FIG. 3.
Purification of 32P-labeled Gag particles. (A) Sedimentation analyses of 32P-labeled particles released from HIV-1 gag-expressing cells. BHK-21 cells were labeled with [32P]orthophosphate for 40 h and then infected with SFV-C/Pr55gag vectors. After these steps, labeling was continued, and released particles were collected and separated as described in the legend to Fig. 2A. The isolated particles were solubilized in hot SDS and analyzed for 32P-labeled SDS-phospholipid (SDS-PL) mixed micelles by SDS-PAGE (20%). Labeled RNA at the top of the gels and free orthophoshate (Pi) at the bottom are indicated. (B) Sedimentation analyses of 32P-labeled particles released from NPflu-expressing cells. Labeling of cells and analyses of particles were as described for panel A. (C) Quantification of 32P-labeled SDS-lipid mixed micelles from panels A and B. PL, phospholipids; PSL, photostimulated luminescence. (D) EM analysis of isolated particles. Particles in fractions 13 to 15 of the separation procedure shown in Fig. 2A were pooled, concentrated by centrifugation at the tip of a centrifuge tube, embedded in gelatin, processed for sectioning, and analyzed by EM. Note the ∼100-nm particles with an apparent Gag layer.