Single-cycle infection assay.

A single-cycle infection assay was performed as described previously (50). Briefly, HIV-1 or SIV pseudoviruses were generated via the cotransfection of HEK293T cells with an Env-expressing plasmid and a backbone plasmid, pSG3Δenv, that encodes an Env-defective, luciferase-expressing HIV-1 genome. Culture supernatants were harvested 48 h after transfection, and 50% tissue culture infectious doses (TCID₅₀) were determined in TZM-bl cells. To measure the antiviral activity of inhibitors, peptides were prepared in 3-fold dilutions, mixed with 100 TCID₅₀ of viruses, and then incubated for 1 h at room temperature. The mixture was added to TZM-bl cells (10⁴ cells/well), and the cells were incubated for 48 h at 37°C. Luciferase activity was measured by using a luminescence counter (Promega), and IC₅₀s were calculated as described above.