRV in PBMC without that method is unlikely to find something.

    Then Coffin says that they could not find any DNA sequences in mice that match what Alter/Lo have found. And that they have a good assay to screen for contamination with mouse DNA. So if we have not heard Coffin or the BWG, that he's a member of, say "it's contamination", that's good news.
    On the other hand, using that assay they seem to not have been able to find XMRV in prostate cancer, so i feel like it's not really sure wheter it's working in real samples.

    The next one is about what was reported in newspaper articles where they quoted the researcher as saying his work could be "potentially explosively important" and then there's one about the DERSE cells. If Ruscetti has them, i hope he shares that with the WPI. Would be one more way to rule out contamination, i think, but that's just my layman's understanding. I think with what they know now about XMRV and the DERSE cell lines, it should be possible to develop at least some form of treatment and also measure it's success. So with more money, more personnel and some more time, we should see some good results, i hope.
  4. lancelot

    lancelot Senior Member

    southern california
    Now we are getting somewhere! Good job Dr. Coffin!
  5. omerbasket

    omerbasket Senior Member

    Correct me if I'm wrong, but I think that the people who are named in the study about this assay are not the people who are named in the study about not finding XMRV in 800 cases of prostate cancer - and also there is no evidence that the study that didn't find XMRV in 800 cases of prostate cancer used the assay described in Coffin et al. study.

    So - perhaps there is not other hand here...;)
  6. eric_s

    eric_s Senior Member

    Switzerland/Spain (Valencia)
    You are right, it's not the same people, they're just all working for the DRP. To me it sounded as if it's the same assay, but i don't know. I don't need any other hands :D

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