The finding that syncytia induction can be observed in cells expressing XMRV Env truncation mutants prompted us to further quantitatively measure their membrane fusion activities using a flow cytometry-based cell-cell fusion assay adapted from our previous studies on JSRV [18, 19, 45] . In this assay, the effector 293T/GFP cells were transfected with Env-encoding plasmids, and the target 293 cells were labeled with a red-fluorescent dye, CMTMR. Consistent with the syncytia formation data, the XMRV Env wildtype and CT635 were unable to induce cellcell fusion as evidenced by no fluorescent dye transfer (,1.8%, similar to the No-Env background), whereas truncation at the putative R-peptide cleavage site or further upstream towards the MSD, i.e., CT624 or CT613, induced apparent cell-cell fusion (4,5%) (p,0.05) ( Fig. 4B and 4C ). Again, CT609 showed a relatively low cell-cell fusion activity (,3%), and the tailless CT608 and CT606 mutants were incapable of inducing fusion with background signals (Fig. 4B and 4C ). We also examined the fusion activities of all constructs in 293 cells overexpressing the XPR1 receptor (293/XPR1), but observed only modest increases for CT624, CT613 and CT609 (,10-20%). The titers of XMRV wildtype and mutants in 293/XPR1 cells were approximately 5fold higher than those in the parental 293 cells, and the overexpression of XPR1 in 293/XPR1 cells was confirmed by flow cytometry using the soluble XMRV SU (data not shown). The differential fusion activities of CT624, CT613 and CT609 could be due to their different levels of Env expression on the cell surface or/and intrinsic fusogenicity. To distinguish these possibilities, we transfected effector 293T cells with different amounts of plasmid DNA encoding individual truncated Envs, and determined their cell-cell fusion activities and SU surface expression in parallel. As shown in Figure 5A , the fusion profiles of CT624 and CT613 were almost identical, as evidenced by their similar slopes (,0.095 and ,0.010, respectively, R 2 = 0.97-0.99). In contrast, CT609 exhibited a slightly decreased slope (,0.068, R 2 = 0.93), implying that its reduced fusogenicity relative to CT624 and CT613 cannot be fully attributable to its low level of surface expression. We further performed cell-cell fusion using different co-culture periods, i.e., 0, 2, 4, and 8 h, and again observed faster fusion kinetics for CT624 and CT613 (Fig. 5B ) as compared to CT609, further confirming that additional truncation of XMRV Env beyond the R peptide cleavage site does not increase fusion activity as we had seen for JSRV Env and that CT609 has an intrinsically relatively low fusogenicity. The expression of XMRV Env on the 293T cell surface was measured using anti-FLAG and flow cytometry. Fluorescence geometric means were normalized to XMRV Env (100%). Shown are the averages of 3 independent experiments 6 S.D. XMRV Env: XMRV Env tagged with a FLAG sequence only at the N-terminus. All truncations were also tagged similarly with an N-terminal FLAG. F-XMRV-F: an XMRV Env construct that is tagged by FLAG sequences on both N-and C-termini. Mock: untransfected 293T cells. doi:10.1371/journal.pone.0033734.g003