Infectivity and entry of Env cytoplasmic domain mutants. (A) Single round infectivity. Env-pseudotyped SG3ΔEnv viruses produced in 293T cells and p24-normalized, were used to infect TZM-bl indicator cells. After a 48 h incubation, the cell mixtures were lysed and luciferase activity was assayed. In this assay infection with virus pseudotyped with WT Env yielded 2.7 × 105 DLU and ∆Env virions an average of 1.65 × 104 DLU (B) Virus-cell fusion assay. Env CD mutants in NL4-3, pseudotyped with pCMV-BlaM-Vpr, were produced in 293T cells and subjected to gradient ultracentrifugation. Resuspended viral pellets were then normalized using p24 ELISA assays and used to infect TZM-bl indicator cells. The CCF2-AM fluorescent dye was loaded into the cells and incubated for 16 h at room temperature. The data represents results from three independent experiments conducted in triplicate. The error bars represent the standard deviation of the means. WT virus induced blue fluorescence in 5.48% of the 25,000 cells analyzed, a mock infected background of 0.46% blue cells was subtracted from all values.