Figure 3. Effect of Rev on Gag-Pol Particle Formation.

Viral particles were pelleted from the supernatant of cells transfected in the presence (+) or absence (-) of an HIV-1 rev expression plasmid with codon-optimized gag-pol expression plasmids of SIV (Sgp^syn) and HIV-1 (Hgp^syn) (A) or a wild-type HIV-1 gag-pol expression plasmid (UTRgp-RRE) (B). Western blot analyses were performed with pelleted particles and a CA-specific monoclonal antibody (upper panel). To control for transfection efficiency, a GFP expression plasmid had been included during transfection, and lysates of transfected cells were analyzed using an anti-GFP antibody (lower panel).

(C) Cytoplasmic and particle-associated vector RNA copy numbers were determined in cell cultures transfected with different amounts of the V^H vector plasmid in the presence or absence of Rev expression plasmid. In addition, pelletable p24CA levels were determined and used to calculate the packaging efficiency as particle-associated vector RNA copies per nanogram p24CA.