In-Depth Investigation of Archival and Prospectively Collected Samples Reveals No Evidence for XMRV Infection in Prostate Cancer

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.     

Identification of XMRV infection-associated microRNAs in four cell types in culture

Introduction

XMRV is a gammaretrovirus that was thought to be associated with prostate cancer (PC) and chronic fatigue syndrome (CFS) in humans until recently. The virus is culturable in various cells of human origin like the lymphocytes, NK cells, neuronal cells, and prostate cell lines. MicroRNAs (miRNA), which regulate gene expression, were so far not identified in cells infected with XMRV in culture.

Methods

Two prostate cell lines (LNCaP and DU145) and two primary cells, Peripheral Blood Lymphocytes [PBL] and Monocyte-derived Macrophages [MDM] were infected with XMRV. Total mRNA was extracted from mock- and virus-infected cells at 6, 24 and 48 hours post infection and evaluated for microRNA profile in a microarray.

Results

MicroRNA expression profiles of XMRV-infected continuous prostate cancer cell lines differ from that of virus-infected primary cells (PBL and MDMs). miR-193a-3p and miRPlus-E1245 observed to be specific to XMRV infection in all 4 cell types. While miR-193a-3p levels were down regulated miRPlus-E1245 on the other hand exhibited varied expression profile between the 4 cell types.

Discussion

The present study clearly demonstrates that cellular microRNAs are expressed during XMRV infection of human cells and this is the first report demonstrating the regulation of miR193a-3p and miRPlus-E1245 during XMRV infection in four different human cell types.     

Long-term infection and vertical transmission of a gammaretrovirus in a foreign host species

Increasing evidence has indicated natural transspecies transmission of gammaretroviruses; however, viral-host interactions after initial xeno-exposure remain poorly understood. Potential association of xenotropic murine leukemia virus-related virus (XMRV) in patients with prostate cancer and chronic fatigue syndrome has attracted broad interests in this topic. Although recent studies have indicated that XMRV is unlikely a human pathogen, further understanding of XMRV xenoinfection would allow in vivo modeling of the initial steps of gammaretroviral interspecies transmission, evolution and dissemination in a new host population. In this study, we monitored the long-term consequences of XMRV infection and its possible vertical transmission in a permissive foreign host, wild-derived Mus pahari mice. One year post-infection, XMRV-infected mice showed no notable pathological changes, while proviral DNA was detected in three out of eight mice. XMRV-infected mice remained seropositive throughout the study although the levels of gp70 Env- and p30 capsid-specific antibodies gradually decreased. When vertical XMRV transmission was assessed, no viremia, humoral immune responses nor endogenization were observed in nine offspring from infected mothers, yet one offspring was found PCR-positive for XMRV-specific sequences. Amplified viral sequences from the offspring showed several mutations, including one amino acid deletion in the receptor binding domain of Env SU. Our results therefore demonstrate long-term asymptomatic infection, low incidence of vertical transmission and limited evolution of XMRV upon transspecies infection of a permissive new host, Mus pahari.     

Absence of XMRV and closely related viruses in primary prostate cancer tissues used to derive the XMRV-infected cell line 22Rv1

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.     

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis

Xenotropic murine leukemia virus-related virus (XMRV) is a virus generated under artificial conditions by the recombination of 2 murine leukemia virus (MLV) proviruses, PreXMRV-1 and PreXMRV-2, during the in vivo passage of human prostate cancer cells in athymic nude mice. The molecular etiology of XMRV infection has not been characterized and its implication in human prostate cancer progression remains equivocal. As a step toward resolving this issue we developed an in vitro enzymatic assay system to characterize XMRV protease (PR)-mediated cleavage of host-cell proteins. Enzymatically-active XMRV PR protein was synthesized using a wheat-germ cell-free system. By monitoring cleavage activity of XMRV PR by AlphaScreen and 2-color immunoblot analyses, we revealed that the catalytic activity of XMRV PR is selectively blocked by the HIV PR inhibitor, Amprenavir, and identified several human tumor suppressor proteins, including PTEN and BAX, to be substrates of XMRV PR. This system may provide an attractive means for analyzing the function of retrovirus proteases and provide a technology platform for drug screening.     

