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. Author manuscript; available in PMC: 2017 Aug 15.
Published in final edited form as: J Immunol. 2016 Jul 6;197(4):1399–1407. doi: 10.4049/jimmunol.1600541

Structure of the Adenovirus Type 4 (Species E) E3-19K/HLA-A2 Complex Reveals Species-Specific Features in MHC I Recognition

Lenong Li *, Bernard D Santarsiero †, Marlene Bouvier *
PMCID: PMC4975982  NIHMSID: NIHMS793624  PMID: 27385781

Abstract

Adenoviruses (Ads) subvert MHC class I antigen presentation and impair host anti-Ad cellular activities. Specifically, the Ad-encoded E3-19K immunomodulatory protein targets MHC class I molecules for retention within the endoplasmic reticulum (ER) of infected cells. Here, we report the x-ray crystal structure of the Ad type 4 (Ad4) E3-19K of species E bound to HLA-A2 at 2.64 Å resolution. Structural analysis shows that Ad4 E3-19K adopts a tertiary fold that is shared only with Ad2 E3-19K of species C. A comparative analysis of the Ad4 E3-19K/HLA-A2 structure with our x-ray structure of Ad2 E3-19K/HLA-A2 identifies species-specific features in HLA-A2 recognition. Our analysis also reveals common binding characteristics that explain the promiscuous, and yet high-affinity, association of E3-19K proteins with HLA-A and -B molecules. We also provide structural insights into why E3-19K proteins do not associate with HLA-C molecules. Overall, our study provides new information into how E3-19K proteins selectively engage with MHC I to abrogate antigen presentation and counteract activation of CD8+ T-cells. The significance of MHC I antigen presentation for controlling viral infections, and the threats of viral infections in immunocompromised patients, underline our efforts to characterize viral immunoevasins such as E3-19K.

Keywords: Adenovirus, immune evasion mechanism, immunoevasin, MHC class I

Introduction

Human adenoviruses (Ads) are common pathogens that comprise at least 70 types (Ad1-Ad70) classified into seven species (A–G) (1–3). Ads cause a variety of clinical symptoms involving the gastrointestinal, upper respiratory, and ocular systems (4). Although healthy adults can generally control the virus, a large number of infected individuals develop persistent lifelong infections. In immunocompromised individuals, such as children, transplant recipients, and AIDS patients, Ad infections can be severe and even fatal (5–9). The ability of Ad to persist in infected cells presupposes that the virus is capable of interfering with host antiviral cellular immune responses. Consistent with this, it was shown that the E3-19K protein, the most abundantly expressed protein of the early transcription unit 3 of Ads, modulates host immune functions (10,11). E3-19K binds to and retains MHC class I molecules within the endoplasmic reticulum (ER) of infected cells (10–14). Specifically, the ER-lumenal domain of E3-19K associates with the ER-lumenal domain of MHC I (15–22), while the transmembrane domain of E3-19K and the dilysine motif in its cytoplasmic tail provide signals for sequestration of the E3-19K/MHC I complex within the ER (12–14, 23). As a consequence, Ad-infected human and mouse cells have an impaired ability to present MHC I-associated viral peptides, making infected cells less sensitive to lysis by CTLs (15, 24–28). In animal models, it was shown that E3-19K-mediated effects alter host inflammatory responses to the virus (29, 30). Taken together, in vitro and in vivo data strongly support an immunomodulatory role for E3-19K during human Ad infections.

We determined previously the three-dimensional structure of the Ad type 2 (Ad2) of species C E3-19K/HLA-A2 complex (31). The structure showed that Ad2 E3-19K adopts a novel tertiary fold and uses a new binding surface on HLA-A2. Here, we set out to characterize how the structure of E3-19K and its mode of interaction with HLA-A2 vary between different Ad species. This is an intriguing question given that all E3-19K proteins characterized thus far were shown to have an MHC I-binding function, but yet their ER-lumenal domains share only ~35% sequence homology across Ad species. Furthermore, we and others showed that the affinities of E3-19K proteins for a given MHC I molecule of the HLA-A and -B loci vary significantly across Ad species and that, interestingly, E3-19K proteins do not associate with HLA-C molecules (16, 32–34). The locus-specific recognition and downregulation of MHC I molecules by other viral immunomodulatory proteins was reported previously (35–39). Presumably, such strategies allow virally infected cells to escape recognition by CD8+ T-cell receptors, while also preventing activation of NK cell inhibitory receptors that recognize HLA-C molecules (missing “self”) (37, 39).

