XMRV, or xenotropic murine leukemia virus (MLV)-related virus, is a novel gammaretrovirus originally identified in studies that analyzed tissue from prostate cancer patients in 2006 and blood from patients with chronic fatigue syndrome (CFS) in 2009. However, a large number of subsequent studies failed to confirm a link between XMRV infection and CFS or prostate cancer. On the contrary, recent evidence indicates that XMRV is a contaminant originating from the recombination of two mouse endogenous retroviruses during passaging of a prostate tumor xenograft (CWR22) in mice, generating laboratory-derived cell lines that are XMRV-infected. To confirm or refute an association between XMRV and prostate cancer, we analyzed prostate cancer tissues and plasma from a prospectively collected cohort of 39 patients as well as archival RNA and prostate tissue from the original 2006 study. Despite comprehensive microarray, PCR, FISH, and serological testing, XMRV was not detected in any of the newly collected samples or in archival tissue, although archival RNA remained XMRV-positive. Notably, archival VP62 prostate tissue, from which the prototype XMRV strain was derived, tested negative for XMRV on re-analysis. Analysis of viral genomic and human mitochondrial sequences revealed that all previously characterized XMRV strains are identical and that the archival RNA had been contaminated by an XMRV-infected laboratory cell line. These findings reveal no association between XMRV and prostate cancer, and underscore the conclusion that XMRV is not a naturally acquired human infection.

The 22Rv1 cell line is widely used for prostate cancer research and other studies throughout the world. These cells were established from a human prostate tumor, CWR22, that was serially passaged in nude mice and selected for androgen independence. The 22Rv1 cells are known to produce high titers of xenotropic murine leukemia virus-related virus (XMRV). Recent studies suggested that XMRV was inadvertently created in the 1990's when two murine leukemia virus (MLV) genomes (pre-XMRV1 and pre-XMRV-2) recombined during passaging of the CWR22 tumor in mice. The conclusion that XMRV originated from mice and not the patient was based partly on the failure to detect XMRV in early CWR22 xenografts. While that deduction is certainly justified, we examined the possibility that a closely related virus could have been present in primary tumor tissue. Here we report that we have located the original prostate tumor tissue excised from patient CWR22 and have assayed the corresponding DNA by PCR and the tissue sections by fluorescence in situ hybridization for the presence of XMRV or a similar virus. The primary tumor tissues lacked mouse DNA as determined by PCR for intracisternal A type particle DNA, thus avoiding one of the limitations of studying xenografts. We show that neither XMRV nor a closely related virus was present in primary prostate tissue of patient CWR22. Our findings confirm and reinforce the conclusion that XMRV is a recombinant laboratory-generated mouse virus that is highly adapted for human prostate cancer cells.

ABSTRACT Xenotropic murine leukemia-related virus (XMRV) was identified in association with human prostate cancer and chronic fatigue syndrome. To examine the infection potential, kinetics, and tissue distribution of XMRV in an animal model, we inoculated five macaques with XMRV intravenously. XMRV established a persistent, chronic disseminated infection, with low transient viremia and provirus in blood lymphocytes during acute infection. Although undetectable in blood after about a month, XMRV viremia was reactivated at 9 months, confirming the chronicity of the infection. Furthermore, XMRV Gag was detected in tissues throughout, with wide dissemination throughout the period of monitoring. Surprisingly, XMRV infection showed organ-specific cell tropism, infecting CD4 T cells in lymphoid organs including the gastrointestinal lamina propria, alveolar macrophages in lung, and epithelial/interstitial cells in other organs, including the reproductive tract. Of note, in spite of the intravenous inoculation, extensive XMRV replication was noted in prostate during acute but not chronic infection even though infected cells were still detectable by fluorescence in situ hybridization (FISH) in prostate at 5 and 9 months postinfection. Marked lymphocyte activation occurred immediately postinfection, but antigen-specific cellular responses were undetectable. Antibody responses were elicited and boosted upon reexposure, but titers decreased rapidly, suggesting low antigen stimulation over time. Our findings establish a nonhuman primate model to study XMRV replication/dissemination, transmission, pathogenesis, immune responses, and potential future therapies.

