Figure 3

Influence of Vif on the DSB susceptibility of HIV-1 VLP assembly in Sf9 cells. Sf9 cells were coinfected with equal MOI (5 PFU/cell) of two baculoviruses expressing Pr55Gag and Vif, respectively. 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 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. Blots were reacted with anti-Vif primary antibody and secondary phosphatase-labelled antibody, followed by anti-Gag primary antibody and secondary peroxidase-labelled antibody. (a), WCL. (*), Asterisk marks posttranslationally modified Gag precursor (ubiquitinated and/or phosphorylated). This Gag species was not included in the quantification of Pr55Gag polyprotein. (b), VLP. Molecular mass of prestained markers (m) are indicated in kiloDaltons (kDa) on the left side of panels (a) and (b). (c), Quantification of Gag and Vif proteins in WCL (IC-Gag, intracellular Gag; IC-Vif, intracellular Vif) and extracellular VLP, using SDS-PAGE and radio-immunoblotting. Gag and Vif protein contents were quantified by autoradiography of immunoblots reacted with anti-Gag and anti-Vif rabbit primary antibodies and ³⁵S-labelled secondary anti-rabbit IgG antibody. After autoradiography of the blots, bands of Pr55Gag and Vif proteins were excised and their radioactive content determined by liquid scintillation spectrometry. Results were expressed as percentage of control, untreated samples, which was attributed the 100% value. Mean of three separate experiments ± standard deviation.