The cell-cell fusion assay was performed as described previously [19, 45] . Briefly, effector 293T/GFP cells were transfected with plasmid DNA encoding XMRV Env or CT truncation mutants using Lipofectamine 2000 (Invitrogen). Twenty-four hours later, cells were washed with PBS and detached using PBS containing 5 mM EDTA. Target 293, 293/XPR1, CHO, or CHO/XPR1 cells were detached using PBS-5 mM EDTA and labeled with 3.5 mM CMTMR in serum-free media for 30 min at 37uC, washed, incubated for an additional 30 min at 37uC in fresh media and washed 3 times with media. Effector cells and target cells were cocultured on 24-well plates for the indicated time periods. Cell-cell fusion was measured by flow cytometry using FACSCalibur (BD Bioscience, Missauga, Canada). The surface expression of XMRV Env in the 293T/GFP cells was measured by flow cytometry using the anti-FLAG antibody and anti-mouse IgG coupled to PE.