Antiviral activities of A3H WT and mutants. (A) Western blot analysis showing the amounts of A3H WT and mutants in HIV-1vif (−) virions and 293T cells. 293T cells were transfected with HIV-1vif (−) and A3H WT or mutant plasmids at a 1:1 ratio (1 μg each for determination of expression in cell extracts and 8 μg each for determination of A3H in viral lysates). Cell extracts (10 μg of total protein) as well as viral lysates (8 μl of viral pellet resuspended in 200 μl of loading buffer) were subjected to Western blot analysis. Viral lysates were probed with antibodies to the N-terminal FLAG tag of A3H and HIV-1 CA; cell extracts were probed with antibodies to the N-terminal FLAG tag of A3H, HIV-1 Gag (Pr55gag), and tubulin. Controls: Left side, EMPTY refers to HIV-1vif (−) and empty vector (pTR600); Right side, EMPTY refers to empty vector alone; WT, refers to A3H plasmid DNA alone. (B-D) Antiviral activity was determined as described in Methods. The gray and black bars respresent transfection with 0.1 μg or 1 μg of the indicated A3H plasmid, respectively.