Figure 4

RNA binding activities of CD1 and CD2 domain mutants of A3G. (A) Effect of CD1 mutations on the ability of A3G to bind cellular RNA. 293T cells were co-transfected with pF-A3G and empty vector, or pF-A3G and A3G-CY, or pF-A3G and fourfold higher amounts of H65R-CY or F70A-CY DNA compared to pF-A3G. An anti-FLAG antibody was used to co-immunoprecipitate F-A3G and associated proteins in the presence or absence of RNase A treatment. The F-A3G, A3G-CY, H65R-CY, and F70A-CY proteins were detected by Western blot using an anti-A3G antibody. (B) A3G-A3G interactions between F-A3G and untagged A3G proteins. 293T cells were co-transfected with F-A3G and empty vector, F-A3G and fourfold higher amounts of untagged A3G DNA or F-A3G and fourfold higher amounts of untagged H65R mutant of A3G DNA. Co-IP assays were performed as described in Fig. 4A. (C) Effect of N-terminal NY and CY tags on A3G-A3G interactions. 293T cells were co-transfected with F-A3G and NY-A3G or CY-A3G. Co-IP assays were performed as described in Fig. 4A. (D) Effect of CD2 mutations on ability of A3G to bind to RNA. 293T cells were co-transfected with pF-A3G and empty vector, or pF-A3G and A3G-CY, or pF-A3G and fourfold higher amounts of H257R-CY, and C288S-CY DNA compared to pF-A3G. Co-IP assays were performed as described in Fig. 4A.