Characterization, Mapping, and Distribution of the Two XMRV Parental Proviruses

Abstract

Xenotropic murine leukemia virus-related virus (XMRV) was previously reported to be associated with human prostate cancer and chronic fatigue syndrome. Our groups recently showed that XMRV was created through recombination between two endogenous murine retroviruses, PreXMRV-1 and PreXMRV-2, during the passaging of a prostate tumor xenograft in nude mice. Here, multiple approaches that led to the identification of PreXMRV-2, as well as the distribution of both parental proviruses among different mouse species, are described. The chromosomal loci of both proviruses were determined in the mouse genome, and integration site information was used to analyze the distribution of both proviruses in 48 laboratory mouse strains and 46 wild-derived strains. The strain distributions of PreXMRV-1 and PreXMRV-2 are quite different, the former being found predominantly in Asian mice and the latter in European mice, making it unlikely that the two XMRV ancestors could have recombined independently in the wild to generate an infectious virus. XMRV was not present in any of the mouse strains tested, and among the wild-derived mouse strains analyzed, not a single mouse carried both parental proviruses. Interestingly, PreXMRV-1 and PreXMRV-2 were found together in three laboratory strains, Hsd nude, NU/NU, and C57BR/cd, consistent with previous data that the recombination event that led to the generation of XMRV could have occurred only in the laboratory. The three laboratory strains carried the Xpr1 n receptor variant nonpermissive to XMRV and xenotropic murine leukemia virus (X-MLV) infection, suggesting that the xenografted human tumor cells were required for the resulting XMRV recombinant to infect and propagate.

Broad-scale phylogenomics provides insights into retrovirus–host evolution

“…Historically, gammaretroviruses have attracted significant attention due to their occurrence in several vertebrates being linked with disease symptoms such as malignancies, immunosuppression, and neurological disorders (6). Interest has been stimulated further by concerns regarding the possibility of cross-species transmission (7,8), particularly to humans via xenotransplantation (9) and by subsequently disputed hypotheses that a novel gammaretrovirus related to MLV was the causative agent of human disease (10). The potential of gammaretroviruses to switch host species is indicated both by close phylogenetic relationship between viruses in distantly related host taxa (7) and by experimental cross-species infections performed in vitro and in vivo (11)(12)(13).…”

Generation of Multiple Replication-Competent Retroviruses through Recombination between PreXMRV-1 and PreXMRV-2

“…Previously, it was determined by our groups that the Hsd nude mouse population (which is maintained as an outbred stock) harbors integrated copies of PreXMRV-1 and/or PreXMRV-2, with 53% of the mice being positive for both PreXMRV-1 and PreXMRV-2 (22). To determine the levels of expression of these proviruses in mouse tissues, total RNA was extracted from the blood and spleen of three Hsd nude mice positive for both proviruses, three Hsd nude mice positive for PreXMRV-2 only, and two BALB/cAnNHsd mice negative for both proviruses.…”

“…Construction of plasmid clone PreXMRV-1 was previously described (18). For the construction of plasmid clone PreXMRV-2, two overlapping halves of the provirus were amplified using flanking primers (C12-1F, C12-4R [22]) and provirus primers (M19F [5=-TGGCCTTACT GAAAGCTCTCTTCC-3=], 4438R [5=-TACTGAGTCTCTTGGGCAGG A-3=]). The two halves were digested by the SalI restriction enzyme, and the full-length provirus was cloned into pCR-Blunt (Invitrogen).…”

“…Total DNA from mouse tail snips from 10 Hsd nude mice were prescreened for the presence of PreXMRV-1 and PreXMRV-2 using PCR primer pairs XF6 and C3-4R for PreXMRV-1 and PCR primer pairs C12_1f and 129_1r for PreXMRV-2, as previously described (22). Three mice that were positive for PreXMRV-2 only and 3 mice that were positive for both PreXMRV-1 and PreXMRV-2 were then sacrificed, and total RNA was extracted from the blood and spleens of these 6 animals, as well as two negative-control mice (BALB/cAnNHsd) obtained from Harlan Laboratories.…”

“…These findings have been refuted by numerous independent laboratories, debunking any disease association with XMRV (18)(19)(20)(21). We previously reported that XMRV was a laboratory-derived virus which resulted from recombination between two endogenous proviruses, PreXMRV-1 and PreXMRV-2 (18,22), present in Harlan Sprague-Dawley (Hsd) nude mice, in which a human prostate tumor (CWR22) was repeatedly passaged as a xenograft. During this process, progeny of these two proviruses recombined to form a replication-competent xenotropic MLV that was able to spread throughout the human tumor tissue present in the mouse.…”