IN 2011, THE INTERNATIONAL YEAR OF Chemistry, we pause to celebrate the achievements of chemistry and the fi eld’s contributions to the well-being of humankind (1). In doing so, it seems appropriate to recognize the history of the chemical elements, the building blocks of chemistry. The commonly used names of chemical elements have been presented and explained in detail over the years (2–4). However, some erstwhile names are much more diffi cult to fi nd. Who remembers the name policium, proposed by the discoverers of element 110? They suggested the name because the telephone number of the police in Germany is 110 (5). Today we refer to this element as darmstadtium. What about pandemonium and delirium? Seaborg and co-workers proposed these names for the elements known today as americium and curium, respectively, because of the tremendous diffi culties they faced in preparing them (6, 7). Mendeleev suggested the prefi xes eka and dwi (meaning one and two in Sanskrit, to indicate that a given element is one or two rows below the stated element) (8). If the prefi xes had been adopted, germanium would be known as ekasilicon (9) and gallium would be ekaaluminium (10). Accordingly, Otto Hahn and co-workers named neptunium ekarhePartial Retraction

Xenotropic Murine Leukemia Virus-related Retrovirus (XMRV) was identified from prostate cancer tissue using DNA based ViroChip technology as well as in a cohort of chronic fatigue syndrome patients. To delineate the infection dynamics and dissemination in vivo, we have thus far infected 7 healthy rhesus macaques and 2 pigtailed macaques. Results show that XMRV induces a chronic and clinically silent infection that is nevertheless persistent and susceptible to reactivation in vivo. While XMRV seems rapidly cleared from the blood circulation in healthy macaques, XMRV protein positive CD4+ T cells were detected in all lymphoid organs throughout infection. However, among all organs subjected to in situ detection, mucosal sites overall and sexual organs showed markedly higher frequencies of XMRVgag positive cells, including gastrointestinal mucosa, pulmonary environment and organs from the reproductive tract. Of interest, the lineage of cells that were XMRV positive markedly differed among the different sites, including CD4+ T cells in the GI mucosa, alveolar macrophages in the lung, epithelial cells in the prostate, seminal gland, vagina and cervix and interstitial cells in the testes. The latter were consistently observed during acute and chronic infection, suggesting the potential for sexual transmission of XMRV. In fact, a single atraumatic mucosal exposure with a high dose of XMRV virus into the urethra resulted in infection of 1 out of 4 macaques providing proof of concept that such transmission is possible. However, additional work is needed to fully investigate potential modes of XMRV infection.

ABSTRACT We analyzed xenotropic murine leukemia virus-related virus (XMRV) integration site sequences previously identified from human prostate tissues for single-nucleotide polymorphisms (SNPs) to discriminate between patient and potential cell line sources of the proviruses. The SNPs of two integration sites were identical to those in cell lines but not the patients, whereas the data on the remaining 12 integration sites were inconclusive. Our results provide direct evidence for contamination during analysis of XMRV integration sites.

The gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), replicates to high titers in some human cell lines and is able to infect non-human primates. To determine whether APOBEC3 (A3) proteins restrict XMRV infections in a non-human primate model, we sequenced proviral DNA from peripheral blood mononuclear cells of XMRV-infected rhesus macaques. Hypermutation characteristic of A3DE, A3F and A3G activities was observed in the XMRV proviral sequences in vivo. Furthermore, expression of rhesus A3DE, A3F, or A3G in human cells inhibited XMRV infection and caused hypermutation of XMRV DNA. These studies show that some rhesus A3 isoforms are highly effective against XMRV in the blood of a non-human primate model of infection and in cultured human cells.

Xenotropic murine leukemia virus-related virus (XMRV) was first reported in 2006 in a study of human prostate cancer patients with genetic variants of the antiviral enzyme, RNase L. Subsequent investigations in North America, Europe, Asia, and Africa have either observed or failed to detect XMRV in patients (prostate cancer, chronic fatigue syndrome-myalgic encephalomyelitis (CFS-ME), and immunosuppressed with respiratory tract infections) or normal, healthy, control individuals. The principal confounding factors are the near ubiquitous presence of mouse-derived reagents, antibodies and cells, and often XMRV itself, in laboratories. XMRV infects and replicates well in many human cell lines, but especially in certain prostate cancer cell lines. XMRV also traffics to prostate in a nonhuman primate model of infection. Here, we will review the discovery of XMRV and then focus on prostate cancer-related research involving this intriguing virus.

