FIG. 4.

Gradient analysis of HIV-1 vRNPs isolated directly from virions. Concentrated virions were incubated in the presence of 0.5% Triton X-100 for 3 h at 37°C. Following incubation, the sample was loaded onto a 20-to-70% (wt/wt) sucrose gradient and subjected to ultracentrifugation at 100,000 × g for 16 h. Fractions were collected from the top of the gradient. (A) Proteins were precipitated with trichloroacetic acid and analyzed by Western blotting. (B) RNA was immobilized on nitrocellulose and viral RNA content was determined by hybridization with a ³²P-labeled HIV-1 probe, followed by phosphorimager quantification. Fraction densities were determined by refractometric analysis of sucrose concentrations.