FIG. 3.
Replication of HIV-1 R71- and T71-Nef variants in cocultures of imDCs and autologous PBMC. (A) Average RT activities obtained after triplicate infection with the indicated HIV-1 Nef variants. (B) Average RT values ± standard deviations for the T71 and R71 groups of HIV-1 Nef variants. ImDCs (5 × 104) were isolated as described (14) and incubated with the R5-tropic HIV-1 NL4-3 005pf135 V3 Env variant expressing the indicated R71- or T71-Nefs for 90 min. The cells showed the characteristic features of imDCs, e.g., high expression of MHC-I and MHC-II, moderate expression of CD14, and relatively low expression of CD80, CD83 and CD86 compared to maDCs. Subsequently, the cells were washed twice with fresh medium to remove unbound virus and incubated overnight. One day after infection 2 × 105 nonactivated autologous PBMC were added to the cultures. Infections were performed in triplicate with virus stocks containing 2.5 ng p24 antigen. Virus production in the culture supernatant was determined by RT assay (19). The results were confirmed in an independent experiment.