Figure 3From: Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNAA3G 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.Back to article page