Figure 6

Intact catalytic site is required for ZfL2-2 inhibition by the human A3A protein. HeLa cells were co-transfected with 1 μg of target ZfL2-2 plasmid and (A) 1 μg of plasmid encoding the A3A effector or its E72A, CC101,106AA, F75L and F95L mutants, (B) 2, 1, 0.5, 0.1 and 0.01 μg of A3A and its catalytic site mutants E72A and CC101,106AA, or (C) 2, 0.5 and 0.1 μg of A3A and its F75L and F95L mutants. Neomycin resistant colonies, formed after G418 selection, were fixed, stained and counted, and relative retrotransposition efficiency was calculated by setting the value obtained for cells co-transfected with the retrotransposon plasmid and an empty vector at 100%. The reported cytidine deaminase activity (CDA), or lack thereof, of each protein is designated with + or – (1, ref. [49]; 2, ref. [68]). Western blotting was performed using extracts from 293T cells transiently expressing HA epitope-tagged proteins. GAPDH protein levels were used as loading controls. Data are the means ± SD of at least three independent experiments. ***P < 0.001, **P < 0.01, *P < 0.05, t-test.