Human Cytomegalovirus US2 Endoplasmic Reticulum-Lumenal Domain Dictates Association with Major Histocompatibility Complex Class I in a Locus-Specific Manner

Abstract

The human cytomegalovirus-encoded US2 glycoprotein targets endoplasmic reticulum-resident major histocompatibility complex (MHC) class I heavy chains for rapid degradation by the proteasome. We demonstrate that the endoplasmic reticulum-lumenal domain of US2 allows tight interaction with class I molecules encoded by the HLA-A locus. Recombinant soluble US2 binds properly folded, peptide-containing recombinant HLA-A2 molecules in a peptide sequence-independent manner, consistent with US2's ability to broadly downregulate class I molecules. The physicochemical properties of the US2/MHC class I complex suggest a 1:1 stoichiometry. These results demonstrate that US2 does not require additional cellular proteins to specifically interact with soluble class I molecules. Binding of US2 does not significantly alter the conformation of class I molecules, as a soluble T-cell receptor can simultaneously recognize class I molecules associated with US2. The lumenal domain of US2 can differentiate between the products of distinct class I loci, as US2 binds several HLA-A locus products while being unable to bind recombinant HLA-B7, HLA-B27, HLA-Cw4, or HLA-E. We did not observe interaction between soluble US2 and either recombinant HLA-DR1 or recombinant HLA-DM. The substrate specificity of US2 may help explain the presence in human cytomegalovirus of multiple strategies for downregulation of MHC class I molecules.

Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2

“…The C␣ rms deviation for HLA-A2͞Tax in the absence (18) and presence of US2 is Ϸ0.7 Å, similar to the value of 0.6 Å between the two free HLA-A2͞Tax complexes in an asymmetric unit (18). Indeed, a T cell receptor can still interact with the US2͞HLA-A2 complex (16).…”

“…The DR␤ and DM␤ chains most closely resemble the HLA-A2 region bound by US2, although the DR␣ and DM␣ chains are the subunits destabilized by US2 (30). In addition, incubation of recombinant US2 with either soluble HLA-DR or HLA-DM does not produce a gel shift by native electrophoresis (16). Other portions of the US2 and͞or DR͞DM molecules not contained in the soluble constructs may account for degradation of HLA-DR or HLA-DM.…”

“…Such locus specificity may allow HCMV-infected cells to avoid lysis by natural killer cells, which eliminate cells that have reduced surface expression of HLA-C, -E, or -G (28). Whereas incubation of soluble US2 with several recombinant HLA-A locus products produces a gel shift in native electrophoresis, several HLA-B complexes, HLA-Cw4 and HLA-E, do not shift in the presence of US2 (16). Sequence alignments of consensus sequences for class I locus products reveal that US2 binds to a relatively conserved region of class I heavy chains (Fig.…”

“…US2 and HLA-A2 form a 1:1 complex in solution (16) and are present in a 1:1 ratio in the crystal. However, packing in the crystal lattice causes each US2 molecule to make significant contacts with two HLA-A2͞Tax complexes at distinct locations ( Fig.…”

“…Conserved hydrophobic residues are blue, predicted N-linked glycosylation sites are orange, and cysteines predicted to form the A to G strand disulfide bond are magenta. US2 core residues (Ͼ90% buried, determined by using SPDBV (48) are indicated by F. with HLA-A locus products in vitro (16). We crystallized US2 refolded in the presence of recombinant of the HLA-A2͞human T lymphotropic virus type I Tax peptide complex.…”