Title

Adenoviral protein E3-19K and immune system evasion-the relationship between E3-19K and MHC class I

Date of Completion

January 2008

Keywords

Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The E3-19K protein from human adenoviruses retains class I MHC molecules in the endoplasmic reticulum. As a consequence, the cell surface expression of class I molecules is suppressed, allowing Ads to evade immune surveillance. ^ We have developed an expression system for the endoplasmic reticulum lumenal domain (residues 1 to 100) of adenovirus type 2 E3-19K tagged with a C-terminal His6 sequence in baculovirus-infected insect cells. 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 MHC 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 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. ^ Using native gel electrophoresis, gel filtration chromatography, and surface plasmon resonance, we show that soluble E3-19K associates with HLA-A and -B molecules; equilibrium dissociation constants were in the nanomolar range and ∼2.5-fold higher affinity for HLA-A relative to HLA-B molecules. Among the alleles of the HLAA locus examined, HLA-A*3101 associated ∼15-fold less avidly with soluble E3-19K. Soluble E3-19K interacted only very weakly with HLA-Cw*0304, and no interaction with HLA-Cw*0401 could be detected under identical conditions. ^ Together, our results suggest that Ads have evolved to exploit the late and early stages of the class I antigen presentation pathway. Moreover, a link may exist between host genetic factors and the susceptibility of individuals to Ad infections. ^