Nef-induced CD4 and major histocompatibility complex class I (MHC-I) down-regulation are governed by distinct determinants: N-terminal alpha helix and proline repeat of Nef selectively regulate MHC-I trafficking

The Nef protein of primate lentiviruses triggers the accelerated endocytosi s of CD4 and of class I major histocompatibility complex (MHC-I), thereby d own-modulating the cell surface expression of these receptors. Nef acts as a connector between the CD4 cytoplasmic tail and intracellular sorting path ways both in the Golgi and at the plasma membrane, triggering the de novo f ormation of CD4-specific clathrin-coated pits (CCP). The downstream partner s of Nef in this event are the adapter protein complex (AP) of CCP and poss ibly a subunit of the vacuolar ATPase. Whether Nef-induced MHC-I down-regul ation stems from a similar mechanism is unknown. By comparing human immunod eficiency virus type 1 (HIV-1) Nef mutants for their ability to affect eith er CD4 or MHC-I expression, both in transient-transfection assays and in th e context of HIV-1 infection, it was determined that Nef-induced CD4 and MH C-I down-regulation constitute genetically and functionally separate proper ties. Mutations affecting only CD4 regulation mapped to residues previously shown to mediate the binding of Nef to this receptor, such as W57 and L58, as well as to an AP-recruiting dileucine motif and to an acidic dipeptide in the C-terminal region of the protein. In contrast, mutation of residues in an alpha-helical region in the proximal portion of Nef and amino acid su bstitutions in a proline-based SH3 domain-binding motif selectively affecte d MHC-I down-modulation, Although both the N-terminal alpha-helix and the p roline-rich region of Nef have been implicated in recruiting Src family pro tein kinases, the inhibitor herbimycin A did not block MHC-I down-regulatio n, suggesting that the latter process is not mediated through an activation of this family of tyrosine kinases.