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

• Published in: Molecular immunology 2011-01, Vol.48 (4), p.532-538
• Main Authors: Fu, Jie, Bouvier, Marlene
• Format: Article
• Language: English
• Subjects: Adenoviridae; Adenovirus; Adenovirus E3 Proteins - chemistry; Adenovirus E3 Proteins - metabolism; Amino Acid Sequence; amino acid sequences; Amino Acids; Antigen presentation; binding capacity; Chymotrypsin - metabolism; Conserved Sequence; conserved sequences; cytotoxic T-lymphocytes; E3-19K; endoplasmic reticulum; Endoplasmic Reticulum - metabolism; histocompatibility antigens; Histocompatibility Antigens Class I - metabolism; HLA-A Antigens - metabolism; HLA-A11 Antigen; host-pathogen relationships; Humans; Immune evasion mechanisms; Immunomodulation; Immunomodulatory proteins; major histocompatibility complex; MHC class I molecules; Molecular Sequence Data; Mutagenesis, Site-Directed; pathogenesis; Peptide Fragments - metabolism; Persistent viruses; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; protein-protein interactions; Solutions; Structure-Activity Relationship; structure-activity relationships; tyrosine; Tyrosine - metabolism; viral proteins
• ISSN: 0161-5890; 1872-9142
• DOI: 10.1016/j.molimm.2010.10.017

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 Tyr93, but not Tyr88, critically modulates the cell-surface expression of MHC class I molecules. Taken together, these results indicate that the sequence comprising residues 89–93 (M89SKQY93), and in particular Tyr93, 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.