(A) The effect of viperin was measured by the infectious virus yield. 293T cells co-transfected with 200 ng of HIVLai or HIVLaiΔNef with serial dilutions of human viperin was titered by infecting TZM.BL indicator cells. The infectivity readout by β-galactosidase activity measured in relative light units (RLU) is shown on the left, and the respective viruses were normalized to β-galactosidase activity in the absence of viperin as shown on the right. Error bars indicate standard deviations from three infection replicates; this data are representative of five independent experiments. (B) Western blot analysis was performed on the cell-free virus and cellular extracts, and probed with α-p24 antibody. Viperin expression in the cellular extracts is shown, and actin was probed as a loading control. This blot is representative of four independent experiments. (C) The effect of Viperin on the specific infectivity of virus particles was calculated. HIVLai or HIVLaiΔNef virus from 293T cells co-transfected with or without Viperin (700 ng) was titered by infecting TZM.BL indicator cells as shown on the left. Virus release in the cell-free supernatant was quantified by p24 ELISA as shown in the middle. The specific infectivity was calculated as ratio of β-galactosidase activity (RLU) over the amount of p24 (ng/ml), as shown on the right. Error bars indicate standard deviations of triplicate infections; the data are representative of at least three independent experiments. (D) Virus yield from 293T cells co-transfected with a combination of Tetherin (50 ng) or Viperin (700 ng) was titered on TZM.BL cells. Fold inhibition was calculated in comparison to virus yield in absence of Tetherin/Viperin. Error bars indicate standard deviations of triplicate infections; the data are representative of three independent experiments.