XMRV abstracts from the 11th Symposium on Antiviral Drug Resistance Hershey, PA, Nov. 7-10- 2010. http://antiviralresistance.org/ProgramBook_SADR10.pdf Oral Presentations http://antiviralresistance.org/abstract25_2010.pdf INEFFICIENT REPLICATION AND SPREAD OF XENOTROPIC MURINE LEUKEMIA VIRUS-RELATED VIRUS IN HUMAN PERIPHERAL BLOOD MONONUCLEAR CELLS 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. http://antiviralresistance.org/abstract43_2010.pdf LOOKING FOR XMRV IN PROSTATE CANCER 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. http://antiviralresistance.org/abstract44_2010.pdf PROBING THE RELATIONSHIP OF XMRV AND MLV 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 DNA. Poster Presentations http://antiviralresistance.org/abstract_poster5_2010.pdf POSTER 5 BIOCHEMICAL, STRUCTURAL, AND INHIBITION STUDIES OF XMRV REVERSE TRANSCRIPTASE 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 inhibitors. http://antiviralresistance.org/abstract_poster44_2010.pdf POSTER 44 DEVELOPMENT OF A GFP-INDICATOR CELL LINE FOR THE DETECTION OF XMRV 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, USA 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.