Figure 4

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 [51] 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.