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Comparative Study
. 2005 Nov;79(21):13317-25.
doi: 10.1128/JVI.79.21.13317-13325.2005.

The endoplasmic reticulum lumenal domain of the adenovirus type 2 E3-19K protein binds to peptide-filled and peptide-deficient HLA-A*1101 molecules

Affiliations
Comparative Study

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 et al. J Virol. 2005 Nov.

Abstract

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.

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Figures

FIG. 1.
FIG. 1.
Characterization of E3-19K. (A) SDS-PAGE (15%) analysis showing purification of the ER lumenal domain of E3-19K from High Five cell culture: lane 1, culture supernatant 70 h postinfection; lane 2, crude eluate from Ni-NTA column using a buffer containing 200 mM imidazole; and lane 3, E3-19K after purification by gel filtration chromatography. (B) Analysis of purified E3-19K by MALDI mass spectrometry showing three distinct regions: a single peak at 12.75 kDa and two clusters of peaks between 13.48 and 13.94 kDa and between 14.51 and 15.04 kDa. (C) SDS-PAGE (15%) analysis of purified E3-19K after incubation with an excess of PNGase F under denaturing conditions at 37°C for 16 h: lane 1, E3-19K without added PNGase F; and lane 2, E3-19K incubated with PNGase F. (D) Native PAGE (10%) analysis of purified E3-19K showing a single, compact band. The gel was run at 4°C in a buffer system consisting of 60 mM Tris plus 40 mM CAPS (pH 9.4).
FIG. 2.
FIG. 2.
Gel filtration analysis of purified E3-19K showing a single symmetric peak. Analysis was carried out on a Superdex 200 HR 10/30 column at 10°C in 20 mM Tris plus 150 mM NaCl (pH 7.5).
FIG. 3.
FIG. 3.
Circular dichroism. (A) Far-UV CD spectrum (200 to 250 nm) of E3-19K recorded at 10°C. (B) Thermal denaturation curve of E3-19K obtained by monitoring the change in CD signal at 213 nm. In both panels A and B, a 1-mm path-length cuvette was used with a protein concentration of 0.4 mg/ml in 2 mM HEPES plus 150 mM NaCl (pH 7.5). (C) Near-UV CD spectra (250 to 320 nm) of E3-19K recorded at 10 and 80°C. A 1-cm path-length cuvette was used with a protein concentration of 0.8 mg/ml in 2 mM HEPES plus 150 mM NaCl (pH 7.5).
FIG. 4.
FIG. 4.
Complex formation between E3-19K and HLA-A11 molecules. Samples of E3-19K (14 μg) and HLA-A11 molecules (20 μg; 2:1 molar ratio) were incubated in 20 mM Tris plus 150 mM NaCl (pH 7.5; buffer contained 10% glycerol for peptide-deficient HLA-A11 molecules) on ice for 30 min, followed by the addition of native gel loading buffer (50 mM Tris, 0.1% bromophenol blue, 10% glycerol [pH 6.8]). Samples were immediately loaded onto the native PAGE gel (10%): lane 1, E3-19K; lane 2, peptide-filled HLA-A11 molecules (HLA-A11/Nef); lane 3, E3-19K/HLA-A11/Nef complex; lane 4, peptide-deficient HLA-11 molecules; and lane 5, E3-19K/peptide-deficient HLA-A11 complex. The gel was run at 4°C in a buffer system consisting of 25 mM Tris plus 200 mM glycine (pH 8.3). Given that the pI of E3-19K (pI = 8.85) is higher than the pH of the buffer system (pH 8.3), uncomplexed E3-19K (lane 1) does not penetrate into the gel under these conditions due to its overall positive charge (refer to Fig. 1C for analysis of uncomplexed E3-19K on native PAGE gel).
FIG. 5.
FIG. 5.
Sedimentation velocity analysis of E3-19K/HLA-A11 complexes. Sedimentation velocity experiments showing complex formation between E3-19K and peptide-filled HLA-A11 molecules (curve A, E3-19K; curve B, HLA-A11/Nef; and curve C, E3-19K and HLA-A11/Nef mixed 1:1 at 15 μM) (top panel) and peptide-deficient HLA-A11 molecules (curve A, E3-19K [same data as in the top panel for comparison purposes]; curve B, peptide-deficient HLA-A11 molecules; and curve C, E3-19K and peptide-deficient HLA-A11 molecules mixed 1:1 at 11 μM) (bottom panel). Curves for the dilution series are not shown.
FIG. 6.
FIG. 6.
E3-19K stabilizes peptide-deficient HLA-A11 molecules. (A) SDS-PAGE (15%) analysis of products from thermolysin digests of peptide-deficient HLA-A11 molecules and of E3-19K/peptide-deficient HLA-A11 complex carried out at 15, 25, 37, and 45°C (for E3-19K/peptide-deficient HLA-A11 complex only). Lanes C1 and C2 represent proteins without added thermolysin and loaded at the same concentration as in other lanes. Digests were carried out at the indicated temperatures for 15 min at enzyme/substrate ratio of 1:100 in 20 mM Tris, 150 mM NaCl, 10% glycerol, and 2 mM CaCl2 (pH 7.5). (B) Quantitative analysis of the band corresponding to HLA-A11 heavy chain in panel A at different digest temperatures.
FIG. 7.
FIG. 7.
E3-19K does not block binding of antigenic peptides. E3-19K/HLA-A11 complexes were assembled and analyzed as described in Fig. 4. Lane 1, peptide-deficient HLA-A11 molecules; lane 2, peptide-deficient HLA-A11 molecules incubated with a 50-fold molar excess of Nef peptide on ice for 30 min; lane 3, E3-19K/peptide-deficient HLA-A11 complex; lane 4, E3-19K/peptide-deficient HLA-A11 complex incubated with a 50-fold molar excess of Nef peptide on ice for 30 min; lane 5, preformed E3-19K/HLA-A11/Nef complex. The native PAGE gel (10%) was run at 4°C in 25 mM Tris plus 200 mM glycine (pH 8.3).

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