Figure 5

Viral receptor down-regulation in HIV-infected MDMs. (A) MDMs prepared from an HIV-negative donor were infected with wt or variant viruses as indicated. The cells were stained with 7-AAD and allophycocyanin-Cy7 anti-human CD4 mAb (Biolegend, CA) and phycoerythrin-Cy7-conjugated anti-human CCR5 mAb (BD Biosciences) followed by intracellular staining with antibody against p24 Gag. In flow cytometric analysis, cells negative for 7-AAD and positive for p24 Gag were gated and analyzed for their fluorescence intensity for CD4 and CCR5. (B, C) The same experiment as above was done by using 3 additional HIV-negative donors. The relative down-regulation activity of wt Nef and its various variants toward CCR5 (panel B) and CD4 (panel C) is presented, with the wt Nef activity set to 100%. Data represent the means ± SD of all 4 donors, and statistical analysis was performed based on ANOVA with multiple comparisons vs. wt (Bonferroni t-test). n.s., not significant.