TY - JOUR
T1 - 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
AU - Fu, Jie
AU - Bouvier, Marlene
N1 - Funding Information:
This work was supported, in whole or in part, by NIH/NIAID (Grant AI045070) and the University of Illinois at Chicago Center for Clinical and Translational Science (Award number UL1RR029879 from the National Center for Research Resources). The contribution of L. Visvabharathy in preparing E3-19K(1-93) is acknowledged.
PY - 2011/1
Y1 - 2011/1
N2 - 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.
AB - 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.
KW - Adenovirus
KW - Antigen presentation
KW - E3-19K
KW - Immune evasion mechanisms
KW - Immunomodulatory proteins
KW - MHC class I molecules
KW - Persistent viruses
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U2 - 10.1016/j.molimm.2010.10.017
DO - 10.1016/j.molimm.2010.10.017
M3 - Article
C2 - 21094528
AN - SCOPUS:78650613873
SN - 0161-5890
VL - 48
SP - 532
EP - 538
JO - Molecular Immunology
JF - Molecular Immunology
IS - 4
ER -