Figure 5
Counteracting effect of packaging-defective mutant Vif sub C on the DSB inhibition of HIV-1 VLP assembly. Sf9 cells were coinfected with two baculoviruses at equal MOI of each (5 PFU/cell), one expressing Pr55Gag, the other expressing the double substitution, packaging-defective mutant Vifsub C. Cells were treated with increasing concentrations of DSB in DMSO for 30 h at 18 h pi, as indicated on top of panels (a) and (b), and on the x-axis of panel (c). Cells were harvested at 48 h pi, and whole cell lysates (WCL) and extracellular VLP analyzed by SDS-PAGE and immunoblotting, using anti-Vif primary antibody and secondary phosphatase-labelled antibody, followed by anti-Gag primary antibody and secondary peroxidase-labelled antibody. (a), WCL; (b), VLP. Note the low level of Vif protein in VLP, consistent with the packaging-defective phenotype of Vifsub C [50]. (m), prestained molecular mass markers; (kDa), kiloDaltons. (c), Quantification of Pr55Gag and Vif protein content of VLP, performed by autoradiography of immunoblots with anti-Gag and anti-Vif rabbit antibodies and 35S-labelled secondary anti-rabbit IgG antibody, as described in the legend to Fig. 3 (c). Results were expressed as percentage of control, untreated samples, which was attributed the 100% value. Mean of three separate experiments ± standard deviation.