title: Determinants of the endoplasmic reticulum (ER) lumenal-domain of the adenovirus serotype 2 E3-19K protein for association with and ER-retention of major histocompatibility complex class I molecules.
abstract: The E3-19K immunomodulatory protein from adenoviruses (Ads) inhibits antigen presentation by major histocompatibility complex (MHC) class I molecules. As a result, the ability of Ad-specific cytotoxic T lymphocytes (CTLs) to lyse infected cells is suppressed. The ER-lumenal domain of E3-19K is subdivided into a variable (residues 1 to ∼78/81) and conserved (residues ∼79/82 to 98) region followed by a linker (residues 99-107). Using molecular and cellular approaches, we characterized in detail the properties of the ER-lumenal domain of E3-19K that enable it to target MHC class I molecules. Proteolysis of recombinant serotype 2 E3-19K (residues 1-100) (with six His residues) generated a large N-terminal (residues 1-88) and a small C-terminal fragment (residues 94-100) in solution. Neither of these fragments associates with HLA-A*1101 as shown by a native gel band-shift assay. In contrast, the N-terminal 1-93 residues of Ad2 E3-19K exhibited the same binding affinity to HLA-A*1101 as E3-19K. Using a site-directed mutational analysis and flow cytometry, we show that Tyr(93), but not Tyr(88), critically modulates the cell-surface expression of MHC class I molecules. Taken together, these results indicate that the sequence comprising residues 89-93 (M(89)SKQY(93)), and in particular Tyr(93), in the conserved region of E3-19K is critical for its immunomodulatory function. Residues 89-93 likely form a linker or loop in E3-19K. Overall, our data provide novel insights into the structure of E3-19K and identify key determinants for association with and ER-retention of its cellular target protein. This knowledge is important for our understanding of the molecular basis of Ad pathogenesis.
authors.affiliation: Department of Microbiology and Immunology, University of Illinois at Chicago, College of Medicine, 835 S Wolcott, Chicago, IL 60612, United States.
authors.forename: Jie
authors.initials: J
authors.lastname: Fu
authors.forename: Marlene
authors.initials: M
authors.lastname: Bouvier
mesh_terms: D017503:Adenovirus E3 Proteins / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids; D002918:Chymotrypsin / Q000378:metabolism; D017124:Conserved Sequence; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D015234:HLA-A Antigens / Q000378:metabolism; D059870:HLA-A11 Antigen; D015395:Histocompatibility Antigens Class I / Q000378:metabolism*; D006801:Humans; D056747:Immunomodulation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012996:Solutions; D013329:Structure-Activity Relationship; D014443:Tyrosine / Q000378:metabolism
chemical_list: D017503:Adenovirus E3 Proteins; D000596:Amino Acids; D015234:HLA-A Antigens; C515740:HLA-A*11:01 antigen; D059870:HLA-A11 Antigen; D015395:Histocompatibility Antigens Class I; D010446:Peptide Fragments; D012996:Solutions; D014443:Tyrosine; D002918:Chymotrypsin