Severe restriction of xenotropic murine leukemia virus-related virus replication and spread in cultured human peripheral blood mononuclear cells

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). We and others have shown that host restriction factors APOBEC3G (A3G) and APOBEC3F (A3F), which are expressed in human PBMCs, inhibit XMRV in transient-transfection assays involving a single cycle of viral replication. However, the recovery of infectious XMRV from human PBMCs suggested that XMRV can replicate in these cells despite the expression of APOBEC3 proteins. To determine whether XMRV can replicate and spread in cultured PBMCs even though it can be inhibited by A3G/A3F, we infected phytohemagglutinin-activated human PBMCs and A3G/A3F-positive and -negative cell lines (CEM and CEM-SS, respectively) with different amounts of XMRV and monitored virus production by using quantitative real-time PCR. We found that XMRV efficiently replicated in CEM-SS cells and viral production increased by >4,000-fold, but there was only a modest increase in viral production from CEM cells (<14-fold) and a decrease in activated PBMCs, indicating little or no replication and spread of XMRV. However, infectious XMRV could be recovered from the infected PBMCs by cocultivation with a canine indicator cell line, and we observed hypermutation of XMRV genomes in PBMCs. Thus, PBMCs can potentially act as a source of infectious XMRV for spread to cells that express low levels of host restriction factors. Overall, these results suggest that hypermutation of XMRV in human PBMCs constitutes one of the blocks to replication and spread of XMRV. Furthermore, hypermutation of XMRV proviruses at GG dinucleotides may be a useful and reliable indicator of human PBMC infection.     

Identification of replication competent murine gammaretroviruses in commonly used prostate cancer cell lines

A newly discovered gammaretrovirus, termed XMRV, was recently reported to be present in the prostate cancer cell line CWR22Rv1. Using a combination of both immunohistochemistry with broadly-reactive murine leukemia virus (MLV) anti-sera and PCR, we determined if additional prostate cancer or other cell lines contain XMRV or MLV-related viruses. Our study included a total of 72 cell lines, which included 58 of the 60 human cancer cell lines used in anticancer drug screens and maintained at the NCI-Frederick (NCI-60). We have identified gammaretroviruses in two additional prostate cancer cell lines: LAPC4 and VCaP, and show that these viruses are replication competent. Viral genome sequencing identified the virus in LAPC4 and VCaP as nearly identical to another known xenotropic MLV, Bxv-1. We also identified a gammaretrovirus in the non-small-cell lung carcinoma cell line EKVX. Prostate cancer cell lines appear to have a propensity for infection with murine gammaretroviruses, and we propose that this may be in part due to cell line establishment by xenograft passage in immunocompromised mice. It is unclear if infection with these viruses is necessary for cell line establishment, or what confounding role they may play in experiments performed with these commonly used lines. Importantly, our results suggest a need for regular screening of cancer cell lines for retroviral "contamination", much like routine mycoplasma testing.     

Absence of XMRV in Peripheral Blood Mononuclear Cells of ARV-Treatment Na?ve HIV-1 Infected and HIV-1/HCV Coinfected Individuals and Blood Donors

Background

Xenotropic murine leukemia virus-related virus (XMRV) has been found in the prostatic tissue of prostate cancer patients and in the blood of chronic fatigue syndrome patients. However, numerous studies have found little to no trace of XMRV in different human cohorts. Based on evidence suggesting common transmission routes between XMRV and HIV-1, HIV-1 infected individuals may represent a high-risk group for XMRV infection and spread.

