FIG. 3.

An N-terminal alpha-helix and the proline repeat element of Nef are required for efficient MHC-I down-regulation in HIV-1-infected cells. Two-dimensional FACS analysis of HIV-1-infected CEM-GFP cells. The horizontal axis shows phycoerythrin-conjugated anti-HLA-ABC (MHC-I). The vertical axis shows GFP fluorescence. The LTR-GFP reporter responds to Tat expression by increasing GFP production, thus allowing selection of GFP-positive infected cells for analysis. Cells were infected by coculture with virus-producing 293T cells. Wild-type and mutant Nef alleles were expressed within the context of the R9 virus, which expresses all HIV-1 accessory genes. A total of 2,000 cells were collected per condition. For the histograms, GFP-positive (infected) cells were collected for analysis of the MHC-I levels in infected cells; 5,000 cells were collected per condition. The results are representative of two independent experiments.