Figure 3

Disrupting L domain-1 increases the effect of the S40F mutation on budding. COS-1 cells were transfected with plasmids expressing HA-tagged HIV-1 Gag-P7L (panels A1 and A2) or P7L-S40F (panels B1 and B2) and prepared for examination by electron microscopy as described in Methods. Panels A2, and B2: Samples were labeled with primary rabbit anti-CA antibody that was tagged with 15 nm gold-conjugated mouse anti-rabbit secondary antibody (bars in panels A1 and A2 measure 100 nm; bars in panels B1 and B2 measure 500 nm). Panel C, Quantitative analysis of particle morphology. 200 gold-tagged VLPs were counted and the frequency of spherical versus extended structures were determined (n =2). Panel D, Western Analysis. VLPs and cell lysates from the COS-1 cells transfected with HA-tagged HIV-1 Gag-P7L (lane 1), P7L-S40F (lane 2) were analyzed by Western blotting and gag related proteins identified by monoclonal HA antibody. Panel E, the relative VLP release efficiencies [VLP/(VLP + Gag from cell lysate)] were determined as described in Methods.