Methodology/Principal Findings

DNA was isolated from the peripheral blood mononuclear cells (PBMCs) of 179 HIV-1 infected treatment naïve patients, 86 of which were coinfected with HCV, and 54 healthy blood donors. DNA was screened for XMRV provirus with two sensitive, published PCR assays targeting XMRV gag and env and one sensitive, published nested PCR assay targeting env. Detection of XMRV was confirmed by DNA sequencing. One of the 179 HIV-1 infected patients tested positive for gag by non-nested PCR whereas the two other assays did not detect XMRV in any specimen. All healthy blood donors were negative for XMRV proviral sequences. Sera from 23 HIV-1 infected patients (15 HCV⁺) and 12 healthy donors were screened for the presence of XMRV-reactive antibodies by Western blot. Thirteen sera (57%) from HIV-1⁺ patients and 6 sera (50%) from healthy donors showed reactivity to XMRV-infected cell lysate.

Conclusions/Significance

The virtual absence of XMRV in PBMCs suggests that XMRV is not associated with HIV-1 infected or HIV-1/HCV coinfected patients, or blood donors. Although we noted isolated incidents of serum reactivity to XMRV, we are unable to verify the antibodies as XMRV specific.     

Acutely Transforming Retrovirus Expressing Nras Generated from HT-1080 Fibrosarcoma Cells Infected with the Human Retrovirus XMRV

Virus from HT-1080 fibrosarcoma cells infected with the human retrovirus XMRV (xenotropic murine leukemia virus-related virus) can induce rare foci of transformation in rat 208F fibroblasts. Characterization of three such foci revealed that one produced an acutely transforming virus at a high titer. The virus consists of a mutant Nras cDNA from the HT-1080 cells inserted into a retroviral vector (added to the HT-1080 cells as a marker for infection) in place of internal vector sequences. These results show that XMRV can generate acutely transforming viruses at a low rate, as is typical of other replication-competent retroviruses, and reveal the potential for transforming virus contamination of retroviral vectors made from transformed cell lines.XMRV (xenotropic murine leukemia virus-related virus) has been associated with prostate cancer (19, 20) and chronic fatigue syndrome (12), although some researchers fail to detect XMRV in other populations with these diseases (4, 8). XMRV is found integrated into human genomic DNA from prostate cancer samples, indicating that it is indeed a human retrovirus and not a laboratory contaminant (3, 9). Because of the potential role of XMRV in prostate cancer, we previously tested XMRV for transforming activity in fibroblast and epithelial cell lines. Although XMRV is a simple retrovirus that does not carry a host-derived oncogene, there is precedence for transformation by retroviral Env genes (21, 22). However, transfection of XMRV proviral DNA or viral envelope expression vectors into 208F rat fibroblasts did not result in transformation, and infection of most cell types tested with XMRV did not induce transformation (13). In contrast, infection of 208F cells with XMRV did result in rare transformed foci suggestive of oncogene activation by XMRV. Characterization of cells from three transformed foci produced by infection of 208F cells with virus from HTX cells (a pseudodiploid subclone of HT-1080 fibrosarcoma cells [18]) infected with XMRV and the LAPSN retroviral vector (included as a marker for infection) revealed that all produced XMRV and that one produced a highly active transforming virus (13).     

Xpr1 is an atypical G-protein-coupled receptor that mediates xenotropic and polytropic murine retrovirus neurotoxicity

