Fig 3.

Fate of the wild-type and mutant SIVmac239 capsids in infected cells. Cf2Th cells were incubated at 37°C with equivalent concentrations of VSV-G-pseudotyped viruses, based on RT units. At the indicated times, the infected cells were mechanically disrupted, and their cytosolic fractions were layered atop a 50% sucrose column, saving some to measure the “input.” After centrifugation, a “soluble” capsid sample was removed from near the top of the column, and the pelletable capsid was collected at the bottom of the tube following column removal. Input, soluble, and pelletable fractions were loaded onto a 4 to 12% Bis-Tris gel, Western blotted, and probed with anti-SIVmac251 polyclonal antibody. (A) The results of a single experiment are shown, with the fate of capsid measured at 16 h postinfection (p.i.). (B) Three independent infections were assayed for pelletable capsid at 16 h postinfection. The amounts of pelletable capsid were determined by densitometric analysis of Western blots, adjusted for input and normalized to the signal for WT SIVmac239, which was set at 100%. The A87E and A87D mutants had significantly more pelletable capsid than wild-type SIVmac239 (*, P < 0.01). (C) Time course of the fate of the capsid in infected cells. After the initiation of infection, the amounts of input and pelletable fractions containing CA were assayed every 4 h until 28 h postinfection. A parallel infection of recombinant WT SIVmac239 without a VSV-G glycoprotein (no Env) was used as a negative control. Data from one of two independent experiments, which generated similar results, are shown.