FIG. 4.

Effects of a positively charged residue in the Nef AC on the downregulation of MHC-I. (A) CEM T cells transduced with vector alone (LXSN), wild-type SF2Nef, or SF2Nef with a lysine substitution for each of the individual core components of the Nef AC (⁶²KEEE⁶⁵, ⁶²EKEE⁶⁵, ⁶²EEKE⁶⁵, and ⁶²EEEK⁶⁵). Cells were stained for surface MHC-I and CD4 and were analyzed by flow cytometry. A representative example is shown from one of four independent experiments. (B) The top panel represents the effect of a basic residue in the Nef AC on the activation of PAK2. PAK2 activity was assayed from extracts of transduced CEM. The PAK2 activity of SF2Nef was set to 100. The negative control represents cells transduced with vector (LXSN). The bottom panel is a Western blot analysis of Nef expression in these extracts. (C) The average of four independent experiments was used to determine the effect of lysine substitutions in the AC on the percentage of cells downregulating either MHC-I (left) or CD4 (middle) or the activation of PAK2 by Nef (right), and the effects of the mutants compared to that of wild-type SF2Nef were analyzed by Student's t test. FITC, fluorescein isothiocyanate; PE, phycoerythrin.