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011280 XMRV INFECTION INDUCES HOST GENES THAT REGULATE INFLAMMATION AND CELLULAR PHYSIOLOGY Michael Lee, Elona Gusho, Jaydip Das Gupta, Eric Klein, and Robert Silverman Michael LeeMichael Lee Cleveland, OH More articles by this author , Elona GushoElona Gusho Cleveland, OH More articles by this author , Jaydip Das GuptaJaydip Das Gupta Cleveland, OH More articles by this author , Eric KleinEric Klein Cleveland, OH More articles by this author , and Robert SilvermanRobert Silverman Cleveland, OH More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.372AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES XMRV is a novel human retrovirus associated with prostate cancer. Although other gammaretroviruses cause cancer in animals, it remains unknown if XMRV is a cause of disease. However, indirect or direct modes of carcinogenesis by XMRV have been suggested depending on whether the virus was found in stroma or malignant epithelium. To gain insight into the possible role of XMRV in these diseases we have identified genes that are induced in response to XMRV infection. METHODS Prostate cancer cell line DU145 was infected for 8, 24, 48 and 120h with XMRV. A comparison to uninfected DU145 cells cultured for the same periods of time served as controls. A population of total RNA was isolated using Qiagen RNeasy Mini Kit followed by digestion of DNA with DNAse treatment. XMRV infections at the different time points were monitored using real-time RT-PCR for env XMRV RNA. The RNA samples were analyzed for gene expression using Sentrix humanRef-8 v3 expression bead chips from Illumina (Cleveland Clinic Genomics Core). To verify the results obtained by the array experiment, we determined induction of a subset of the regulated genes. Total RNA was reverse transcribed to cDNA using iScript Select cDNA Synthesis Kit from Bio-Rad (random primers method). Induction of selected genes by XMRV infection was verified by qPCR (Relative Quantification) from the cDNA pool using SYBR Green master mix. Fold-induction at each time point for the individual mRNAs was determined. In addition, pathway predictions were determined using Ingenuity Systems (content version 3002) software for genes induced by more than 2-fold following XMRV infection. RESULTS In gene expression profiling, we observed maximal gene induction between 24 and 48 h post-infection. For example, the pro-inflammatory cytokine IL8 gene, a potential contributing factor to androgen independent growth of late-stage prostate cancer, was consistently induced by XMRV infection by up to 6-fold. Of the XMRV induced genes, pathway analysis indicated 10 genes are implicated in cell morphology, 11 genes in cellular development, 12 genes in cell-to-cell signaling and interaction, 11 genes in cellular movement and 13 genes in cellular growth and proliferation. CONCLUSIONS The chemokine IL-8 is one of the most highly induced genes in response to XMRV infection of prostate cancer cell line DU145. XMRV induction of the 30 host genes identified in this study suggests a profound effect of the virus on fundamental cellular physiology and inflammation. These findings could be relevant to the possible pathogenic effects on XMRV in prostate cancer. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e113 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Michael Lee Cleveland, OH More articles by this author Elona Gusho Cleveland, OH More articles by this author Jaydip Das Gupta Cleveland, OH More articles by this author Eric Klein Cleveland, OH More articles by this author Robert Silverman Cleveland, OH More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Background Xenotropic Murine Leukemia Virus-related Virus (XMRV) is a human gammaretrovirus recently identified in prostate cancer tissue and in lymphocytes of patients with chronic fatigue syndrome. To establish the etiologic role of XMRV infection in human disease requires large scale epidemiologic studies. Development of assays to detect XMRV-specific antibodies would greatly facilitate such studies. However, the nature and kinetics of the antibody response to XMRV infection have yet to be determined. Results Three rhesus macaques were infected with XMRV to determine the dynamics of the antibody responses elicited by infection with XMRV. All macaques developed antibodies to XMRV during the second week of infection, and the predominant responses were to the envelope protein gp70, transmembrane protein p15E, and capsid protein p30. In general, antibody responses to gp70 and p15E appeared early with higher titers than to p30, especially in the early period of seroconversion. Antibodies to gp70, p15E and p30 persisted to 158 days and were substantially boosted by re-infection, thus, were identified as useful serologic markers. Three high-throughput prototype assays were developed using recombinant proteins to detect antibodies to these viral proteins. Both gp70 and p15E prototype assays demonstrated 100% sensitivity by detecting all Western blot (WB) positive serial bleeds from the XMRV-infected macaques and good specificity (99.5-99.9%) with blood donors. Seroconversion sensitivity and specificity of the p30 prototype assay were 92% and 99.4% respectively. Conclusions This study provides the first demonstration of seroconversion patterns elicited by XMRV infection. The nature and kinetics of antibody responses to XMRV in primates were fully characterized. Moreover, key serologic markers useful for detection of XMRV infection were identified. Three prototype immunoassays were developed to detect XMRV-specific antibodies. These assays demonstrated good sensitivity and specificity; thus, they will facilitate large scale epidemiologic studies of XMRV infection in humans.

