VNP Nefs enhance virion infectivity and viral replication. (A) P4-CCR5 (left) and TZM-bl (right) cells were infected with recombinant HIV-1 IRES-EGFP constructs expressing the indicated groups of nef alleles. Infections were performed in triplicate with two independent virus stocks. All panels give average values ± SD. RLU, relative light units. (B) Average levels of p24 antigen and (C) cumulative p24 production by PBMCs over 11 days of culture. PBMCs were either stimulated with PHA (1 μg/ml) for 3 days prior to infection with the X4-tropic HIV-1 NL4-3 clone or an R5-tropic derivative thereof  or infected immediately after isolation and stimulated three days later. Supernatants were collected at 2- or 3-day intervals, and productive HIV-1 infection was assessed by measuring p24 antigen content. The values were derived from three independent experiments. (D) Cumulative virus production in human lymphoid tissue (HLT) infected ex vivo. (E) Depletion of CD4+ T cells in HLT infected ex vivo. Tissues from six donors were infected with X4 HIV-1 NL4-3 expressing the indicated nef alleles, and cumulative p24 production by the tissue blocks over 15 days or CD4+ T-cell depletion at the end of culture was determined as described previously [25, 27]. Refer to the legend to Figure 2 for abbreviations, symbols, color coding and nef alleles analyzed.