IFITM Proteins Restrict Viral Membrane Hemifusion
Figure 2
Expression of IFITM proteins does not affect binding of JSRV Env to cells expressing the Hyal2 receptor, nor does it perturb receptor-mediated priming for fusion activation.
(A) Examination of JSRV Env binding to cells expressing IFITMs. HTX cells stably expressing indicated IFITM proteins were incubated with purified JSRV SU-human IgG Fc proteins at 4°C; following incubation with FITC conjugated anti-human Fc antibody, cells were analyzed by flow cytometry. Representative histograms from one typical experiment are shown. Arrow indicates the secondary antibody alone control. (B) Quantitative analysis of JSRV Env binding data shown in (A). The fluorescence intensities (geometric means) obtained from (A) were averaged and normalized to those of mock controls. The means ± SD of at least three independent experiments are shown. (C) Examination of the JSRV pseudovirion binding to cells expressing IFITMs. HTX cells expressing IFITM proteins were incubated with purified JSRV pseudovirions containing MLV Gag-YFP to allow virus binding. Cells were washed, fixed and analyzed by flow cytometry. “Control” indicates cells incubated with MLV Gag-YFP pseudovirions in the absence of JSRV Env. Representative flow cytometry profiles are shown. (D) Quantitative analysis of the JSRV pseudovirion binding experiments shown in (C). The fluorescence intensities of three independent experiments were averaged and plotted. (E) Expression of IFITM proteins on the surface of HTX cells. The expression was examined by an anti-FLAG antibody and analyzed by flow cytometry. (F) Quantitative analysis of IFITM expression on the cell surface shown in (E). Values are the means ± SD of at least five independent experiments. (G and H) Examination of the effect of IFITMs on JSRV SU shedding. 293T cells were co-transfected with plasmids encoding FLAG tagged-JSRV Env and FLAG-tagged IFITMs. Cells were metabolically labeled and chased in the presence of indicated amounts of sHyal2. Cell lysates and culture media were harvested and immunoprecipitated with anti-FLAG beads. Samples were resolved by SDS-PAGE and subjected to autoradiograthy. (G) Expression of JSRV Env and IFITM in transfected cells. Env: the full length of JSRV Env; SU: surface subunit; TM: transmembrane subunit. (H) Shedding of JSRV SU into culture medium. Note the increased SU shedding in cells expressing JSRV Env with increasing amounts of sHyal2; no significant differences in shedding among cells expressing IFITM and mock controls were observed. The relative intensities of signals for shed SU were calculated by setting the signals of the mock control without sHyal2 stimulation as 1.0; three independent experiments were used for the quantification.