FIG. 4.

CA disassembly of WT and IN mutant viruses in vitro and in vivo. (A) Western blots illustrating disassembly of CA from the viral core. Gradient fractions corresponding to viral cores were pooled and placed at either 4°C or 37°C for 0, 60, or 120 min. Samples were then subjected to ultracentrifugation at 100,000 × g for 20 min at 4°C. The pellets were resuspended in 2% SDS sample buffer for Western blot analysis, and the blots were probed with a monoclonal anti-CA antibody. The blots were analyzed by chemiluminescence of the horseradish peroxidase (HRP)-conjugated secondary antibody and are representative of at least 3 independent experiments. (B) Disassembly kinetics of viral cores at 37°C. The Western blots shown in panel A were scanned, and ImageQuant TL software (GE Healthcare) was used to quantify band intensity. Percent disassembly was determined by using the value at time zero as the total p24 amount. The results are the mean values ± standard errors of the mean (SEM) of at least three independent experiments for WT (▴), NL-C130S (▪), and NL-ΔIN (⧫). (C) CA degradation in the cytoplasmic cell lysates of infected cells. HeLa CD4⁺ cells were infected with either WT, NL-C130S, or NL-ΔIN virus for 6 h at 37°C. The CA contents in cytoplasmic lysates were examined by Western blot analysis, and the blots were probed with polyclonal anti-CA antibodies (top). The cytoplasmic CypA content, probed with polyclonal anti-CypA antibody, served as a specificity control and was used to normalize the amount of lysate loaded onto the gel (bottom). The results are representative of two independent experiments.