Lys to Arg substitution mutations conferring A3G resistance to HIV-1 Vif-dependent degradation. (A) WT A3G and mutants. Numbers 1 to 14, shown above the diagram, refer to the 14 Lys residues illustrated in Fig. S2 [i.e., at positions 63, 76, 79, 99, 113, 141, 180, 249, 270, 297, 301, 303, 334, and 344 (K1–14), respectively (position numbers indicated below the figure)]. Five mutants have Arg substitutions in K1–9, 1–4, 5–9, 8–14, and 10–14 and were designated as K (1–9)R, K(1–4)R, K (5–9)R, K(8–14)R, and K(10–14)R, respectively. (B) and (C) Vif-dependent degradation of A3G mutants. 293T cells were transfected with or without pcDNA-HVif plus the indicated A3G expression plasmid. The amounts of cellular A3G from cells transfected with or without the Vif-expressing plasmid were compared by Western blot analysis using anti-A3G antibody. In (B), D128K, a defective A3G that does not bind Vif, was used as a positive control. (D) Effect of increasing levels of Vif expression on degradation of WT A3G and A3G mutant K(10, 11, 12, 13)R. Two micrograms of each A3G expression plasmid was used. For Vif expression, pcDNA-HVif/pcDNA 3.1 (-) plasmids (8 μg) were used at the following ratios: 8:0 (lanes 1, 8, 15); 1:1 (lanes 2 and 9); 3:5 (lanes 3 and 10); 1:3 (lanes 4 and 11); 1:15 (lanes 5 and 12); 1:79 (lanes 6 and 13); and 0:8 (lanes 7 and 14). Anti-ß-tubulin antibody was used as a control.

Effect of incorporation of the K(10, 11, 12, 13)R mutant (S-A3G) into WT or vif-deficient virions on infectivity in a single-cycle replication assay. (A) 293T cells were cotransfected with pcDNA-HVif and either empty (-), WT (W), or S-A3G (S) plasmids having the Lys-free MycHIS tag, using the procedures described in Materials and Methods. The MycHIS-tagged proteins were analyzed either directly (Left) as “Total Cell Lysate” or following pull-down (binding of the HIS tag to TALON resin) (Right) by immunoblotting with the indicated antibodies. (B) Virus infectivity; 293T cells were cotransfected with (i) pNL4–3 and either WT (▴) or S-A3G (●) expression plasmids or (ii) pNL4–3vif(-) and either WT (■) or S-A3G (crosses) expression plasmids, using 0, 0.1, 1, or 2 μg of DNA. Where appropriate, the amounts of DNA added for each of the expression plasmids were adjusted with empty vector pcDNA 3.1 (-) to a total of 2 μg. Infectivity values are given relative to the values for 0 μg of the expression plasmids and were set at 100%. (C) Analysis of intracellular expression (Cell) and incorporation of WT and S-A3G into virions (Virus) by Western blot. Virions were produced by transfection of pNL4–3 WT/pNL4–3vif(-) plus either A3G WT, S-A3G expression plasmid or pcDNA 3.1(-) (shown as “no”). The ratios of pNL4–3 WT to pNL4–3vif(-) were as follows. 1:0 (lanes 1, 2 and 8), 1:1 (lanes 3 and 9), 1:3 (lanes 4 and 10), 1:7 (lanes 5 and 11), 1:79 (lanes 6 and 12), and 1:0 (lanes 7 and 13). The intracellular levels of β-tubulin and Vif and the CA level in virions were detected by anti-β-tubulin, anti-Vif, and anti-CA antibodies, respectively.

Effect of a MycHIS tag at the C-terminal end of A3G. WT A3G (W) or S-A3G (S) without a tag (No), with a MycHIS tag or the Lys-free MycHIS tag were expressed in the presence or absence of Vif in 293T cells. A3G was detected by Western blot with anti-A3G antibody (Anti-A3G), anti-Myc tag monoclonal antibody (Anti-Myc), or anti-(His₆) tag antibody (Anti-HIS).

Region of A3G involved in ubiquitination around Lys residues 297, 301, 303, and 334 and Vif binding mapped onto the structural model. (A) Side and 90°-rotated (counterclockwise or clockwise) views of the model in surface representation. Lys residues 297, 301, 303, and 334 are shown in blue, all of the other Lys in cyan, the C-terminal end of A3G in green, and residues D128, P129, and D130 responsible for Vif-binding in red. (B) Cartoon illustration of the A3G/Vif/ElonginB/C/Cul-5/Rbx2/E2 complex. The ubiquitination sites on the A3G substrate are marked in blue and the Vif binding site in orange.