Expression of XMRV Env, Gag and VLP.

<p>(A) Western blot analysis of XMRV gag expression. HeLa cells were infected with Ad5-XMRV (10 MOI) for 24 h and then whole cell lysate (Lane 1) and cell culture media concentrated 100-fold by centrifugation through a 20% sucrose cushion (Lane 2) were subjected to 10% SDS-PAGE and then transferred to PVDF. The blots were probed with anti-Gag mAb R187 and HRP-conjugated goat anti-rat immunoglobulin G antiserum (Southern Biotechnology Associates, Inc.). The masses (kDa) of the molecular weight standards (Std) are shown on the left. The arrows (←) indicate the positions of the Gag precursor at ∼65 kDa (top arrow) and a cleaved, lower molecular mass Gag protein (bottom arrow). (B) Detection of XMRV envelope expression by flow cytometric (left) and Western blot (right) analyses. For flow cytometry, HeLa cells infected as in (A) were stained with mAb 83A25 and fluorescein isothiocyanate-conjugated goat anti-rat immunoglobulin G antiserum. For Western blot analysis, VLP produced by those cells were purified from culture media and probed with mAb 83A25. MAb 83A25 recognizes an epitope located near the carboxyl terminus of Env that common for many MuLVs. (C) Electron microscopy showing VLP production in HeLa cells after 48 hours of infection with Ad5-XMRV (Panels I and II). An infectious XMRV virus is shown budding (arrows) from Du145-C7 cells, a prostate cancer cell line that constitutively produces XMRV (Panels III and IV). The similarities in morphology and size between the VLP and live XMRV particles are in the insets of Panels II and IV.