FIG 2.

The efficiency of SIV-Env incorporation into virions determines IFITM sensitivity. (A) MLV vectors encoding luciferase and bearing escalating amounts of SIV-Env or MLV-Env were produced by transient transfection of 293T cells with equal amounts of vector plasmid and the indicated amounts of Env-encoding plasmids. Equal volumes of the vector preparations were then inoculated onto 293T cells previously transduced to express IFITMs, and luciferase expression in cell lysates was analyzed at 72 h postransduction. The results of a single representative experiment conducted with triplicate samples are shown. Error bars indicate SD. The results were confirmed in three separate experiments. Numbers above bars indicate the averages from four independent experiments for which transduction of control cells was set as 100%. Statistical analysis was carried out for normalized data. (B) sMAGI cells were transfected with the indicated siRNAs, and IFITM3 expression was analyzed by an immunoblot assay employing an IFITM3-specific antibody. Similar results were obtained in two separate experiments. (C) sMAGI cells transfected as described for panel B were transduced with MLV vectors bearing escalating amounts of SIV-Env, and transduction efficiency was determined as described for panel A. In addition, the cells were infected with FLUAV encoding Gaussia luciferase, and luciferase expression in culture supernatants was analyzed at 48 h postinfection. The average from three independent experiments (two for FLUAV) is shown. Error bars indicate SEM.