Sensitivity of A3G single lysine mutants to HIV-1 Vif. A, human A3G amino acid sequence. It has 20 lysines that are underlined and numbered. B, expression profile of A3G lysine mutants in the presence of HIV-1 Vif. Twenty A3G single lysine mutants were created by mutating each lysine (K) to arginine (R). The lysine mutants were coexpressed with HIV-1 Vif in 293T cells, and their expressions were determined by Western blotting. Actin blots served as controls for loading. WT, wild-type.

Lys-free A3G is degraded in proteasomes. A, requirement of a functional proteasomal system for degradation. Wild-type or Lys-free A3G-FLuc reporter constructs were cotransfected with HIV-1 Vif expression vector pNL-A1 or its control, pNL-A1ΔVif. Cells were treated with proteasomal inhibitor MG132 (20 μm) or proteasome inhibitor I (10 μm) and lysosomal inhibitor NH₄Cl (20 mm), chloroquine (100 nm), or bafilomycin (200 nm). Cellular luciferase activities were then determined. B, requirement of a functional polyubiquitylation system for degradation. Wild-type or Lys-free A3G proteins were coexpressed with HIV-1 Vif in the presence of wild-type ubiquitin or mutant ubiquitins (Ub_48A and Ub_63A). A3G and Vif expressions were determined by Western blotting. C, requirement of the Cul5 E3 ligase for degradation. Wild-type or Lys-free A3G-FLuc fusion proteins were coexpressed with Vif proteins in the presence of Cul5ΔNedd8 (ΔNedd) or Cul5ΔRbx1 (ΔRbx). Cellular luciferase activities were then determined as before. Error bars in A and C represent S.D. of at least three independent experiments.

Half-life of Lys-free Vif and A3G. A, pulse-chase radiolabeling of Vif. 293T cells were transiently transfected with the wild-type or Lys-free Vif expression vector. Cells were pulse-labeled for 30 min and then incubated for the indicated time points. After immunoprecipitation of lysates with anti-Vif antibody, proteins were separated by SDS-PAGE, and the gels were subjected to autoradiography. The relative intensity of the bands was quantified, normalized to 100, and graphed. B, pulse-chase radiolabeling of A3G. GST-tagged wild-type or Lys-free A3G was transiently expressed in 293T cells in the presence or absence of Vif. Cells were pulse-chased as above, and proteins were isolated on glutathione-Sepharose beads. Labeled A3G proteins were analyzed and quantified as in A.

Sensitivity of Lys-free A3G mutants to HIV-1 Vif. A, schematic description of A3G expression constructs. The constructs express wild-type or lysine-free A3G proteins either C-terminally fused with FLuc, V5, or HA or N-terminally fused with 3× HA. B, expression of Lys-free A3G in the presence of HIV-1 Vif. The four A3G expression constructs were transfected alone or coexpressed with HIV-1 Vif, and their levels were determined either by measuring luciferase activity or by Western blotting. WT, wild-type. C, polyubiquitylation of Lys-free A3G in vivo. HA-tagged wild-type or Lys-free A3G was coexpressed with HIV-1 Vif and a FLAG-tagged ubiquitin mutant (Ub_48A). Polyubiquitylated proteins were isolated via anti-FLAG-Sepharose beads and visualized by Western blotting using anti-HA antibody. D, anti-HIV-1 activity of Lys-free A3G. Wild-type and vif-deficient HIV-1 luciferase reporter viruses were produced in the presence of the wild-type or Lys-free A3G expression vector, and viral infectivity was determined. The pcDNA3.1 empty vector was used as a control (Ctrl). Error bars represent S.D. in three independent experiments. E, virion packaging of Lys-free A3G. Wild-type and vif-deficient HIV-1 were produced in the presence of wild-type or Lys-free A3G, and virions were purified by ultracentrifugation. Virion-associated proteins were determined by Western blotting using antibodies for V5 or p24^Gag.

Role of Vif polyubiquitylation in A3G degradation. A, polyubiquitylation of HIV-1 Vif protein in vivo. Wild-type or Lys-free (Vif16K/R) HIV-1 Vif proteins were coexpressed with FLAG-tagged Ub_48A. Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum. B, expression of Lys-free Vif. Equal amounts of expression vectors for wild-type (WT) or Lys-free Vif were transfected into 293T cells, and protein expression was determined by Western blotting. C, activity of Lys-free Vif. Wild-type or Lys-free Vif proteins were coexpressed with A3G protein fused with FLuc in 293T cells, and the cellular luciferase activity was determined as before. Error bars represent S.D. in at least three independent experiments. D, interaction of HIV-1 Vif with A3G and Cul5 E3 ligase. Vif proteins were coexpressed with FLAG-tagged A3G or HA-tagged Cul5 or EloC. To detect the interaction between Vif and A3G, proteins were pulled down by anti-FLAG beads and analyzed by Western blotting using anti-FLAG and anti-Vif antibodies. To detect the interaction between Vif and Cul5 or EloC, proteins were immunoprecipitated (IP) by anti-Vif polyclonal antibody and analyzed by Western blotting using anti-Vif and anti-HA antibodies. I, input; P, pull down. E, A3G polyubiquitylation by Lys-free Vif. Wild-type A3G was coexpressed with wild-type or Lys-free Vif in the presence of the FLAG-tagged Ub_48A expression vector. Proteins were isolated on anti-FLAG beads, and polyubiquitylated proteins were visualized by Western blotting using anti-Vif polyclonal antiserum.

Possible model of how Vif targets A3G for proteasomal degradation. Vif interacts with both A3G and a Cul5 E3 ligase. These interactions result in the polyubiquitylation of both Vif and A3G proteins by this enzyme. However, only the polyubiquitin chain of Vif can direct the Vif and A3G complex to proteasomes for degradation.