Since we centrifuged the culture supernatant through 20% sucrose cushions, we assumed that the recovered Gag would be present in pelleted particles. To confirm that the recovered Gag was from VLPs, we observed supernatant samples ( Fig. 3B ) with a transmission electron microscope, and found spherical wt and mutant Gag particles with electron-dense cores in PI-treated transfectant samples (Figs. 3D and 3F). However, mature virions with cone-shaped cores were only detected in non-PI-treated wt transfectant samples (Fig. 3C ). Some vesicles lacking cores were noted, but virion-size particles containing electron-dense cores were not detected in mock-transfected samples, or barely detected in PRWWz transfectant supernatant that had not been treated with PI (Figs. 3E and 3G). These data support the hypothesis that the LZ-incurred assembly defect is PR activity-dependent.