Figure 6

IID-IN mutant virions are not defective for RT activity and in vitro uncoating: A. Graphic representation of exogenous RT activity in IID-IN mutant virions. Viral lysates from 500 ng p24 each of HIV-1_NL4-3 WT and IID-IN mutant virions were tested for in vitro RT activity using a poly-rA template, oligo-dT primer, and dNTPs in the presence of ³²P-dTTP. RT activity was determined by incorporation of radiolabeled dTTP and measured by CPM in each reaction. Data represents average of three independent experiments (Mean +/- SEM). B. Quantitation of CA levels in cores purified from wild type and IID-IN mutant virions. Cores were isolated from concentrated virion suspensions by detergent treatment and density gradient ultracentrifugation, and the levels of core-associated CA were quantified as a percentage of the total CA protein in each gradient. Shown are mean values from three independent experiments; error bars represent SEM. C. Kinetic assay of HIV-1 uncoating in vitro. Purified cores were incubated at 37°C at indicated times prior to pelleting by ultracentrifugation, and the levels of CA in the supernatants and pellets were quantified by p24 ELISA. Shown are mean values from three independent experiments; error bars represent SEM.