The xenotropic murine leukemia virus-related virus (XMRV) has recently been detected in prostate cancer tissues and may play a role in tumorigenesis. It is currently unclear how this virus is transmitted and which factors promote its spread in the prostate. We show that amyloidogenic fragments known as semen-derived enhancer of virus infection (SEVI) originating from prostatic acid phosphatase greatly increase XMRV infections of primary prostatic epithelial and stromal cells. Hybrid simian/human immunodeficiency chimeric virus particles pseudotyped with XMRV envelope protein were used to demonstrate that the enhancing effect of SEVI, or of human semen itself, was at the level of viral attachment and entry. SEVI enhanced XMRV infectivity but did not bypass the requirement for the xenotropic and polytropic retrovirus receptor 1. Furthermore, XMRV RNA was detected in prostatic secretions of some men with prostate cancer. The fact that the precursor of SEVI is produced in abundance by the prostate indicates that XMRV replication occurs in an environment that provides a natural enhancer of viral infection, and this may play a role in the spread of this virus in the human population.

Xenotropic murine leukemia virus-related virus (XMRV) is a new human gammaretrovirus identified in prostate cancer tissue from patients homozygous for a reduced-activity variant of the antiviral enzyme RNase L. Neither a casual relationship between XMRV infection and prostate cancer nor a mechanism of tumorigenesis has been established. To determine the integration site preferences of XMRV and the potential risk of proviral insertional mutagenesis, we carried out a genome-wide analysis of viral integration sites in the prostate cell line DU145 after an acute XMRV infection and compared the integration site pattern of XMRV with those found for murine leukemia virus and two human retroviruses, human immunodeficiency virus type 1 and human T-cell leukemia virus type 1. Among all retroviruses analyzed, XMRV has the strongest preference for transcription start sites, CpG islands, DNase-hypersensitive sites, and gene-dense regions; all are features frequently associated with structurally open transcription regulatory regions of a chromosome. Analyses of XMRV integration sites in tissues from prostate cancer patients found a similar preference for the aforementioned chromosomal features. Additionally, XMRV integration sites in cancer tissues were associated with cancer breakpoints, common fragile sites, microRNA, and cancer-related genes, suggesting a selection process that favors certain chromosomal integration sites. In both acutely infected cells and cancer tissues, no common integration site was detected within or near proto-oncogenes or tumor suppressor genes. These results are consistent with a model in which XMRV may contribute to tumorigenicity via a paracrine mechanism.

RNase L, a principal mediator of innate immunity to viral infections in higher vertebrates, is required for a complete IFN antiviral response against certain RNA stranded viruses. dsRNA produced during viral infections activates IFN-inducible synthetases that produce 5'-phosphorylated, 2',5'-oligoadenylates (2-5A) from ATP. 2-5A activates RNase L in a wide range of different mammalian cell types, thus blocking viral replication. However, 2-5A has unfavorable pharmacologic properties; it is rapidly degraded, does not transit cell membranes, and leads to apoptosis. To obtain activators of RNase L with improved drug-like properties, high-throughput screening was performed on chemical libraries by using fluorescence resonance energy transfer. Seven compounds were obtained that activated RNase L at micromolar concentrations, and structure-activity relationship studies resulted in identification of an additional four active compounds. Two lead compounds were shown to have a similar mechanistic path toward RNase L activation as the natural activator 2-5A. The compounds bound to the 2-5A-binding domain of RNase L (as determined by surface plasmon resonance and confirmed by computational docking), and the compounds induced RNase L dimerization and activation. Interestingly, the low-molecular-weight activators of RNase L had broad-spectrum antiviral activity against diverse types of RNA viruses, including the human pathogen human parainfluenza virus type 3, yet these compounds by themselves were not cytotoxic at the effective concentrations. Therefore, these RNase L activators are prototypes for a previously uncharacterized class of broad-spectrum antiviral agents.