FIG. 3.

Downregulation of cell surface MHC-I and CD4 molecules by patient-derived Nef proteins. T1 cells were infected by pseudotyped HIV vectors expressing nef sequences derived from the study subjects and downregulation of cell surface HLA A*02 and CD4 molecules was analyzed using flow cytometry. (A) In the upper panel, viable T1 cells were selected by examining forward and side scatter properties. In the lower panel, infected cells (M1 region) were detected by expression of mCD24 marker. The shaded area represents mock-infected T1 cells in a representative histogram. (B) Representative plots demonstrate HLA- A*02 (horizontal axis) and CD4 (vertical axis) downregulation in T1 cells (gated) infected by vectors expressing Nef from wild-type HIV (positive control) and four study subjects: the twins, 1-05 and 1-06, 1-03, and 1-10. Negative controls include LL>AA mutant (loss of CD4 downregulation), M20A mutant (loss of MHC-I downregulation), and a ΔNef mutant. Numbers represent percentages of cells in each quadrant of total infected T1 cells. (C, D) Histograms demonstrate functional loss of both HLA A*02 (C) and CD4 (D) downregulation by Nef from 1-03 and 1–10, functional loss of CD4 downregulation (D) in Nef from 1-06, and retained functions of Nef from 1-05. These results are representative of at least three independent experiments for each study subject.