We recently reported identification of a previously undescribed gammaretrovirus genome, xenotropic murine leukemia virusrelated virus (XMRV), in prostate cancer tissue from patients homozygous for a reduced activity variant of the antiviral enzyme RNase L. Here we constructed a full-length XMRV genome from prostate tissue RNA and showed that the molecular viral clone is replication-competent. XMRV replication in the prostate cancer cell line DU145 was sensitive to inhibition by IFN-. However, LNCaP prostate cancer cells, which are deficient in JAK1 and RNase L, were resistant to the effects of IFN- against XMRV. Furthermore, DU145 cells rendered deficient in RNase L with siRNA were partially resistant to IFN inhibition of XMRV. Expression in hamster cells of the xenotropic and polytropic retrovirus receptor 1 allowed these cells to be infected by XMRV. XMRV provirus integration sites were mapped in DNA isolated from human prostate tumor tissue to genes for two transcription factors (NFATc3 and CREB5) and to a gene encoding a suppressor of androgen receptor transactivation (APPBP2/PAT1/ARA67). Our studies demonstrate that XMRV is a virus that has infected humans and is susceptible to inhibition by IFN and its downstream effector, RNase L.cancer ͉ RNase L ͉ xenotropic murine leukemia virus-related virus A diverse range of mammalian species are susceptible to infections by viruses from the gammaretrovirus genus of Retroviridae (1). Examples of these simple viruses whose genomes include gag, pro, pol, and env genes only are murine leukemia virus (MLV), feline leukemia virus, koala retrovirus, and gibbon ape leukemia virus. These viruses are responsible for leukemogenesis and other diseases in their respective host species (1-3). However, until recently evidence of authentic infections of humans by gammaretroviruses was lacking. We reported in 2006 identification of viral genomes for a previously undescribed gammaretrovirus, termed xenotropic MLV-related virus (XMRV), in a subset of men with prostate cancer (4). The discovery of XMRV followed investigations of the role of the antiviral enzyme RNase L in hereditary prostate cancer, a disease in which tumors arise in three or more first-degree relatives (5). The human RNase L gene (RNASEL) was initially proposed as a candidate for the hereditary prostate cancer 1 (HPC1) gene based on a positional cloning/candidate gene method (6).RNase L is a regulated endoribonuclease for single-stranded RNA that functions in the IFN antiviral response (7,8). IFN treatment of cells induces a family of 2Ј-5Ј oligoadenylate synthetases that produce 5Ј-phosphorylated, 2Ј-5Ј-linked oligoadenylates (2-5A) from ATP in response to stimulation by viral dsRNA. 2-5A activates the preexisting, latent, and ubiquitous RNase L, resulting in degradation of viral and cellular RNA. Sustained activation of RNase L leads to apoptosis, a function consistent with a role in the suppression of tumor growth (9). Although mice lacking RNase L do not spontaneously develop tumors at higher rates than wild-typ...