Structural analysis of the adenovirus type 2 E3/19K protein using mutagenesis and a panel of conformation-sensitive monoclonal antibodies

Abstract

The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to MHC class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide-MHC complexes to the cell surface and consequently T cell recognition.Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress NK cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine-tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3-13, 15-21 and 41-45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed.Secondary structure prediction suggests that the lumenal domain contains six -strands and an -helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition.

Conserved Amino Acids within the Adenovirus 2 E3/19K Protein Differentially Affect Downregulation of MHC Class I and MICA/B Proteins

“…A similar pattern is seen for mutants E5 (control mutation of a nonconserved amino acid), T14, M87, and W96 (Fig. 3), whereas mutants P9, I37, W52, G55, Y60, V62, and V64 exhibited a 7-22-fold enhanced binding to mAb 3F4 [see also (57)]. This indicates that the 3F4 epitope comprising aa 41-45 and predicted to be on an internal loop between two b-strands becomes exposed by these mutations.…”

“…Thus, we conclude that M87 plays a crucial functional role in several E3/19K molecules and the loss of function is unrelated to the charge alteration. Most importantly, the minimal alterations in mAb binding (57) suggest that the mutation does not induce major conformational changes, indicating that M87 may be directly involved in HLA-I binding. Interestingly, in species D Ads methionine is replaced by leucine.…”

“…Tw1.3 (21) recognizes a discontinous, conformational epitope in Ad2 and Ad5 E3/19K. 3F4, 3A9, and 10A2 are specific for Ad2 E3/ 19K (57). Their epitopes were mapped to aa 3-13 (10A2), 15-21 (3A9), and 41-45 (3F4).…”

“…As the extent of HLA complex formation is dependent on the amount of E3/19K expressed, which is mainly determined by the transfection efficiency, the expression level of E3/19K was controlled with the C-tail serum. To obtain preliminary information about potential conformational changes, E3/19K was also precipitated with the conformation-sensitive mAb Tw1.3 that recognizes a discontinuous epitope (21,57). Examples of directly precipitated E3/19K using C-tail serum and Tw1.3 as well as coprecipitated E3/19K are shown in Fig.…”

“…2C). For example, although mutants I37 and V64 no longer bound Tw1.3, all mutants from F77 to P98 representing the so-called "conserved domain" were recognized with similar binding efficiency as Wt (56,57).…”