FIG 3.

Cell-surface localization of SERINC5 F412A and its incorporation into HIV-1 virions. (A) Restriction activity of WT and F412A mutant of SERINC5-iHA against HIV-1 NL43_ΔEnvΔNef pseudotyped with Env_NL that had been produced in JTAg-SERINC^−/− cells. (B) JTAg-SERINC^−/− cells transfected with the genes encoding GFP alone (a), WT SERINC5-GFP (green) (b), or the F412A mutant (c) of SERINC5-GFP (green) were visualized under a fluorescence microscope. DAPI was used to stain cellular nuclei (in blue). (C) JTAg-SERINC^−/− cells transfected with empty vector or genes encoding WT and F412A mutant of SERINC5-iHA were stained with anti-HA antibody and analyzed for cell surface expression by flow cytometry. (D) The cell surface expression of WT and F412A SERINC5-iHA was analyzed in the presence of GFP only or Nef_SF2-GFP. Fold reduction of MFI of SERINC5-iHA in GFP-gated JTAg-SERINC^−/− cells was shown. (E) Immunoblots showing total cellular expression and virion incorporation of the WT and the F412A mutant of SERINC5-iHA in HEK293T and JTAg-SERINC^−/− producer cells. A mock-infected control was included. The virion incorporation level is depicted in the graphs (right). Data are means ± SD from 3 independent experiments.