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. 2014 Sep;88(17):9893–9908. doi: 10.1128/JVI.01318-14

FIG 2.

FIG 2

Effect of Vif2 multiple-alanine substitutions on its activity against A3G, A3F, and A3H-hapII. (A) Alignment of Vif1 and Vif2 N-terminal 62 amino acids. The locations of amino acids replaced by alanines to generate multiple-alanine substitution mutants 1 to 19 are indicated with brackets below the sequence. (B) Effects of multiple alanine mutations in Vif2 on HIV-1Δvif infectivity in the presence of A3G, A3F, or A3H-hapII in HEK293T cells. Viruses were produced from HEK293T cells that were transfected with pHDV-EGFP, expression plasmids for VSV-G, A3G, A3F, and A3H-hapII, and either empty vector, WT Vif2, or mutant Vif2. Amino acid substitutions are shown for the multiple-alanine-substitution mutants, numbered 1 to 19. Dashed lines, inhibition of virus infectivity by A3G, A3F, or A3H-hapII in the absence of Vif2. Error bars indicate standard errors of the means (SEMs) of the results from three infectivity experiments. All comparisons were made on the basis of the results for the no-Vif2 controls for each A3 protein using two-tailed Student's t test. A P value of <0.05 was considered statistically significant (*). The F box labels indicate Vif2 mutants that had no activity against A3F but retained activity against A3G and/or A3H-hapII. The G box label indicates the Vif2 mutant that had no activity against A3G but retained activity against A3F and A3H-hapII. The H box label indicates the Vif2 mutant that had no activity against A3H-hapII but retained activity against A3G and A3F. Mutants 2 to 4, 7, 12, and 16 to 19 lost activity against all three A3 proteins. (C) Effects of multiple alanine mutations in Vif2 on HIV-1Δvif infectivity in the presence of A3G, A3F, or A3H-hapII in CEM2n cells. Viruses were produced from CEM2n cells that were nucleofected with pHDV-EGFP and either empty vector, WT Vif2, or the indicated multiple-alanine mutants of Vif2 that specifically blocked A3F, A3G, or A3H-hapII. Relative infectivity was determined for two independent virus stocks by infection of TZM-bl cells with p24 CA-normalized virus and quantitation of luciferase activity at 72 h postinfection. The infectivity in the absence of Vif2 was set equal to 1.0. All comparisons were made on the basis of the results for the Vif2 control for using two-tailed Student's t test. A P value of <0.05 was considered statistically significant (*).