Expression of IFITM proteins or treatment of cells with IFN suppresses syncytia formation induced by JSRV Env and IAV HA.

<p>(<b>A</b>) 293/LH2SN cells stably expressing the indicated IFITM proteins were transfected with plasmid DNA encoding JSRV Env, IAV HA or 10A1 MLV Env with the R peptide deleted (10A1 Env R⁻); cells were treated with a pH 5.0 buffer for 1 min (for JSRV Env and IAV HA) or left untreated (for 10A1 MLV Env) and analyzed for syncytium formation using fluorescence microscopy. Note the stronger inhibition of IFITM1 relative to that of IFITM2 and 3 for JSRV Env and IAV HA; no apparent inhibition was observed for 10A1 MLV Env. (<b>B</b>) Expression of IFITM proteins in 293/LH2SN cells was determined by immunoblotting with an anti-FLAG antibody. β-actin was used as a loading control. (<b>C</b>) Expression of IFITM proteins does not downregulate the JSRV Env expression on the cell surface. 293T cells were co-transfected with plasmid DNAs encoding FLAG-tagged JSRV Env (at both the N- and C-termini) plus indicated wildtype IFITMs. Cells were incubated with an anti-FLAG antibody on ice, and the surface expression of JSRV SU was determined by flow cytometry. A second antibody alone was used control. (<b>D</b>) 293/LH2SN cells were transfected with plasmids encoding JSRV Env, IAV HA, or 10A1 MLV Env with the R peptide deleted; 6 h after transfection, cells were treated with indicated amounts of IFN-α2b or medium for 24 h. Cells were exposed to a pH 5.0 buffer for 1 min (for JSRV Env and IAV HA) or left untreated and examined for syncytia formation.