Xenotropic murine leukemia virus-related virus (XMRV) was first identified in human prostate cancer tissue and was later found in a high percentage of humans with chronic fatigue syndrome (CFS). While exploring potential disease mechanisms, we found that XMRV infection induced apoptosis in SY5Y human neuroblastoma cells, suggesting a mechanism for the neuromuscular pathology seen in CFS. Several lines of evidence show that the cell entry receptor for XMRV, Xpr1, mediates this effect, and chemical cross-linking studies show that Xpr1 is associated with the Gβ subunit of the G-protein heterotrimer. The activation of adenylate cyclase rescued the cells from XMRV toxicity, indicating that toxicity resulted from reduced G-protein-mediated cyclic AMP (cAMP) signaling. Some proteins with similarity to Xpr1 are involved in phosphate uptake into cells, but we found no role of Xpr1 in phosphate uptake or its regulation. Our results indicate that Xpr1 is a novel, atypical G-protein-coupled receptor (GPCR) and that xenotropic or polytropic retrovirus binding can disrupt the cAMP-mediated signaling function of Xpr1, leading to the apoptosis of infected cells. We show that this pathway is also responsible for the classic toxicity of the polytropic mink cell focus-forming (MCF) retrovirus in mink cells. Although it now seems clear that the detection of XMRV in humans was the result of sample contamination with a recombinant mouse virus, our findings may have relevance to neurologic disease induced by MCF retroviruses in mice.     

Investigation of xenotropic murine leukemia virus-related virus (XMRV) in human and other cell lines

Xenotropic murine leukemia virus-related virus (XMRV) was discovered in human prostate tumors and later in some chronic fatigue syndrome (CFS) patients. However, subsequent studies have identified various sources of potential contamination with XMRV and other murine leukemia virus (MLV)-related sequences in test samples. Biological and nucleotide sequence analysis indicates that XMRV is distinct from known xenotropic MLVs and has a broad host range and cell tropism including human cells. Therefore, it is prudent to minimize the risk of human exposure to infection by evaluating XMRV contamination in cell lines handled in laboratory research and particularly those used in the manufacture of biological products. Nested DNA PCR assays were optimized for investigating XMRV gag and env sequences in various cell lines, which included MRC-5, Vero, HEK-293, MDCK, HeLa, and A549, that may be used in the development of some vaccines and other cell lines broadly used in research. The sensitivity of the DNA PCR assays was <10 copies in approximately 1.8 x 10⁵ cells equivalent of human DNA. The results indicated the absence of XMRV in the cell lines tested; although in some cases DNA fragments identified as cellular sequences were seen following the first round of PCR amplification with the env primer pair.     

Toll-like receptor (TLR) 3 as a surrogate sensor of retroviral infection in human cells

The toll-like receptor (TLR)-7 has been shown to sense the retroviral infection. However, a surrogate sensor has been implicated. We examined whether retrovirus serves as a TLR3 ligand in human cells by utilizing cell lines LNCaP and PC-3 lacking TLR7, and the xenotropic murine leukemia virus-relamoted virus (XMRV) insensitive to human tripartite motif-containing (TRIM) 5, a newly characterized pattern recognition receptor (PRR). A dominant-negative TLR3 or a chemical inhibitor of TLR3 attenuated the XMRV-induced IP-10/CXCL10 expression, a marker of TLR3 response. These data clearly indicated that retroviral infection exemplified by XMRV activates the TLR3 signal in human cells.     

Early events in retrovirus XMRV infection of the wild-derived mouse Mus pahari

A novel gammaretrovirus, xenotropic murine leukemia virus-related virus (XMRV), has been identified in patients with prostate cancer and in patients with chronic fatigue syndromes. Standard Mus musculus laboratory mice lack a functional XPR1 receptor for XMRV and are therefore not a suitable model for the virus. In contrast, Gairdner's shrew-mice (Mus pahari) do express functional XPR1. To determine whether Mus pahari could serve as a model for XMRV, primary Mus pahari fibroblasts and mice were infected with cell-free XMRV. Infection of cells in vitro resulted in XMRV Gag expression and the production of XMRV virions. After intraperitoneal injection of XMRV into Mus pahari mice, XMRV proviral DNA could be detected in spleen, blood, and brain. Intravenous administration of a green fluorescent protein (GFP) vector pseudotyped with XMRV produced GFP(+) CD4(+) T cells and CD19(+) B cells. Mice mounted adaptive immune responses against XMRV, as evidenced by the production of neutralizing and Env- and Gag-specific antibodies. Prominent G-to-A hypermutations were also found in viral genomes isolated from the spleen, suggesting intracellular restriction of XMRV infection by APOBEC3 in vivo. These data demonstrate infection of Mus pahari by XMRV, potential cell tropism of the virus, and immunological and intracellular restriction of virus infection in vivo. These data support the use of Mus pahari as a model for XMRV pathogenesis and as a platform for vaccine and drug development against this potential human pathogen.     

