The endoplasmic reticulum lumenal domain of the adenovirus type 2 E3-19K protein binds to peptide-filled and peptide-deficient HLA-A*1101 molecules.
E3-19K is a type I membrane glycoprotein expressed by adenoviruses (Ads) to modulate host antiviral immune responses. We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of Ad type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. In this system, recombinant E3-19K is secreted into the culture medium. A characterization of soluble E3-19K by analytical ultracentrifugation and circular dichroism showed that the protein is monomeric and adopts a stable and correctly folded tertiary structure. Using a gel mobility shift assay and analytical ultracentrifugation, we showed that soluble E3-19K associates with soluble peptide-filled and peptide-deficient HLA-A*1101 molecules. This is the first example of a viral immunomodulatory protein that interacts with conformationally distinct forms of class I major histocompatibility complex molecules. The E3-19K/HLA-A*1101 complexes formed in a 1:1 stoichiometry with equilibrium dissociation constants (Kd) of 50 +/- 10 nM for peptide-filled molecules and of about 10 microM for peptide-deficient molecules. A temperature-dependent proteolysis study revealed that the association of E3-19K with peptide-deficient HLA-A*1101 molecules stabilizes the binding groove. Importantly, our studies showed that peptide-deficient HLA-A*1101 molecules sequestered by E3-19K are capable of binding antigenic peptides and maturing into peptide-filled molecules. This firmly establishes that E3-19K does not block binding of antigenic peptides. Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway.
Liu
Hong
H
University of Connecticut, School of Pharmacy, 69 North Eagleville Rd., U-3092, Storrs, CT 06269, USA.
Stafford
Walter F
WF
Bouvier
Marlene
M
D000256:Adenoviridae / Q000276:immunology*; D017503:Adenovirus E3 Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016367:Baculoviridae / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004721:Endoplasmic Reticulum; D015234:HLA-A Antigens / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D066298:In Vitro Techniques; D010446:Peptide Fragments; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012995:Solubility
D017503:Adenovirus E3 Proteins; D015234:HLA-A Antigens; D010446:Peptide Fragments; D011994:Recombinant Proteins