FIG. 3.

Nef acts on the AP-2-dependent pathway to induce Ii chain upregulation. (A and B) HeLa-CIITA or HeLa-P4 cells were cotransfected with pSUPER CTL or pSUPER AP-2-μ2 and limiting amounts of pDsRed2-C1. Two days later, cells were transfected with the bicistronic plasmid Nef NL4-3 or Nef-STOP. After 24 h, cells were collected, stained for surface Ii chain or CD4 by using specific MAbs and Alexa 647-labeled secondary antibodies, and analyzed by flow cytometry. Doubly positive cells (DsRed⁺ GFP⁺) were gated and analyzed for FL4 staining. Within each group, corresponding to pSUPER CTL or pSUPER AP-2-μ2, MFI values obtained in FL4 with Nef-expressing cells were normalized with MFI from the respective control cells (Nef-STOP-transfected cells) and expressed as the degrees of Ii chain upregulation (A) or CD4 downregulation (B) (n-fold). Data from three independent experiments were used to calculate means and their standard errors. (C) HeLa cells stably expressing CD8-LL or CD8-AA chimeras were transfected with siRNA against pSUPER AP-2-μ2. After 72 h, the cells were harvested, labeled for cell surface CD8 by using a MAb and anti-mouse Alexa 647 antibody, and analyzed by flow cytometry. The mean FL4 fluorescence of siRNA-treated cells was normalized with the fluorescence of the respective mock-treated controls (CTRL) and is expressed as the degree of CD8 upregulation (n-fold). (D) The expression of a CD8 chimera containing a dileucine signal of endocytosis does not induce cell surface accumulation of the Ii chain. Indicated doses of plasmids encoding CD8-LL or CD8-AA were introduced into HeLa-CIITA cells by electroporation. Twenty-four hours later, cells were collected, stained for surface Ii chain, and analyzed by flow cytometry. MFI values and their means obtained in three independent experiments are shown.