Abstract: The association of retroviruses with human diseases has long been studied. As an essential step during the life cycle, retrovirus integrates the DNA copy of its RNA genome into the host chromosome. Target site selection of retrovirus integration is important for transcription of the integrated provirus. It is also important for the host cell because integration near or within a required gene may alter the normal biological activity and transform the host cell into a pathologic state. Retrovirus integration into the host genome can occur at many sites throughout the chromosomes but the site selection is not completely random. Availability of the human genome sequences has enabled large-scale sequence-based surveys of retrovirus integration sites. Until now, the integration sites of nine retroviruses, including human immunodeficiency virus type 1(HIV-1), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV), murine leukemia virus (MLV), foamy virus (FV), Porcine endogenous retrovirus (PERV), Human T-lymphotropic virus (HTLV) and avian sarcoma and leucosis virus (ASLV) have been published, and much of attention has been drawn to understand biological significances of retrovirus integration sites. Genome-wide analysis of retroviral integration sites requires the collection and analysis of a large library of integration site sequences. Such analyses are time consuming and labor intensive using conventional means. We have developed an efficient and high throughput method (Int-tag assay) to clone and sequence a large number of independent integration sites without selection or bias. The new assay involves the use of a modified HIV-1 (HIV-Mme) containing a type IIS restriction site, Mme I, at the U5 end of LTR. By utilizing Mme I enzyme, the assay produces a 46-47 by DNA fragment (Int-tag) consisting of 19 or 20 bp of cellular DNA sequences flanked by 25 bp of viral end and 2 by linker sequence. Ligation mediated serial analysis enables high throughput reading of Int-tag sequences. Int-tag assay along with control assays validated 19-20 by information is useful for integration site analysis. We have conducted integration site analysis for a recently identified Xenotropic murine leukemia virus-related virus (XMRV). XMRV is a previously undescribed gammaretrovirus that infects human cells. A strong correlation of XMRV infection with prostate cancer formation in patients who are homozygous for a RNase L variant has been shown. In this study, 472 XMRV integration sites in a human prostate cell line DU 145 were sequenced and compared to other retroviruses. Integration site selection of XMRV shares a largely conserved pattern with that of MLV and FV, but it also shows significantly higher preference for transcription start sites compared to the other retroviruses. The result suggests that XMRV integration into the genome may have strong genotoxicity for the host cell. Viruses have long been studied for the association with cancer. Recent identification of XMRV infection in prostate tissue from patients with variants in RNASEL, an innate antiviral agent and also one of hereditary prostate cancer genes, suggested a viral etiology in these patients. In this study, 16 XMRV integration sites in prostate cancer tissues from 8 patients who are homozygous for the RNASEL variant R462Q were sequenced and analyzed. The chromosomal regions hosting the 16 integration sites have a high correlation with prostate cancer development. Gene ontology study and cancer database search for the genes nearest to the 16 sites also showed a strong correlation with mechanisms involved in cancer formation. For comparison, genes near XMRV integration sites in acute infection and human cancer genes in Affymetrix G110 array were used. The results suggest a potential causative role of XMRV integration in prostate cancer formation.