APOBEC3G and APOBEC3F Require an Endogenous Cofactor to Block HIV-1 Replication
Figure 5
Anti-HIV activity of A3F/A3G proteins expressed in trans from a MuLV-based vector.
(A) Stable transduction of CEM-T4 and CEM-SS cells by MuLV-based vector. An A3F, A3G, A3GE259Q, or GFP gene with an HA tag was inserted into the pMSCVneo vector, and recombinant MuLV was produced. CEM-T4 and CEM-SS cells were then infected with these viruses, and stably infected cells were selected by G418 treatment. The expression of A3F/A3G proteins in each individual cell line was determined by Western blotting. (B) A3G subcellular localization. CEM-T4 and CEM-SS cells stably transduced with A3G were fixed with formaldehyde and then stained with a mouse anti-A3G monoclonal antibody and a rabbit anti-MOV10 polyclonal antibody. A3G was visualized using Alexa Fluor 488–conjugated secondary antibody (green), and MOV10 was visualized by Alexa Fluor 594–conjugated antibody (red). The cells were also stained with Hoechst 33342 to visualize nuclei (blue). Areas of overlap between A3G and MOV10 appear as yellow. (C) Replication kinetics of HIV-1 in stably transduced cell lines. CEM-T4 and CEM-SS cells stably transduced with A3G, A3GE259Q, A3F, or GFP were infected with the same amount of wild-type or vif-deficient HIV-1. Viral growth curves were determined by measuring p24Gag in the supernatants using ELISA.