A3G-3A is sensitive to HIV-1 Vif. (A) HeLa cells were transfected with vectors expressing vif-deficient pNL4-3 (3 μg each) along with 1.5 μg each of pcDNA-A3G-3A (lanes 1–2, 5–6) or pcDNA-Apo3G (lanes 3–4. 7–8) as well as 1.5 μg pcDNA-hVif (+) or 1.5 μg empty pcDNA3.1 vector DNA (-). Cells and virus-containing supernatants were collected 24 h post-transfection. Total cell lysates 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-probed with a Vif-specific monoclonal antibody (Vif) followed by an HIV-positive patient serum (CA). Proteins are identified on the right. (B) Virus-containing supernatants from panel A were normalized for equal reverse transcriptase activity and used to infect LuSIV indicator cells to  determine 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 1). The infectivity of the remaining viruses was calculated relative to the control virus. Error bars reflect standard deviations from triplicate independent infections.