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Journal of Virology, November 2005, p. 13317-13325, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13317-13325.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

The Endoplasmic Reticulum Lumenal Domain of the Adenovirus Type 2 E3-19K Protein Binds to Peptide-Filled and Peptide-Deficient HLA-A*1101 Molecules

Hong Liu,¹ Walter F. Stafford,² and Marlene Bouvier^1*

University of Connecticut, School of Pharmacy, Storrs, Connecticut 06269,¹ Boston Biomedical Research Institute, Watertown, Massachusetts 02472²

Received 24 May 2005/ Accepted 27 July 2005

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 (K_d) of 50 ± 10 nM for peptide-filled molecules and of about 10 µM 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.

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* Corresponding author. Mailing address: University of Connecticut, School of Pharmacy, 69 North Eagleville Rd., U-3092, Storrs, CT 06269. Phone: (860) 486-4355. Fax: (860) 486-4998. E-mail: bouvier{at}uconnvm.uconn.edu.

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Journal of Virology, November 2005, p. 13317-13325, Vol. 79, No. 21
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.21.13317-13325.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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