No detection of XMRV in blood samples and tissue sections from prostate cancer patients in Northern Europe

Background

We recently published the rare detection of xenotropic murine leukemia virus-related virus (XMRV) (1/105) in prostate cancer (PCA) tissue of patients in Northern Europe by PCR. The controversial discussion about the virus being detected in PCA tissue, blood samples from patients suffering from chronic fatigue syndrome (CFS), as well as from a significant number of healthy controls prompted us to deepen our studies about detection of XMRV infection applying different detection methods (PCR, cocultivation and immunohistochemistry [IHC]).

Methodology/Principal Findings

Peripheral blood mononuclear cells (PBMCs) from 92 PCA and 7 healthy controls were isolated, PHA activated and cocultivated with LNCaP cells for up to 8 weeks. Supernatant of these cells was applied to a reporter cell line, DERSE-iGFP. Furthermore, the PBMCs and cocultivated LNCaP cells were tested for the presence of XMRV by PCR as well as Western Blot analysis. While all PCR amplifications and Western Blot analyses were negative for signs of XMRV infection, DERSE-iGFP cells displayed isolated GFP positive cells in three cases. In all three cases XMRV presence could not be confirmed by PCR technology. In addition, we performed XMRV specific IHC on PCA tissue sections. Whole tissue sections (n = 20), as well as tissue microarrays (TMA) including 50 benign prostate hyperplasia (BPH), 50 low grade and 50 high grade PCA sections and TMAs including breast cancer, colon cancer and normal tissues were stained with two XMRV specific antisera. XMRV protein expression was not detected in any cancer sections included. One BPH tissue displayed XMRV specific protein expression in random isolated basal cells.

Conclusion

We were unable to conclusively detect XMRV in the blood from PCA patients or from healthy controls and there is no conclusive evidence of XMRV protein expression in PCA, breast cancer and colon cancer tissue sections tested by IHC staining.     

新型人类逆转录病毒XMRV研究进展

异嗜性鼠白血病病毒相关病毒(xenotropic murine leukemia virus-related virus,XMRV)是迄今发现的第一种可以感染人类的r逆转录病毒。XMRV最初于2006年在RNase L基因缺陷型的前列腺癌组织中首次被鉴定,其序列与鼠科白血病病毒(murine leukemia virus,MLV)十分相似。目前,北美、欧洲和亚洲的多个研究机构在人类前列腺癌和慢性疲劳综合征(chronic fatigue syndrome,CFS)患者中检测到XMRV。但不同研究间结果差异很大,XMRV感染与人类疾病之间的相关性尚不明确。该文综述了目前XMRV的相关研究进展,包括与人类疾病的关系、XMRV的基本特征、病理生理学可能的机制等方面,并就今后研究趋势和注意问题进行了讨论。     

Restricted replication of xenotropic murine leukemia virus-related virus in pigtailed macaques

