A panel of HIV-1ΔNef strains was tested for their sensitivity to Viperin overexpression. (A) Virus release was assayed by p24 ELISA quantification of cell-free supernatant forty-eight hours after co-transfection of 293T cells. Error bars indicate standard deviations of three replicates and are representative of three independent experiments. (B) Western blot analysis was performed on the cell-free virus and cellular extracts of (A). HIV-1 Gag expression was assessed by α-p24 antibody. Viperin expression in the cellular extracts is shown, and Actin was probed as a loading control. This blot is representative of at least three independent experiments. (C) Single-cycle infectious virus yield of VSV-G pseudotyped primate lentiviruses from 293T cells co-transfected with serial titration of Viperin and was titered on TZM.BL indicator cells. Proviruses were deleted of the nef gene. The infectivity readout by β-galactosidase activity was measured in relative light units (RLU). (D) The effect of Viperin on retroviruses was measured by co-transfecting 293T cells with MLV, FIV or HIVLaiΔNef with serial titrations of Viperin. Viruses were pseudotyped with VSV-G and titered by infecting HeLa cells. The expression of virus-encoded GFP was quantified by flow cytometry. This analysis is representative of at least three independent experiments. (E) Schematic of chimeric proviruses of HIVLai and HIVNL4-3 highlighting the breakpoint and features in the non-coding region of the provirus. Single-cycle infectious virus yields from co-transfected 293T cells were assayed by titering on TZM.bl reporter cells. Error bars indicate standard deviations of four infection replicates, and the data are representative of at least three independent experiments.