The 17th Conference on Retroviruses and Opportunistic Infections
New Studies on XMRV Presented at the 2010 Conference on Retroviruses and Opportunistic Infections
February 16-19, 2010
Moscone Center West, San Francisco
The 17th Conference on Retroviruses and Opportunistic Infections (CROI 2010) is a scientifically focused meeting of the world’s leading researchers working to understand, prevent and treat HIV/AIDS and its complications.
From the CROI 2010 Program Schedule: http://zika.retroconference.org/croi-2010-pocket-program.pdf
Dr. Stephen Goff
Friday, February 19, 2010
Session 38: Plenary
Mouse to Man? XMRV and Human Disease
Howard Hughes Medical Institute
Columbia University College of Physicians and Surgeons
Objectives: This session is directed to clinicians and scientists interested in the newly discovered xenotropic murine leukemia virus-related virus (XMRV). This group will include those interested in prostate cancer in addition to chronic fatigue syndrome. It will also include those interested in human retroviral diseases in general and the transmission of animal retroviruses to the human population. It is assumed that participants are familiar with the basic aspects of retrovirus replication. At the completion of the session, participants will be knowledgeable about the history of XMRV virus and the linkage to human disease.
Session 41: Oral Abstracts
Moderators: Vinay Pathak, NCI-Frederick, Maryland and Jonathan Stoye, National Institute of Medical Research, London, UK
Cellular Determinants Required for Infection of XMRV, a Novel Retrovirus Associated with Human Familial Prostate Cancer
S. Bhosle, S Suppiah, R Arnold, Y Liang, J Blackwell, J Petros, D Liotta, E Hunter, R Molinaro and Hinh Ly
Emory University, Atlanta, Georgia
Background: The newly identified retrovirus, the xenotropic MuLV-related virus XMRV, has recently been shown to be strongly associated with familial prostate cancer in human. While that study showed evidence of XMRV infection exclusively in the prostatic stromal fibroblasts, a recent study found XMRV protein antigens mainly in malignant prostate epithelial cells.
Methods: To help understand the mechanisms behind XMRV infection, we show that prostatic fibroblast cells express Xpr1, a known receptor of XMRV, but its expression is absent in other cells of the prostate (ie, epithelial and stromal smooth muscle cells). We also show for the first time that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry.
Results: While we found strong evidence to support XMRV infection of prostatic fibroblasts via Xpr1, we learned that XMRV was indeed capable of infecting cells that did not necessarily express Xpr1, such as those of the prostatic epithelial and smooth muscle origins. Further studies suggest that the expression of Xpr1 and certain genotypes of the RNASEL gene, which could restrict XMRV infection, may play important roles in defining XMRV tropisms in certain cell types.
Conclusions: Collectively, our data reveal important cellular determinants required for XMRV infection of human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.
Host Regulation of XMRV in Prostate Cancer
B Dong and Robert Sliverman
Cleveland Clinic, Cleveland, Ohio
Background: XMRV is a human gammaretrovirus associated with prostate cancer and chronic fatigue syndrome. While it is unknown if XMRV is an etiological agent of these diseases, other gammaretroviruses cause cancer and neurological disease in animals. Virus-host interaction studies could provide insight on the pathogenic consequences of XMRV infections as well as a means for controlling infections. Here we investigated roles of IFN pathways and androgen on XMRV infections in prostate cancer cells.
Methods: DU145 cells were treated with varying doses of human IFN-beta before and after infection with XMRV. Viral protein levels were determined in Western blots and viral release was monitored by RT activity in conditioned media. Effects of RNase L were determined by decreasing expression with siRNA. Reporter plasmids were constructed containing wild type and glucocorticoid response element (GRE) mutant versions of the XMRV U3 region. Effects of dihydrotestosterone (DHT) on viral transcription were determined in prostate cancer cells containing (LNCaP) or lacking (DU145) androgen receptor. LNCaP or DU145 cells grown in hormone-depleted media were infected with XMRV and treated with DHT, or with anti-androgens, casodex, or flutamide, for 2 days. Virus replication was monitored by RT assays.