Although xenotropic murine leukemia virus-related virus (XMRV) has been previously linked to prostate cancer and myalgic encephalomyelitis/chronic fatigue syndrome, recent data indicate that results interpreted as evidence of human XMRV infection reflect laboratory contamination rather than authentic in vivo infection. Nevertheless, XMRV is a retrovirus of undefined pathogenic potential that is able to replicate in human cells. Here we describe a comprehensive analysis of two male pigtailed macaques (Macaca nemestrina) experimentally infected with XMRV. Following intravenous inoculation with >10(10) RNA copy equivalents of XMRV, viral replication was limited and transient, peaking at ≤2,200 viral RNA (vRNA) copies/ml plasma and becoming undetectable by 4 weeks postinfection, though viral DNA (vDNA) in peripheral blood mononuclear cells remained detectable through 119 days of follow-up. Similarly, vRNA was not detectable in lymph nodes by in situ hybridization despite detectable vDNA. Sequencing of cell-associated vDNA revealed extensive G-to-A hypermutation, suggestive of APOBEC-mediated viral restriction. Consistent with limited viral replication, we found transient upregulation of type I interferon responses that returned to baseline by 2 weeks postinfection, no detectable cellular immune responses, and limited or no spread to prostate tissue. Antibody responses, including neutralizing antibodies, however, were detectable by 2 weeks postinfection and maintained throughout the study. Both animals were healthy for the duration of follow-up. These findings indicate that XMRV replication and spread were limited in pigtailed macaques, predominantly by APOBEC-mediated hypermutation. Given that human APOBEC proteins restrict XMRV infection in vitro, human XMRV infection, if it occurred, would be expected to be characterized by similarly limited viral replication and spread.     

Fibrils of Prostatic Acid Phosphatase Fragments Boost Infections with XMRV (Xenotropic Murine Leukemia Virus-Related Virus), a Human Retrovirus Associated with Prostate Cancer

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.Viruses are etiologic agents of various human cancers, including cervical carcinoma (caused by human papillomavirus), Kaposi''s sarcoma (caused by human herpesvirus 8), hepatocellular carcinoma (caused by hepatitis B virus and hepatitis C virus), and adult T-cell leukemia (caused by human T-cell leukemia virus type 1) (6). Genetic and epidemiologic evidence suggests that prostate cancer may also have an infectious etiology, although a causative agent has not been identified (4, 12). The gammaretrovirus xenotropic murine leukemia virus-related virus (XMRV) is a candidate human tumor virus based on its association in human prostate tumors with a reduced-activity variant of the antiviral gene, RNASEL (also known as the hereditary prostate cancer 1 gene or HPC1) (17) and because it is a member of a viral family known to cause leukemias and lymphomas in different mammalian species (8). Interferon, through its effector RNase L, potently inhibits XMRV replication (5). XMRV integration sites in human prostate cancer tissues were mapped to cancer breakpoints, common fragile sites, micro-RNA genes, and cancer-related genes (11). Many of these genes are implicated directly or indirectly in prostate cancer and metabolic pathways that affect prostate cancer, including androgen signaling. XMRV has also been observed in prostate tissue from a nonfamilial prostate cancer patient and in an individual without prostate cancer (7). The possible role of XMRV in prostatic cancer raises questions about its ability to infect the prostate and the route of viral transmission.Recently, it has been shown that fragments of prostatic acid phosphatase (PAP), an abundant nonspecific protein phosphatase produced by the prostate (18) and secreted in semen in large quantities (about 2 mg/ml) (16), form amyloid fibrils that drastically enhance human immunodeficiency virus type 1 (HIV-1) infection (14). The fibrils of PAP_248-286, termed semen-derived enhancer of virus infection (SEVI), enhanced the infectious virus titer by several orders of magnitude by capturing HIV-1 virions and promoting their attachment to target cells. The ability of SEVI to promote the interaction between virions and the cell surface is independent of the viral glycoprotein and hence is not restricted to HIV-1, although subsequent fusion between the viral and cellular membranes still required gp120, CD4, and an appropriate coreceptor (14). A recent study indicates that the positive charges on SEVI (pI = 10.21) promote infectivity by neutralizing negative-charge repulsion between HIV particles and the cell surface (15).Because SEVI originates from the prostate (the organ from which XMRV infection was discovered [17]) and promotes viral attachment in a relatively nonspecific manner, we sought to determine its effect on XMRV infection. Here we demonstrate that XMRV infectivity is greatly enhanced by SEVI or human semen and that XMRV RNA is detectable in expressed prostatic secretions (EPS) from human tumor-bearing prostates.