FIG 4.

Effect of single alanine substitutions in APOBEC3-specific regions of Vif2 on its activity against A3G and A3F. (A) Vif2 mutant rescue of HIV-1Δvif infectivity in the presence of A3G and A3F. Viruses were produced and infectivity was determined as described in the legend to Fig. 2. Data are plotted as relative infectivity levels, with the level in the absence of any A3 protein being set equal to 100%. Dashed lines, inhibition by A3G or A3F in the absence of Vif2. Error bars indicate standard errors of the means (SEMs) of the results from three to four infectivity experiments performed with independent virus stocks. All comparisons were made on the basis of the results for no-Vif2 controls for each A3 protein using two-tailed Student's t test. A P value of <0.05 was considered statistically significant (*). Boxes indicate mutants which retained significant activity against A3G but not A3F (solid) or retained significant activity against A3F but very little activity against A3G (dotted). (B) Effects of Vif2 on the degradation of A3G and A3F. Cell lysates of the viral producers for which the results are presented in panel A were analyzed by Western blotting using anti-FLAG or anti-α-tubulin antibodies. The results are from one representative experiment of three independent experiments. (C) Summary of Vif2 mutational analysis. The alignment of the Vif1 and Vif2 N-terminal 60 amino acids is shown. Dots, amino acid identity; dashes, gaps; fuchsia boxes, multiple-alanine mutants or single-amino-acid-substitution mutants that lost activity against A3F but not A3G and/or A3H-hapII; green box labeled G, G48 single-amino-acid-substitution mutant that lost activity against A3G but not A3F; blue box, multiple-alanine-substitution mutant that lost activity against A3H-hapII but not A3G or A3F; gray box, multiple-alanine-substitution mutants that lost activity against A3G and A3F but not A3H-hapII; black boxes, multiple-alanine-substitution mutants that lost activity against A3G, A3F, and A3H-hapII.