Cooperative binding of the class I major histocompatibility complex cytoplasmic domain and human immunodeficiency virus type 1 Nef to the endosomal AP-1 complex via its mu subunit

Abstract

Human immunodeficiency virus type 1 Nef provides immune evasion by decreasing the expression of major histocompatibility complex class I (MHC-I) at the surfaces of infected cells. The endosomal clathrin adaptor protein complex AP-1 is a key cellular cofactor for this activity, and it is recruited to the MHC-I cytoplasmic domain (CD) in the presence of Nef by an uncharacterized mechanism. To determine the molecular basis of this recruitment, we used an MHC-I CD-Nef fusion protein to represent the MHC-I CD/Nef complex during protein interaction assays. The MHC-I CD had no intrinsic ability to bind AP-1, but it conferred binding activity when fused to Nef. This activity was independent of the canonical leucine-based AP-binding motif in Nef; it required residue Y320 in the MHC-I CD and residues E62-65 and P78 in Nef, and it involved the mu but not the gamma/sigma subunits of AP-1. The impaired binding of mutants encoding substitutions of E62-65 or P78 in Nef was rescued by replacing the Y320SQA sequence in the MHC-I CD with YSQL, suggesting that Nef allows the YSQA sequence to act as if it were a canonical mu-binding motif. These data identify the mu subunit of AP-1 (mu1) as the key target of the MHC-I CD/Nef complex, and they indicate that both Y320 in the MHC-I CD and E62-65 in Nef interact directly with mu1. The data support a cooperative binding model in which Nef functions as a clathrin-associated sorting protein that allows recognition of an incomplete, tyrosine-based mu-binding signal in the MHC-I CD by AP-1.

FIG. 1.

Nef-mediated down-regulation of MHC-I and direct binding to AP-1. (A) Nef-mediated modulation of MHC-I and CD4. T cells (SupT1 line) were transfected with plasmids expressing native Nef and green fluorescent protein (as a transfection marker). The next day, the cells were stained with antibodies to MHC-I-A2 (HLA-A2) and CD4, fixed, and analyzed by three-color flow cytometry. The histograms show cell numbers versus fluorescence intensities for the green fluorescent protein-positive (transfected) cells. “Mock” represents cells transfected with an empty plasmid. Vertical lines represent gates set using an isotype control antibody. The inset numbers are the mean fluorescence intensities of the entire (green fluorescent protein-positive) population. Alanine substitutions are indicated at the left. (B) GST pulldown of intact AP-1 from cytoplasmic lysates. GST proteins were produced in Escherichia coli, bound to glutathione-agarose beads, and quantified by electrophoresis and staining with Coomassie blue. Equal amounts of GST proteins were bound to the beads and incubated with cytoplasmic lysates from HeLa cells; the beads were washed, and the bound proteins were analyzed by SDS-PAGE and immunoblotting using an antibody to the γ subunit of AP-1. The “Input” lane was loaded with an aliquot of cytoplasmic lysate equal to 2% of the amount used in the pulldowns. The blot was also stained with Ponceau red to assess the loading of the GST fusion proteins. (C) Quantification of three independent GST pulldown experiments performed as described for panel B. The bands were quantified using ImageJ; the units are arbitrary. Error bars represent the standard deviations. WT, wild type.

FIG. 2.

GST pulldown of intact AP-1 from cytoplasmic lysates by use of chimeric proteins in which the MHC-I CD is attached to the N terminus of Nef (CD-Nef). (A) Schematic of the GST-MHC-I CD-Nef fusion protein. Key residues mutated herein are indicated. (B) GST pulldown of intact AP-1 from cytoplasmic lysates of HeLa cells. The assay was performed as described in the legend to Fig. 1. “LL/AA” indicates substitution of alanine for leucines 164 and 165 in the Nef ExxxLφ motif.

FIG. 3.

GST pulldown of intact AP-1 from cytoplasmic lysates by use of mutated MHC-I CD-Nef chimeras. Nef residues were mutated in the context of the LL164/165AA sequence to reveal their role in the LL-independent AP-1 binding activity of the MHC-I CD/Nef complex. The assay was performed as described in the legend to Fig. 1, except that the blot was probed with antibodies to both the γ and μ subunits of AP-1 to confirm that the interactions detected are with the intact AP-1 complex.

