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

Abstract

The Nef protein of primate lentiviruses triggers the accelerated endocytosis of CD4 and of class I major histocompatibility complex (MHC-I), thereby down-modulating the cell surface expression of these receptors. Nef acts as a connector between the CD4 cytoplasmic tail and intracellular sorting pathways both in the Golgi and at the plasma membrane, triggering the de novo formation of CD4-specific clathrin-coated pits (CCP). The downstream partners of Nef in this event are the adapter protein complex (AP) of CCP and possibly a subunit of the vacuolar ATPase. Whether Nef-induced MHC-I down-regulation stems from a similar mechanism is unknown. By comparing human immunodeficiency virus type 1 (HIV-1) Nef mutants for their ability to affect either CD4 or MHC-I expression, both in transient-transfection assays and in the context of HIV-1 infection, it was determined that Nef-induced CD4 and MHC-I down-regulation constitute genetically and functionally separate properties. 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 substitutions in a proline-based SH3 domain-binding motif selectively affected MHC-I down-modulation. Although both the N-terminal alpha-helix and the proline-rich region of Nef have been implicated in recruiting Src family protein kinases, the inhibitor herbimycin A did not block MHC-I down-regulation, suggesting that the latter process is not mediated through an activation of this family of tyrosine kinases.

Antigen Presentation and the Ubiquitin‐Proteasome System in Host–Pathogen Interactions

“…Nef itself contains a C-terminal dileucine motif that recruits a subunit of the tetrameric adaptor protein complex-2 (AP-2), a component of clathrin-coated pits at the cell membrane. Thus, Nef connects CD4 to AP-2 on clathrin-coated pits, triggering rapid CD4 endocytosis (Jin et al, 2004b(Jin et al, , 2005Mangasarian et al, 1999;Piguet et al, 1998Piguet et al, , 1999a. Nef can bind not only the AP-2 components of clathrin-coated pits, but also the regulatory V1H subunit of the vacuolar proton (H þ ) ATPase.…”

The MHC class I antigen presentation pathway: strategies for viral immune evasion

“…Taken together with previous mutational analyses of the Nef sequence a role for three motifs in MHC class I down-regulation is highlighted: 62 EEEE 65 , 72 PXXP 75 (where X can be any amino acid) and Met 20 within an amphiphatic a-helix. 77,79,80 These motifs are not functionally equivalent but act sequentially to promote MHC class I down-regulation by subverting the ARF6 pathway. 78 Mutation of either 62 EEEE 65 or 72 PXXP 75 prevents the internalization of MHC class I molecules by Nef.…”

The downregulation of CD4 and MHC‐I by primate lentiviruses: a paradigm for the modulation of cell surface receptors

“…The mechanism by which Nef dissociates CD4 from Lck is not clear yet. Nef can bind Lck as well as other protein kinases throngh a proline-rich motif located in its core domain and a more N-terminally located sequence (40-42), However, mutating either one of these determinants does not prevent Nef-indnced CD4 downregulation, even in T cells (39, 43,44). An interaction between Nef and Lck is therefore not a prerequisite for CD4 modulation,…”