Chim3 blocks HIV-1 replication regardless the presence of hA3G. Empty-, F12-Vif–, and Chim3-transduced permissive SupT1 cells were infected in triplicate cultures with the X4 NL4-3 molecular clone (A) or the Δvif HIV-1 (B) at an MOI of 0.1 and 1, respectively. Supernatants of the kinetic of infection were collected every 3 to 4 days, stored at −20°C, and then assessed for RT activity. Values represent mean plus or minus SEM of triplicate cultures. (C) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of HEK-293T cells and used at an MOI of 4 to infect either empty-LV- or Chim3-LV–transduced SupT1 cells. Seventy-two hours after viral challenge, intracellular p24Gag level was evaluated by FACS analysis using an anti-p24Gag Ab on fixed and then permeabilized cells. Values express mean plus or minus SEM percentage of the p24Gag expression of each condition (n = 5) relative to that of wild-type HIV-1 on empty transduced cells (HIV-1 ■). (D) The same viruses of panel C were used to carry out a kinetic of single round infection for the indicated time points on empty and Chim3-transduced SupT1 cells. (E) VSV-G pseudotyped R9Δenv-HIV-1 and R9Δenv-Δvif-HIV-1 were produced by transient transfection of the corresponding molecular clones in either empty or Chim3-transduced HEK-293T cells. SupT1 cells were infected at an MOI of 4. Seventy-two hours after single round infection, 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 indicate mean plus or minus SEM percentage of p24Gag expression of each condition (n = 6) relative to that of wild-type HIV-1 (HIV-1 ■).