Results: IFN treatment inhibited XMRV expression in both acutely-infected and chronically-infected DU145 cells. Levels of XMRV Gag protein in the intact infected cells and RT activity in the conditioned media were down-regulated by IFN-beta. Cells in which siRNA was used to decrease RNase L levels were partially resistant to IFN-beta. Transcription from the XMRV U3 region was stimulated up to 2-fold by DHT, but only in cells containing a functional androgen receptor. Mutations in the GRE of XMRV impaired basal transcription and androgen responsiveness. Furthermore, DHT stimulated XMRV replication by 3-fold, whereas androgen inhibitors suppressed viral growth by 3-fold.
Conclusions: XMRV was discovered in men with a deficiency in the IFN pathway gene encoding RNase L. Our findings show that RNase L is required for a complete IFN antiviral response. We further show the presence of a functional androgen response element (ARE) in the U3 region of the XMRV LTR. XMRV integration into prostate cells could cause androgen-stimulation of pro-inflammatory genes and proto-oncogenes leading to cancer.
Prevalence of XMRV in Prostate Cancer
William Switzer, H Jia, HQ Zheng, S Tang and W Heniene
Centers for Disease Control and Prevention, Atlanta, Georgia
Background: Recently, a xenotropic murine leukemia-related virus (XMRV) was identified by virus-generic microarray and polymerase chain reaction testing in 40% of prostate cancer tissues with the homozygous R462Q (QQ) variant of the antiviral RNase L enzyme. Two subsequent studies confirmed XMRV sequences in prostate cancers at a lower polymerase chain reaction prevalence (1.5% and 6%) independent of R462Q polymorphism. One of the studies also reported higher detection of XMRV proteins by immunohistochemistry in 23% of cancers. To further evaluate the prevalence of XMRV in prostate cancer and better define markers of infection, we developed new molecular and serologic assays and used them to screen tissue and plasma specimens from patients with prostate cancer.
Methods: We developed both a xenotropic murine leukemia virus-based Western blot assay for antibody detection, and new gag, polymerase (pol), and envelope (env) polymerase chain reaction assays for sequence detection. To detect contamination with mouse DNA, we also designed a mouse-specific mitochondrial DNA (mtDNA) polymerase chain reaction test. Following assay validation, prostate tissue (n = 162) and matching plasma (n = 120) from anonymous US prostate cancer patients were tested. Phylogenetic analysis by neighbor-joining methods was used to infer evolutionary relationships. RNase L R462Q polymorphism in all patients was determined using a commercial test.
Results: Of 165 prostate tissues, 2 (1.2%) were positive by pol and env polymerase chain reaction and had undetectable mouse mtDNA. Phylogenetic analysis showed distinct sequences that clustered with other XMRV. Plasma from both persons (5956 and 6203) were negative by reverse transcriptase-polymerase chain reaction indicating absence of viremia. Both patients were Western blot-negative. Plasma from 118 additional polymerase chain reaction-negative patients tested Western blot negative. Of the patients, 45.1% were R462Q RR homozygotes, 45.6% RQ, and 9.3 % QQ. Person 5956 was RR homozygous; patient 6203 was heterozygous (RQ). Both had intermediate-grade tumors based on Gleason scoring.
Conclusions: Our results demonstrate a low prevalence of XMRV sequences in prostate cancer patients that is not likely associated with R462Q RNase L genotype. Infection with XMRV is confirmed by phylogenetic analysis and absence of contaminating mouse DNA. The finding of undetectable antibodies and viremia in 2 patients is noteworthy and may reflect sequestered or cleared infections. More studies are needed to better understand the prevalence and significance of XMRV in prostate cancer or other diseases.
Organ and Cell Lineage Dissemination of XMRV in Rhesus Macaques during Acute and Chronic Infection
Prachi Sharma, S Supplah, R Molinaro, K Rogers, J Das Gupta, R Silverman, J Hackett, Jr., S Devare, G Schochetman and F Villanger
Yerkes National Primate Research Center, Emory University, Atlanta, Georgia and Abbott Diagnostics, Abbott Park, Illinois
Background: Infection with Xenotropic MuLV-related Retrovirus (XMRV), a g-retrovirus, has been identified in association with familial cases of prostate carcinoma and in patients with chronic fatigue syndrome, although an etiological link remains to be established. In light of these studies, the development of an animal model to study XMRV dissemination, tissue tropism and pathogenicity is essential for understanding its role and infection transmission.
