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 (Sgpsyn) and HIV-1 (Hgpsyn) (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 VH 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.