A3G BiFC constructs containing mutations in CD1 or CD2 and their biological activities. (A) Western blotting analysis of lysates of 293T cells and viral lysates produced from cells co-transfected with pHDV-EGFP, pC-HelpΔVif and pHCMV-G and wild-type A3G or A3G BiFC constructs containing mutations in the CD1 (H65R-NY, H65R-CY, C97S-NY, and C97S-CY) or CD2 (H257R-NY, H257R-CY, C288S-NY, and C288S-CY). The cell lysates were also analyzed using anti-tubulin antibody to insure equivalent loading of cell lysate proteins (panel labeled α-tubulin). (B) Effects of CD1 or CD2 mutations on A3G's ability to inhibit HIV-1 replication. 293T cells were co-transfected with wild-type A3G or A3G BiFC constructs along with pHDV-EGFP, pC-HelpΔVif, and pHCMV-G, and the infectivity of the virions produced was determined by flow cytometry analysis of the infected cells for EGFP expression. The proportion of GFP+ cells in the absence of A3G co-transfection was set to 100%. Error bars represent the s.e.m. of three independent experiments. (C) Vif sensitivity of CD1 domain mutants. A3G-CY and CD1 domain mutants H65R-CY, F70A-CY, and Y91A-CY were transfected into 293T cells with and without Vif expression plasmid. A3G fusion proteins were detected by using anti-A3G antibody and HIV-1 Vif was detected using anti-Vif polyclonal antiserum. Anti-tubulin antibody was used to detect tubulin, which served as a loading control.