Figure 4
Genotype and expression of recombinant Vif mutants in Sf9 cells. (A), Sequence alignment of the central and C-terminal domains of HIV-1 Vif proteins, WT and mutants. The zinc binding domain (ZBD) and its three constitutive loops are boxed: loops 1 and 3 are indicated as dark grey boxes, central loop 2 as a lighter grey box. (B), Cellular expression of recombinant Vif proteins, wild-type and mutants, in baculovirus-infected Sf9 cells. Sf9 cells were infected with baculoviruses (MOI 5) expressing different forms of Vif, as indicated on top of the panel, and harvested at 48 h pi. Whole cell lysates were analyzed by SDS-PAGE and immunoblotting, using anti-Vif primary antibody and secondary peroxidase-labelled antibody. The full-length ZBD mutants VifC133S and Vif116V show an aberrant electrophoretic mobility, as they migrate with a higher apparent molecular weight compared to Vifwt (23 kDa), and a higher sensitivity to proteolysis, as evidenced by the discrete bands of lower molecular weight breakdown products. Note the propensity of the Vif protein of triple mutant Vifsub CΔ170 (20 kDa) to dimerize (Vifx2; 40 kDa).