Figure 6

The C-x1-C-x1-H-x19-C motif is crucial and predicted to be a zinc binding loop. (A) Alignment of partial BIV Vif sequences with primate lentiviral Vifs by BioEdit. (B) 293 T cells (0.5 × 10⁶) were co-transfected with 30 ng HA-tagged btA3Z2-Z3 and 200 ng cmyc-tagged BIV Vif or BIV Vif mutants H102L, C111S, C113S, H115L, C134S, H149L or C111S/C113S. At 48 h after transfection, the cells were harvested for Western blotting using anti-HA, anti-cmyc and anti-tubulin antibodies. (C and D) 293 T cells (1 × 10⁶) were co-transfected with 1 μg pNL4-3ΔVif plus 15 ng VR1012, or HA-tagged btA3Z2-Z3 and 100 ng cmyc-tagged BIV Vif, BIV Vif C111S/C113S or VR1012. The virus infectivity was assayed by the MAGI assay. Virus infectivity was set to 100% in the absence of btA3Z2-Z3. (D) Western blot was performed on the cell lysates from (C) to show the producer cell levels of btA3Z2-Z3 protein (anti-HA), BIV Vif/BIV Vif C111S/C113 (anti-cmyc) and tubulin. (E) 293 T cells (5 × 10⁶) were co-transfected with 10 μg cmyc-tagged btCul2 and 6 μg HA-tagged BIV Vif or 6 μg BIV Vif C111S/C113S. At 48 h after transfection, cells were immunoprecipitated with HA beads, followed by SDS-PAGE and immunoblot analysis using an anti-HA antibody and an anti-cmyc antibody. (F and G) Panels A and B are views of the alpha carbon ribbon and differ from each other by 90 degrees. Residues which likely participate in the coordination of zinc ions are shown and labeled. All infection experiments were repeated three times.