Viperin has direct antiviral activity against HIV-1 in MDMs and other cells. (A) Time of siRNA addition for optimal knockdown of viperin in MDMs. MDMs infected with HIV-1 were transfected using lipofectamine RNAiMAX with siRNA specific to viperin on days 0, 2, and 3 pi. Viperin mRNA expression was then measured on days 2, 3, 4, and 5 after transfection. Data are percentage levels of viperin mRNA after siRNA-treated and nontreated HIV-1–infected MDMs (the no siRNA result was standardized to 100%). (B) Knockdown of viperin in MDMs enhances HIV-1 DNA expression. DNA was extracted on days 6 and 13 from infected MDMs (MOI = 0.25) treated with specific siRNA to viperin (HIV + viperin siRNA) or not (HIV) on day 3 pi. HIV LTR-gag was quantified by quantitative PCR. The mean data from 3 experiments are shown with SE bars. (C) Viperin exerts dose-dependent inhibition of viral production. HEK293T cells were cotransfected with 3.3 μg of the pWT/BaL proviral HIV-1 DNA and either plasmids encoding WT viperin or the control parental vector pLNCX2 at the same molar ratio of HIV or with decreasing ratio of 1:2, 1:3, 1:9, and 1:27. After 48 hours, supernatants were harvested and assayed for virus release by serial dilutions on TZM-bl indicator cells. Infected TZM-bls were stained blue after the addition of X-Gal and were counted using an Elispot reader. The mean data from 3 experiments are shown with SE bars. (D) Viperin is not induced by HIV-1 in HEK293T cells. HEK293T were transfected with pHEF-VsV-g (NIH AIDS Research and Reference Reagent Program, contributed by Dr Lung-Ji Chang) and pWT/BaL proviral HIV-1 DNA plasmid using polyethylenimine to generate VsV-g pseudotyped pBaL to induce a very high level of infectivity within the cell sheet or mock transfected for 3 days. As a positive control, MDMs were exposed to high levels of HIV-1 (MOI 2) or mock infected for 3 and 4 days. Viperin mRNA expression was determined by quantitative PCR relative to GAPDH.