XMRV is a recently discovered gammaretrovirus, found using RNA-based microarray techniques in tissue samples from prostate cancer patients [10
]. XMRV was detected predominantly in patients who are homozygous for the QQ allele at R462Q in the RNaseL gene, which results in a reduced RNaseL activity and therefore in a diminished IFN-based antiviral defense. Later studies showed XMRV to be an infectious virus for human prostate-derived cells and to be sensitive to RNaseL-mediated inhibition of replication by IFN-β [9
]. The question of whether carcinogenic transformation renders the prostate epithelia cells susceptible to XMRV infection as a bystander virus or whether XMRV is itself a prostate cancer causing agent has not yet been addressed. It was very recently shown that XMRV could be detected in 22Rv1 prostate carcinoma cells originally derived from a primary prostatic carcinoma [17
]. This observation further highlights the need to clarify the participation of XMRV in the etiology of human prostate carcinomas.
As known for many years in other cancers, e.g. HPV in cervical carcinoma or other cancers (reviewed by zur Hausen, 2009 [18
]), infectious agents causing inflammatory (precancerous) lesions are suspected to be involved in the pathogenesis of prostate carcinoma [19
]. An increased susceptibility of prostate epithelia cells to infection with RNA-viruses as a result of the impaired function of RNaseL, resulting in proliferative inflammatory atrophy (PIA), could be an intriguing scenario. These focal areas of epithelial atrophy are presumed to be precursors of prostatic intraepithelial neoplasia and prostate cancer [21
]. A small number of other studies during the last ten years attempting to demonstrate a role for viral infections in the development of PCa have yielded rather inconclusive data [22
If a real correlation between viral infection and prostate cancer exists, new therapeutic or even prophylactic treatments against the development of PCa could be developed by targeting, for example, viral antigens. In this respect, a recent observation that radiolabelled therapeutic monoclonal antibodies specific for HPV or HBV proteins can inhibit subcutaneous tumor development in vivo
by cells expressing these antigens [27
] is of particular interest.
In the present study, we report the testing of 589 DNA and RNA samples from sporadic prostate cancer patients for the RNaseL genotype and for XMRV sequences. Although 76 of our samples (12.9%) displayed the "susceptibility" QQ genotype, consistent with the frequency given in the literature, no XMRV-specific sequences were detected in either the RNA or the DNA from the prostate tumor samples. Given the ratio of approximately 40% positive cases harboring the QQ genotype in the study population of Urisman et al
], one would have expected at least 30 XMRV positive specimens amongst our 76 RNaseL QQ-allele samples.
At least two other studies have looked for XMRV at the nucleic acid level, albeit with a much smaller sample groups. Fischer and coworkers [11
] studied material from 105 German patients with sporadic prostate cancer and found only one individual positive for XMRV by nested RT-PCR, but this individual did not display the QQ RNaseL genotype. Another study carried out in Ireland investigated 139 PCa patients. In 7 QQ patients and two heterozygous RQ samples, no XMRV sequences were detected [12
It should be mentioned that this study cannot completely rule out the possibility of an infection with another gammaretrovirus in these patients. The design of our PCR approach was done in such a way that one primer pair (In-For/In-Rev) binds to conserved regions, allowing amplification of various MLV types including AKV MLV (J01998), MLV DG-75 (AF221065), MoMuLV (NC_001501), MTCR (NC_001702), MCF 1233 MLV (U13766), and Rauscher MuLV (NC_001819). In this PCR setup a specific signal was obtained with the mouse tail DNA as template, indicating that endogenous MLVs were detected. As additional controls we tested the cell lines 22Rv1 (XMRV positive [17
]) and DU145 (XMRV negative [9
]). As expected, 22Rv1 was found to be strongly positive for RNA transcripts and for provirus (with In-For/Deletion-Rev primers), while DU145 was negative in both PCR approaches (data not shown).
We also tested 146 sera samples for XMRV antibodies and found none of them to be positive in ELISA or Western blot analyses. The recombinant XMRV proteins that were used reacted positively with sera from immunized mice. As XMRV is closely related to other murine leukemia viruses and therefore immunogenic in mammalian hosts [28
], an infection which allows the virus to spread to the stroma cells should induce a humoral immune response. The analysis of sera from prostate cancer patients for antibodies could therefore offer a rapid and valid screening method to investigate the involvement of a virus. Obviously the determination of sensitivity and specificity of these ELISA assays is to a certain degree limited, due to the lack of a human anti-XMRV positive control antibody. Nevertheless, the mouse sera were used to demonstrate the suitability of the recombinant antigens as ELISA antigens, even though the titration cannot be used to determine the amount of antibodies in the human sera samples. The failure to detect XMRV proviruses or transcripts in the 30 cases where DNA, RNA and sera samples were all available, is consistent with the negative ELISA results. It is theoretically possible that the tumor environment itself compromises the immune system and inhibits the antibody response to the tumor-associated viral antigens. This seems unlikely since animal studies have demonstrated that tumor diseases do not dramatically suppress systemic immunity [29
]. There was a certain degree of background reactivity to the recombinant Gag proteins, as was also seen in an ELISA using a lysate of ultracentrifuge-concentrated virus as antigen (data not shown). Difficulties with background signals in testing human sera for reactivity to MLV-derived antigens are well known when using whole virion particles as antigen [30
], but this also occurs to a lesser extent when using recombinant proteins [28
]. In general, there was a higher background reactivity against Gag in our 146 PCa and healthy control sera tested; and one serum reacted strongly to the pr65 protein. Upon further testing in Western blot and immunfluorescence assay, this serum showed no specificity for XMRV. It might be possible that antibodies directed against the transmembrane protein p15E were missed due to our choice of the gp70 and the pr65 antigen as targets. In other human retrovirus infections, HIV and HTLV antibodies against this region are detectable. Therefore, it should also be mentioned that before the serological assays using XMRV proteins were established all serum samples were screened for cross-reactivity with recombinant gp70, p15E and p27 [31
] of another gammaretrovirus, the porcine endogenous retrovirus (PERV). All sera were found to be negative for any of these targets despite the obvious sequence homology of XMRV and PERV in the ectodomain of p15E and certain conserved regions in gp70 and p27. Regarding this point, it is also of interest that Furuta et al
], recently reported the detection by Western blot of antibodies specific for the XMRV Gag protein in blood bank samples from prostate cancer patients and healthy donors, but no Env-specific antibodies.