Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner - PubMed (original) (raw)

Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner

B E Gewurz et al. J Virol. 2001 Jun.

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.

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Figures

FIG. 1

Regions of US2 used for expression in E. coli. A schematic diagram of the US2 primary structure is shown at the top, with the single US2 disulfide bond and predicted transmembrane domain indicated. The series of US2 deletion mutants constructed for expression in E. coli are displayed beneath. The extent to which the polypeptides can be isolated from E. coli and refolded to yield soluble material is indicated to the right of each US2 mutant. NA, not applicable.

FIG. 2

The US2 ER-lumenal domain is sufficient for association with class I molecules. (A) Reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis of A21–275 (lane 1), β2m1–99 (lane 2), and US215–140 (lane 3) inclusion body material (10 μg each). The proteins were visualized by using Coomassie brilliant blue R-250 (Sigma). Numbers at left are molecular masses in kilodaltons. (B) Superimposed Superdex 75 gel filtration chromatograms of soluble US215–140, HLA-A2/Tax, and HLA-A2/Tax plus US215–140. The magnitude of the shift between the peak of the HLA-A2/Tax/US215/140 complex and free HLA-A2/Tax indicates 1:1 complex stoichiometry. The elution positions of molecular weight standards are shown above the chromatogram. (C) The HLA-A2/Tax/US215–140 complex associates with sufficient affinity to be further purified by Mono Q anion-exchange chromatography. Protein from the peak fraction was separated by nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining.

FIG. 3

US215–140 binds to five distinct HLA-A2/peptide complexes. US215–140 (15 μg) gel shifts HLA-A2 complexes (7.5 μg) containing the following peptides: group 1, LLFGYPVYV; group 2, ILKEPVHGV; group 3, ELAGIGILTV; group 4, ALGIGILTV; group 5, FLPSDFFPSV.

FIG. 4

The US2 ER-lumenal domain associates selectively with class I molecules. (A) US215–140 gel shifts HLA-A2 and HLA-Aw68. US215–140 (15 μg) was incubated with HLA-A2 and HLA-Aw68 complexes (10 μg each) as indicated, and proteins were separated by native gel electrophoresis. (B) US215–140 does not interact with similar affinity with molecules of other class I loci. US215–140 (15 μg) was incubated with HLA-B7, HLA-B27, HLA-Cw4, and HLA-E (10 μg each) as indicated and separated by native gel electrophoresis. (C) Superimposed Superdex 75 gel filtration chromatograms of HLA-B7 and HLA-B7 plus US215–140. (D) Superimposed Superdex 75 gel filtration chromatograms of US215–140, HLA-B27, and HLA-B27 plus US215–140. The presence of US215–140 does not alter the elution position of either HLA-B7 or HLA-B27. The elution positions of molecular weight standards are shown above the chromatograms in panels C and D.

FIG. 5

US215–140 and a TCR can simultaneously interact with the same HLA-A2 molecules. US215–140 (15 μg), HLA-A2/Tax (17 μg), and soluble A6 TCR (6.6 μg) were incubated as indicated, and proteins were separated by native gel electrophoresis. TCR alone is shown by the empty arrowhead, HLA-A2/Tax/TCR complex is shown by the asterisk, and HLA-A2/Tax/US215–140/TCR complex is shown by the filled arrowhead.

FIG. 6

US215–140 does not form high-affinity complexes with either HLA-DR or HLA-DM. US215–140 (15 μg) was incubated with HLA-DR (5 μg) or HLA-DM (10 μg) as indicated, and proteins were separated by native gel electrophoresis.

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Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner - PubMed (original) (raw)