Viral replication assays.

Viral replication assays were performed using recombinant HIV-1 mutants harboring single amino acid substitutions, as previously described (59, 66). In brief, recombinant HIV-1_NL4-3-based infectious clones were generated using the QuikChange II XL site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA) and transfected into HEK293T cells by using Attractene transfection reagent (Qiagen). The viruses were harvested at 72 h after the transfection, and viral p24 antigen was measured using a Lumipulse G1200 instrument. Replication of HIV-1 mutants was determined as described previously (59, 66), with minor modifications. Briefly, MT-4 cells (density, 1.5 × 10⁶ cells in 20 ml RPMI medium) were exposed to the HIV-1 mutants with 20 ng/ml of the p24 antigen and were cultured without the antiretroviral agents for 10 days. Fresh MT-4 cells (1.0 × 10⁶) were added on days 4 and 8. The amount of the p24 antigen was measured every 2 days, and the average value of each data point was determined from two independent assays.