Chim3 does not counteract the antiviral action of hA3G, by acting as a true dominant-negative factor. Kinetic of infections of CEM A3.01 cells (A), CD4⁺ T lymphocytes (B), and CD34⁺-derived macrophages (C) infected with either X4 Δvif-HIV-1 (A,B) or R5 Δvif-HIV-1 (C) at an MOI of 0.1. Values represent mean plus or minus SEM of triplicate (A,B) and quintuplicate (C) cultures. (D) Western blot analysis of cell extracts derived from HEK-293T cells transfected with a fixed amount of hA3G-HA and either WT-Vif– or Chim3-expressing plasmids at the indicated amounts of plasmid DNA. Cell extracts were prepared 48 hours after transfection. Membranes were sequentially probed with anti-HA Abs (top panel), anti-Vif (middle panel), and antiactin (bottom panel) Abs. (E) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of the corresponding plasmids in either empty or Chim3-transduced HEK-293T cells, in the presence of hA3G expression plasmid. SupT1 cells were infected at an MOI of 4. Seventy-two hours after viral challenge, intracellular p24Gag expression was evaluated by FACS analysis (FACSCalibur, BD Biosciences; and FlowJo software, TreeStar) using an anti-p24Gag Ab on fixed and then permeabilized cells. Values represent the mean plus or minus SEM percentage of the p24Gag content of each condition relative to that of wild-type HIV-1 (HIV-1 ■) (n = 6). (F) Western blot analysis of the level of the intracellular (left panel) and intravirion (right panel) hA3G and Vif proteins; 40 μg WCE and 1 μg p24Gag HIV-1 virion equivalent, respectively, were loaded in each conditions. The filter was sequentially probed with the different Abs as indicated.