Figure 1.
XMRV entry is pH-independent and does not require host proteases.
HTX cells were pre-treated with indicated concentrations of (A) ammonium chloride, (B) Bafilomycin A1 (BafA1), (C) leupeptin or (D) cathepsin III inhibitor for 1 h and infected with MoMLV pseudotypes encoding GFP and bearing XMRV Env, MLV 10A1 Env, VSV-G or Ebola GP in the presence of drugs for 6 h before inactivation of remaining virions using citrate buffer. Percentage of GFP-expressing cells was measured by flow cytometry 48 h post-infection and normalized to the infection obtained in the absence of drug set to 100%. Shown are at least the averages of 3 independent experiments ± S.D. ** indicates p<0.01.
Figure 2.
Soluble XMRV SU binds to cells expressing the viral entry receptor, XPR1, and blocks XMRV pseudovirion infection.
(A) Schematic representation of XMRV SU-human immunoglobulin (hIgG) Fc fusion protein. SP: signal peptide. (B) Indicated cell lines were incubated with 5 µg of XMRV SU-hIgG Fc for 4 h and binding was measured using anti-human IgG FITC and flow cytometry. Filled: HTX or CHO cells stained with a FITC-labeled secondary antibody only (HTX/XPR1 or CHO/XPR1 cells gave a similar background signal which was not overlaid); broken line: parental HTX or CHO cells stained with XMRV SU plus secondary antibody; black line: HTX/XPR1 or CHO/XPR1 cells stained with both XMRV SU and secondary antibody. Representative experiments are shown (n = 5). (C, D) HTX cells were pre-bound with indicated amounts of soluble XMRV or JSRV SU for 1 h at 4°C and infected with MoMLV pseudotypes encoding alkaline phosphatase (AP) and bearing XMRV Env (C) or JSRV Env (D) for 6 h before inactivation of remaining virions using citrate buffer. AP foci were counted 72 h post-infection and values were normalized to infection in the absence of soluble protein set to 100%. Shown are the averages of 3 independent experiments ± S.D. * indicates p<0.05; ** indicates p<0.01.
Table 1.
Titers of XMRV Env pseudovirions in CHO and CHO/XPR1 cells.
Figure 3.
C-terminal truncation of XMRV Env induces SU shedding.
(A) Sequence alignment of MoMLV and XMRV Env constructs. MSD: membrane spanning domain. CT: cytoplasmic tail. Arrow: indicates the MoMLV Env R-peptide cleavage site. Underlined: The CT region that differs between XMRV and MoMLV. (B) 293T cells expressing XMRV Env were metabolically labeled for 1 h and chased for 4 h. Env proteins in cells lysates and shed in the culture media were immunoprecipated, resolved by SDS-PAGE and subjected to autoradiography. Band density was measured using the Quantity One software and values were normalized to the intensity of the XMRV Env SU set to 1.0. Representative experiment is shown (n = 2). (C) 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 ± 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.
Figure 4.
C-terminal truncation of XMRV Env in the CT activates its fusion activity.
(A) Syncytium-induction assay. 293 cells were transiently transfected with plasmids encoding the indicated XMRV Env and photographed 24 h post-transfection. Arrows indicate syncytia. (B) Cell-cell fusion. Effector 293T-GFP cells expressing indicated XMRV Env or no envelope (No Env) were co-cultured for 6 h with CMTMR-labeled target 293 cells and analyzed by flow cytometry. Values shown represent the percentages of fused cells. (C) The fusion percentages (GFP+/CMTMR+) of 3 independent experiments performed in duplicate were averaged (±S.D.) and plotted. (D) Binding of XMRV SU to target 293 cells. XMRV SU (2 µg) was bound to cells for 4 h at 4°C, stained with anti-human IgG FITC, and analyzed by flow cytometry. Red: unstained cells. Blue: secondary alone. Green: XMRV SU and secondary. A representative experiment is shown (n = 4).
Figure 5.
Truncation beyond the putative R-peptide cleavage site does not further enhance the XMRV Env fusion activity.
(A) Effect of Env expression on cell-cell fusion. Effector 293T/GFP cells were transfected with different amounts of plasmids encoding indicated XMRV Env, and co-cultured with target CMTMR-labeled 293 cells. Fusion percentages were determined by flow cytometry following 6 h incubation. In parallel, the XMRV Env surface expression was measured by flow cytometry using an anti-FLAG antibody. The obtained fusion percentages were plotted against Env surface expression. A representative experiment with standard errors of triplicate samples is shown (n = 3). (B) Effect of incubation time on cell-cell fusion. Effector 293T/GFP cells expressing indicated XMRV Env were co-cultured with target CMTMR-labeled 293 cells and cell-cell fusion was assessed after different periods of incubation time. A representative experiment with standard errors of triplicate sample is shown (n = 3).
Figure 6.
Cell-cell fusion activities of XMRV Env and mutants in CHO or CHO cells expressing XPR1.
(A) Immunostaining of CHO and CHO/XPR1 cells. Cells were fixed, permeabilized, stained for XPR1 using anti-XPR1 and FITC-coupled secondary antibodies, and counterstained using DAPI. (B) XMRV Env truncation mutants can induce cell-cell fusion in non-permissive CHO cells. Effector 293T/GFP cells expressing indicated XMRV Env were co-cultured for 6 h with target CMTMR-labeled CHO or CHO cells expressing XPR1 (CHO/XPR1) and analyzed for fusion by flow cytometry. Shown are the averages of 3∼6 independent experiments ± S.D. * indicates p<0.05.
Figure 7.
XMRV Env incorporations into an MLV retroviral vector.
293/GP_LAPSN cells were transfected with an XMRV Env-encoding plasmid, and viral particles were purified through ultracentrifugation. Concentrated viruses were subjected to Western blot using an anti-FLAG antibody to detect XMRV SU (upper panel) or an anti-MLV gag antibody to examine MLV Gag (lower panel).
Table 2.
Titers of MLV and HIV-1 vectors bearing XMRV Env and truncation mutants.