FIG. 3.

PF74 destabilizes the HIV-1 capsid. (A) Treatment of HIV-1 with PF74 reduces the level of CA recovered upon purification of HIV-1 cores. Concentrated HIV-1 particles were incubated with or without PF74 then subjected to ultracentrifugation through a layer containing Triton X-100. Fractions were collected from the gradient and analyzed for CA concentrations by ELISA. Shown is the quantity of CA in the fractions of each gradient corresponding to the density of viral cores, expressed as a percentage of the total CA protein in the gradient. (B) Analysis of the effects of 5 and 10 μM PF74 on CA recovery from wild-type and resistant (5Mut) HIV-1 particles. (C) Effects of PF74 on uncoating of HIV-1 cores. Purified wild-type and 5Mut cores were incubated at 37°C in the presence or absence of PF74 (0.25 μM). Wild-type cores were incubated for 30 min, and the 5Mut cores were incubated at 70 min due to their slower uncoating kinetics. After incubation, the extent of uncoating was determined by quantifying the percentage of CA released into soluble form. Shown are the mean values of triplicate parallel determinations from a single experiment. (D) Effect of PF74 on pelletable CA following virus entry into cells. Cultures of CrFK cells were inoculated with VSV-G-pseudotyped HIV-1 in the presence or absence of PF74 (10 μM). Four hours after inoculation, the cells were harvested and lysed, the lysates subjected to ultracentrifugation, and CA in the supernatants and pellets was quantified by ELISA. Shown are the mean values of triplicate parallel determinations, with error bars depicting the standard deviations. All experiments in this figure were performed at least three times, with similar outcomes.