An MHC-I cytoplasmic domain/HIV-1 Nef fusion protein binds directly to the mu subunit of the AP-1 endosomal coat complex.
BACKGROUND
The down-regulation of the major histocompatibility complex class I (MHC-I) from the surface of infected cells by the Nef proteins of primate immunodeficiency viruses likely contributes to pathogenesis by providing evasion of cell-mediated immunity. HIV-1 Nef-induced down-regulation involves endosomal trafficking and a cooperative interaction between the cytoplasmic domain (CD) of MHC-I, Nef, and the clathrin adaptor protein complex-1 (AP-1). The CD of MHC-I contains a key tyrosine within the sequence YSQA that is required for down-regulation by Nef, but this sequence does not conform to the canonical AP-binding tyrosine-based motif Yxxphi, which mediates binding to the medium (micro) subunits of AP complexes. We previously proposed that Nef allows the MHC-I CD to bind the mu subunit of AP-1 (micro1) as if it contained a Yxxphimotif.


RESULTS
Here, we show that a direct interaction between the MHC-I CD/Nef and micro1 plays a primary role in the down-regulation of MHC-I: GST pulldown assays using recombinant proteins indicated that most of the MHC-I CD and Nef residues that are required for the down-regulation in human cells contribute to direct interactions with a truncated version of micro1. Specifically, the tyrosine residue of the YSQA sequence in the MHC-I CD as well as Nef residues E62-65 and P78 each contributed to the interaction between MHC-I CD/Nef and micro1 in vitro, whereas Nef M20 had little to no role. Conversely, residues F172/D174 and V392/L395 of the binding pocket on micro1 for Yxxphi motifs were required for a robust interaction.


CONCLUSIONS
These data indicate that the MHC-I cytoplasmic domain, Nef, and the C-terminal two thirds of the mu subunit of AP-1 are sufficient to constitute a biologically relevant interaction. The data also reveal an unexpected role for a hydrophobic pocket in micro1 for interaction with MHC-I CD/Nef.
Singh
Rajendra Kumar
RK
Department of Medicine, University of California San Diego, La Jolla, California, United States of America. rajendra84@hotmail.com
Lau
David
D
Noviello
Colleen M
CM
Ghosh
Partho
P
Guatelli
John C
JC
D033983:Adaptor Protein Complex mu Subunits / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D015536:Down-Regulation; D011992:Endosomes / Q000378:metabolism*; D015395:Histocompatibility Antigens Class I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
D033983:Adaptor Protein Complex mu Subunits; D000596:Amino Acids; D015395:Histocompatibility Antigens Class I; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1