A3G BiFC constructs and their biological activities. (A) Structures of A3G BiFC constructs A3G-NY, A3G-CY, NY-A3G, and CY-A3G. The YFP N-terminal (NY) and C-terminal (CY) fragments were fused either to the C-terminal end of A3G (A3G-NY and A3G-CY) or the N-terminal end of A3G (NY-A3G and CY-A3G). The glycine-rich hinge regions (thin lines) for N-terminally tagged BiFC constructs is slightly longer than in the C-terminally tagged constructs. The catalytic domains 1 and 2 (CD1 and CD2) are shown as gray boxes. (B) Western blotting analysis of cells co-transfected with HDV-EGFP along with wild-type A3G or A3G BiFC constructs. The A3G protein was detected using a polyclonal anti-A3G antibody. (C) Relative cytidine deaminase activity in lysates of cells co-transfected with wild-type A3G or A3G BiFC constructs as well as pHDV-EGFP, pC-HelpΔVif and pHCMV-G. Total cellular protein (0.3 μg) from each cell lysate was used for determination of enzymatic activity, and the activity in cells transfected with wild-type A3G was set to 100%. Error bars represent the standard error of the mean (s.e.m.) of three independent experiments. (D) Effect of wild-type A3G and A3G BiFC constructs on infectivity of HDV-EGFP. The infectivity of the virions produced in the absence and presence of HIV-1 Vif was determined by flow cytometry analysis of cells infected with the virions. Transfections were also performed in the absence of A3G and Vif, and the proportion of GFP+ cells after infection with HDV-EGFP (23.4% in the absence of Vif, and 28% in the presence of Vif) was set to 100%. Error bars represent the s.e.m. of three independent experiments.