FIG. 4.

Interaction of Nef and the MHC-I CD-Nef chimera with subunits of AP-1 detected using yeast two- and three-hybrid assays. (A) Two-hybrid assay detecting interaction with μ1. Yeast cells were cotransformed with plasmids expressing the indicated proteins fused to the GAL4 DNA-binding domain and a plasmid expressing μ1 fused to the GAL4 activation domain. Cotransformed colonies were selected on plates lacking leucine and tryptophan, pooled, and either patch plated onto solid media containing histidine, followed by replica plating onto solid media containing (+His) or lacking (−His) histidine, or inoculated into liquid media lacking histidine. In each assay format, growth in media lacking histidine reflects an interaction between the indicated protein and μ1. “TGN38” contains a canonical Yxxφ sequence (SDYQRL); “TGN38Y/A” encodes a substitution of alanine for the tyrosine. Replica plates (left) were incubated at 30°C for 4 days. Growth in liquid media (right) was measured as OD₆₀₀. (B) Three-hybrid assay detecting interaction with the γ/σ1 hemicomplex of AP-1. Yeast cells were cotransformed with plasmids expressing the indicated proteins fused to the GAL4 DNA-binding domain, along with σ1 as a “bridge” protein, and a plasmid expressing γ-adaptin fused to the GAL4 activation domain. Cotransformed colonies were selected on plates lacking leucine and tryptophan, pooled, and inoculated into liquid media lacking histidine. Growth in media lacking histidine reflects an interaction between the indicated protein and the γ/σ hemicomplex of AP-1.

FIG. 5.

GST pulldown of μ1 translated in vitro using MHC-I CD-Nef chimeras. RNA encoding μ1 was transcribed using T7 RNA polymerase and translated using rabbit reticulocyte lysates in the presence of [³⁵S]methionine. GST pulldowns were performed as described in the legend to Fig. 1, except that the gels were stained with Coomassie blue and dried before autoradiography. The “Input” lanes were loaded with an aliquot of in vitro translation reaction product equal to approximately 2% of the amount used in the pulldowns. (A) The GST constructs used are the same as those used for Fig. 2. (B) Quantification of three independent GST pulldown experiments performed as described for panel A. The bands were quantified using ImageJ and normalized to an arbitrary value of 1.0 for GST only. Error bars represent the standard deviations. (C) Nef residues were mutated in the context of the LL164/165AA sequence to reveal their role in the LL-independent μ1-binding activity of the MHC-I CD/Nef complex. (D) GST fusion proteins contain Nef alone (without the MHC-I CD) to assess the role of residues in the interaction between Nef and μ1.

FIG. 6.

YSQA-to-YSQL mutation in the MHC-I CD rescues defects in AP-1 binding activity caused by mutation of Nef-E62-65 and -P78. GST pulldown assays using cytoplasmic lysates of HeLa cells were performed as described in the legend to Fig. 1. The blots were probed with an antibody to the γ subunit of AP-1.

FIG. 7.

Hypothetical model of the MHC-I CD/μ/Nef complex. Nef is yellow (18, 30); the μ subunit of AP-1 is shown as a ribbon structure, with blue indicating basic, positively charged residues (22); and residues SYSQAAS of the MHC-I CD are green. Key residues in Nef, the MHC-I CD, and μ1 are indicated. Y320 in the MHC-I CD presumably interacts with D174 in μ1, as in canonical Yxxφ motifs, whereas the hydrophobic pocket formed by μ1 residues V392 and L395, which typically accept the bulky, hydrophobic residue at position Y+3, is likely unoccupied. Nef-P78 and -E62-65 compensate for the lack of the latter interaction. In the case of the E62-65 sequence, binding activity is presumably based on an electrostatic interaction with a positive charge cluster on μ1. The precise role of Nef-P78 is unknown, but it may bind to another subunit of the complex. Structures were generated using DeepView (Swiss-Prot database).