Evaluation of Cellular Determinants Required for In Vitro Xenotropic Murine Leukemia Virus-Related Virus Entry into Human Prostate Cancer and Noncancerous Cells

The newly identified retrovirus—the xenotropic murine leukemia virus-related virus (XMRV)—has recently been shown to be strongly associated with familial prostate cancer in humans (A. Urisman et al., PLoS Pathog. 2:e25, 2006). 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 (R. Schlaberg et al., Proc. Natl. Acad. Sci. U. S. A. 106:16351-16356, 2009). To help elucidate 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 cell lines of the prostate (i.e., epithelial and stromal smooth muscle cells). We also show that certain amino acid residues located within the predicted extracellular loop (ECL3 and ECL4) sequences of Xpr1 are required for efficient XMRV entry. Although we found strong evidence to support XMRV infection of prostatic fibroblast cell lines 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. Collectively, our data reveal important cellular determinants required for XMRV entry into different human prostate cells in vitro, which may provide important insights into the possible role of XMRV as an etiologic agent in human prostate cancer.Prostate cancer is the most common male malignancy in Western countries and the second most common cause of cancer-related deaths in males worldwide (15, 24). The known risk factors for prostate cancer are hormones (i.e., androgens), diet, sex, and race, as well as environmental and genetic factors (27). A recent study suggests that susceptibility to prostate cancer can be influenced by the genetic variations associated with an antagonistic coevolution, which occurs between a specific host locus (RNASEL), known to be involved in antiviral innate immune defense, and a viral pathogen (38). Indeed, several epidemiologic studies have supported the involvement of the RNASEL gene in the prostate cancer etiology (4, 5, 30, 31), whereas other studies do not (9, 22, 34, 43). Some studies have reported that individuals with a single mutated copy of the RNASEL gene have a 50% increased risk for prostate cancer, whereas those with homozygous mutant RNASEL alleles have a 2-fold-increased risk of prostate cancer (5).The RNASEL gene encodes for the RNase L protein, a constitutively expressed latent endoribonuclease, which mediates the interferon-inducible 2-5A system against viral and/or cellular double-stranded RNAs (8, 16, 20, 23, 49, 50). The RNase L “Q” variant allele (R462Q) shows a 3-fold decrease in catalytic activity compared to the wild-type enzyme (5, 44). The possible association of mutant RNASEL alleles with human prostate cancers suggests an enhanced susceptibility of prostate tissues to a viral agent. This hypothesis has led to the recent identification of a new human retrovirus, xenotropic murine leukemia virus (MuLV)-related virus (XMRV), in 40% of prostate cancer patients with the QQ variant alleles of RNASEL compared to 1.5% among heterozygous (RQ) and wild-type (RR) RNASEL carriers (41). XMRV virus infection appears to be susceptible to inhibition by interferon and its downstream effector RNase L protein (7). However, a recent study has provided some evidence to show that XMRV infection is independent of the RNASEL genotype (34), suggesting that population differences and/or other environmental or genetic factors may influence the impact of RNASEL on prostate cancer development.The XMRV genome is 8,185 nucleotides in length and shares up to 95% overall nucleotide sequence identity with known xenotropic MuLVs (41). One receptor for xenotropic MuLVs is Xpr1, a 696-amino-acid protein with multiple transmembrane-spanning domains (2). Expression of this protein in Chinese hamster ovary (CHO) cells that are not known to express Xpr1 endogenously confers an enhanced susceptibility of these cells to xenotropic MuLV infection (2). Infection of hamster and mouse cells with XMRV-like virus that is derived from a prostate cancer cell line (22Rv1) also requires Xpr1 as a receptor (18). Earlier studies have demonstrated the importance of certain residues located within the putative third and fourth extracellular loops (ECL3 and ECL4) of Mus dunni''s Xpr1 in conferring infection by xenotropic MuLVs (25). Furthermore, it has been shown that the specific and common receptor determinants for xenotropic and polytropic murine retroviruses are simultaneously present in discrete domains of a single Xpr1 protein (42). In the present study, we characterized for the first time the important molecular determinants on Xpr1 required for XMRV infection and investigated the role of RNase L in restricting XMRV infection of various human prostate cancer and noncancerous cell lines.