The prostate cancer-associated human retrovirus XMRV lacks direct transforming activity but can induce low rates of transformation in cultured cells

Abstract

The human retrovirus XMRV (xenotropic murine leukemia virus-related virus) is associated with prostate cancer, but a causal relationship has not been established. Here, we have used cultured fibroblast and epithelial cell lines to test the hypothesis that XMRV might have direct transforming activity but found only rare transformation events, suggestive of indirect transformation, even when the target cells expressed the human Xpr1 cell entry receptor for XMRV. Characterization of cells from three transformed foci showed that all were infected with and produced XMRV, and one produced a highly active transforming virus, presumably generated by recombination between XMRV and host cell nucleic acids. Given the sequence similarity of XMRV to mink cell focus-forming (MCF) viruses and the enhanced leukemogenic activity of the latter, we tested XMRV for related MCF-like cytopathic activities in cultured mink cells but found none. These results indicate that XMRV has no direct transforming activity but can activate endogenous oncogenes, resulting in cell transformation. As part of these experiments, we show that XMRV can infect and be produced at a high titer from human HT-1080 fibrosarcoma cells that express TRIM5alpha (Ref1), showing that XMRV is resistant to TRIM5alpha restriction. In addition, XMRV poorly infects NIH 3T3 cells expressing human Xpr1 but relatively efficiently infects BALB 3T3 cells expressing human Xpr1, showing that XMRV is a B-tropic virus and that its infectivity is regulated by the Fv1 mouse locus.

FIG. 1.

Morphology of transformed foci in 208F fibroblasts. 208F and 208F/LhXpr1SN cells seeded the day before at 1 × 10⁵ to 2 × 10⁵ cells per 6-cm dish were infected with XMRV+LAPSN virus (produced by HTX/LAPSN cells infected with virus from 22Rv1 cells) (A, D, E), were transfected with the pSX2Jenv plasmid by using the calcium phosphate method (B, C), or were treated with culture medium only (F). The cells were trypsinized and replated in multiple 6-cm dishes 1 day (A, D to F) or 10 days (B, C) after treatment. Areas with (A to D) and without (E and F) foci are shown. Cell layers were photographed at the indicated times after replating. d, days.

FIG. 2.

Morphology of transformed foci of 208F cells and purification of the transformed cells. Panels A to C show transformed foci at 27 days (focus 1) or 15 days (foci 2 and 3) after plating of XMRV-infected 208F cells (see Table 4 for infection details). Panel D shows the mixed population of transformed and nontransformed cells 1 week after plating of cells from focus 1 that were obtained by using a cloning ring. Panel E shows two colonies of cells produced by plating low numbers of mixed cells from focus 3. The left colony is clearly transformed and resembles the cells in the original focus (C), while the right colony appears to be a colony of untransformed parental 208F cells. Panel F shows a focus of transformed cells (lower-left part of picture) induced by infection of 208F cells with virus from focus 3 cells.