Integrity of the VGF region is required for enhancement of virus infectivity and replication by Nef. Replication competent HIV-1 NL4.3 HSA-IRES-Nef reporter viruses (either Nef WT, AxxA, VGF→AAA, VGF→AGF, VGF→VAF, VGF→VGA, or Stop) were produced in 293 T cells and quantified by p24 measurement (A) Enhancement of virion infectivity by different Nef mutants in HeLa P4R5 LTR-β-galactosidase indicator cells. Percentage β-gal levels were calculated relative to wild-type HIV-1 NL4-3 HSA-IRES Nef. Values are the means of 3 independent experiments, and error bars represent SD from the mean; * indicates P < 0.05, ** indicates P < 0.01 and *** indicates P < 0.001. (B) Replication kinetics in human primary CD4+ T cells. Freshly isolated peripheral blood CD4+ T cells were stimulated for 48 h with IL-2/PHA before infection. After 1, 3, 5, 7, 9 and 11 days of culture, HIV replication was monitored by determining the amount of p24 antigen in the supernatants. Plots show the replication kinetics of the HIV reporter virus in CD4+ T cells derived from 3 different donors.