Methods: We experimentally infected 5 healthy rhesus macaques with XMRV intravenously. At days 6 or 7 (acute infection), 2 macaques were euthanized, as were the others during chronic infection at days 146 and 289 post infection. Extensive tissue collections were done from various organs at necropsy to evaluate both the tissue and cell tropism at various times post infection using FISH to the entire genome and IHC via detection of XMRV gag using a cross reactive monoclonal antibodies to murine spleen focus-forming virus (SFFV).
Results: Both methods were concordant for the detection of XMRV in the various organs tested and showed a wide dissemination of replicating virus even when the plasma viral load was undetectable. Of interest was the finding that isolated lymphoid cells and primarily CD4+ T cells were found positive in most lymphoid organs including spleen, lymph nodes, and gastrointestinal tract, while in lung, XMRV+ cells exhibited a macrophage morphology. The frequency of infected cells appeared to decrease in spleen while increasing in the gastrointestinal tract from acute to chronic infection. XMRV infection was however not restricted to bone marrow derived cells, but showed distinct target specificities in various organs. Using IHC, foci of infected epithelial cells were detected in prostate, seminal vesicles and epididymis while XMRV+ cells in the testes were interstitial. In the lone female animal, XMRV+ epithelial and fibroblast like cells were detected in the vagina and cervix suggesting that the virus may be transmitted sexually. While XMRV dissemination was complete at day 6 post infection, the prostate was positive only during the acute infection in these healthy animals, while other reproductive organs were similarly positive during the chronic phase.
Conclusions: We believe to have established a validated animal model of human XMRV infection, suitable to test its long-term chronic effect, pathogenesis, and immunity and to validate future vaccines. The organ-specific target-cell distribution is intriguing and remains to be studied in more detail.
XMRV: Examination of Viral Kinetics, Tissue Tropism, and Serological Markers of Infection
X Qui, P Swanson, K-C Luk, J Das Gupta, N Onlamoon, R Silverman, F Villanger, S Devare, G Schochetman and John Hackett, Jr.
Abbott Diagnostics, Abbott Park, Illinois and Yerkes National Primate Research Center, Emory University, Atlanta, Georgia
Background: Xenotropic Murine Leukemia Virus-related Retrovirus (XMRV) is a human retrovirus recently discovered in familial prostate cancer tissue using DNA array based Virochip technology. Understanding viral replication kinetics, tissue tropism, and the host immune response is fundamental to establish the etiology of XMRV infection in human disease. Development of serologic assays to detect XMRV-specific antibodies would facilitate epidemiologic studies.
Methods: Five rhesus macaques were inoculated intravenously with XMRV. Blood was collected throughout the course of infection, and tissue from multiple organs was harvested at necropsy. Two macaques were necropsied at day 6 or 7 and one at day 144 post infection. The remaining 2 animals were re-inoculated with XMRV on day 158 and necropsied on day 291. XMRV-specific immunoreactivity was monitored by Western blot using viral lysate. Recombinant env gp70, p15E and gag p30 were utilized to develop serologic assays on the high-throughput automated ARCHITECT instrument system (Abbott Diagnostics).
Results: XMRV inoculation resulted in low transient plasma viremia, although proviral DNA persisted in circulating peripheral blood mononuclear cells for several weeks. Of interest, the earliest leukocyte targets were CD4+ T cells and NK cells followed by CD8+ enriched T and CD20+ enriched B cells (50% positive); CD14+ monocytes were negative. Animals sacrificed at the acute stage showed evidence of viral replication in spleen, lung, lymph nodes and liver. In contrast, sacrifice of 2 animals at 19 weeks post XMRV re-inoculation showed greater dissemination of XMRV DNA and RNA in various organs including the GI and urinary tract as well as in vaginal tissue of the one female. By Western blot analysis, all 3 chronically infected macaques developed antibody responses to env and gag proteins.The serologic assays demonstrated 100% sensitivity by detecting all Western blot positive serial bleeds from the XMRV-infected macaques. Preliminary results showed evidence of detectable reactivity to all 3 antigens in a low proportion (~0.1%) of US blood donors.
Conclusions: These data suggest that lymphocytes are a primary target for replication persistence (low grade replication) of XMRV in the absence of detectable plasma viremia. This study identified specific serological markers useful for detection of antibodies induced by XMRV infection. The prototype antibody assays will facilitate large-scale epidemiological studies.
Videotape of Press Conference Following XMRV Presentations:
Media Article from MedPageToday About XMRV Presentations:
“Secrets of Novel Retrovirus Unfolding”