FIG. 4.

Infectivity of mutant viruses. For each graph, the value of the WT was set at 1. Figures show the averages of results of at least two independent experiments. Error bars represent standard errors. (A) Single-round replication assay. M8166/H1Luc cells (1 × 10⁶) were infected with the same quantity of CA-p24 of WT or mutant viruses pseudotyped with HIV-2 Env. At 24 to 48 h postinfection, cells were lysed and luciferase activity in the cell lysate was measured. (B) CA-p24 production and RNA packaging ability. Quantities of CA-p24 and viral RNA of purified virions were measured with the enzyme-linked immunosorbent assay and the RNase protection assay, respectively. Packaging efficiency was calculated by dividing the quantity of viral RNA by that of CA-p24. (C and D) Viral DNA quantification at early infection steps. A total of 1 × 10⁶ MT-4 cells were infected with the same quantity of CA-p24 of WT or mutant viruses pseudotyped with HIV-2 Env. At 20 h postinfection, total cellular DNA was extracted and treated with DpnI overnight to digest methylated plasmid DNA. An equal amount of DNA was subjected to real-time PCR analysis. R/U5, strong-stop DNAs; U3, first-strand transferred products; Gag, negative-strand late products; 2ndTf, second-strand transferred products; 2LTR, 2-LTR viral circular DNA; Alu, PCR quantification for integrated proviral DNA; Infectivity, M8166/H1Luc cell assay as described for Fig. 4A.