The A3G-3A chimera has antiviral activity. (A) HeLa cells were transfected with vectors expressing vif-deficient pNL4-3 (3 μg each) along with increasing amounts of pcDNA-A3G-3A DNA (lane 1, 1 μg; lane 2, 2 μg; lane 3, 3 μg) or pcDNA-A3G DNA (lane 4, 0.2 μg; lane 5, 0.5 μg, lane 6, 1 μg). Higher amounts of A3G-3A DNA relative to A3G DNA were chosen because A3G-3A was generally expressed at lower levels than A3G. The total amount of transfected DNA in each sample was adjusted to 6 μg using empty pcDNA3.1 vector DNA. Cells and virus-containing supernatants were collected 24 h post-transfection. Total cell lysate and concentrated virus preparations were analyzed by immunoblotting using an A3G-specific rabbit polyclonal antibody (ApoC17) followed by incubation with an HRP-conjugated anti-rabbit antibody (APO). The same blot was subsequently re-blotted with an HIV-positive patient serum (CA). (B) Virus-containing supernatants from panel A were normalized for equal reverse transcriptase activity and used to infect LuSIV indicator cells  for determination of viral infectivity as described in Materials and Methods. Luciferase activity induced by virus produced in HeLa cells in the absence of Vif and A3G was defined as 100% infectivity (lane 7). The infectivity of the remaining viruses was calculated relative to the control virus. Error bars reflect standard deviations from triplicate independent infections.