To advance our understanding of how the amino acid sequences of Ad E3-19K proteins affect their tertiary structures and interaction with MHC I molecules, and to gain insights into the molecular basis for their selective recognition of MHC I, we determined the x-ray crystal structure of Ad4 (species E) E3-19K bound to HLA-A2. An analysis of the structure allows us to identify similarities and differences in the binding modes of Ad4 and Ad2 E3-19K, as well as common and unique contact sites on HLA-A2. Moreover, from our results, we make some predictions on the structures of E3-19K proteins of species B and D, which have not yet been determined. Overall, our study provides new molecular insights into how Ad uses its E3-19K proteins to selectively subvert MHC I antigen presentation, with implications on disabling CD8+ T-cell and NK cell activities.

Materials and Methods

Cloning and protein expression

The cDNA encoding the ER-lumenal domain (residues 1-108) of Ad4 E3-19K was generated by polymerase chain reaction as described previously (33). Plasmids harboring the DNA sequences for HLA-A*0201 heavy chain (residues 1-275) and β2-microglobulin (β2m) (residues 0-99) were kind gifts of Dr. Don C. Wiley (deceased). Ad4 E3-19K, HLA-A2 heavy chain, and β2m were expressed in the Escherichia coli strain BL21(DE3)pLysS (Stratagene, La Jolla, CA) as inclusion bodies. Inclusion bodies were purified from cell pellets and solubilized as described previously (40).

In vitro assembly of HLA-A2/Tax and Ad4 E3-19K/HLA-A2

HLA-A2/Tax was reconstituted from the urea-solubilized inclusion bodies of HLA-A2 heavy chain (1 μM) and β2m (2 μM) in the presence of the Tax peptide (LLFGYPVYV) (10 μM) in an oxidative refolding buffer (40). Stock solutions of purified HLA-A2/Tax (10–30 mg/ml) in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl were kept at −80°C. The Ad4 E3-19K/HLA-A2 complex was assembled using a modified version of this approach, as we described previously (31). Briefly, the urea-solubilized inclusion bodies of Ad4 E3-19K were added at 4°C under rapid stirring, to a final concentration of 1 μM, into an oxidative refolding buffer containing a submolar amount (0.2 μM) of refolded, purified HLA-A2/Tax. The refolding mixture was incubated at 4°C. After 24 hours, the mixture was concentrated in an Amicon stirred cell followed by purification of the mixture on a Superdex 200 HR 10–30 column in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl. The purified Ad4 E3-19K/HLA-A2 complex generated from this rescue refolding strategy was well behaved from analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gel and native PAGE gel. Stock solutions of purified Ad4 E3-19K/HLA-A2 (20–40 mg/ml) in 20 mM Tris-HCl (pH 7.5), 100 mM NaCl were kept at −80°C.

Native gel band-shift assay

Samples of Ad4 and Ad2 E3-19K proteins (13 μg) were incubated on ice with both HLA-A2 and HLA-A2 mutants (20 μg) (2:1 molar ratio) in 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl for 30 minutes. The mixtures were loaded into a native PAGE gel (10%) and run at 4°C in 25 mM Tris-HCl (pH 8.4) and 200 mM glycine. Proteins were visualized with Coomassie blue R-250. Note that the pI of Ad2 E3-19K is 8.85; when pIs of proteins are above the pH of the running buffer (pH 8.4), they do not penetrate into the native gel.

Crystallization and x-ray data collection

Crystallization conditions of the Ad4 E3-19K/HLA-A2 complex were setup with the aid of a Tecan Freedom EVO 200 robot using sitting drops. Complex solution (1 μL, 10 mg/ml) was dispensed into each well of a Corning 96-well plate, mixed with an identical volume of precipitant solution, and equilibrated against 100 μL of reservoir solution. The plates were incubated at 18°C. The initial crystallization condition was found using Crystal Screen Index (Hampton Research) as solution #30, 1.5 M ammonium sulfate, 0.1 M NaCl, and 0.1 M Bis-Tris (pH 6.5). Optimization of the crystallization condition was carried out using the Additive Screen (Hampton Research) and the hanging-drop vapor-diffusion method. Crystals used for data collection were grown by mixing 2 μL of 10 mg/ml protein complex solution with 2 μL of 1.5 M ammonium sulfate, 0.1 M NaCl, 0.1 M Bis-Tris (pH 6.5) and 0.2 μL of 30% 1,6-diaminohexane as additive. Crystals grew over 15 days to dimensions of ~300 × 150 × 100 μm. Prior to data collection, the crystals were soaked for about 1–2 minutes in mineral oil (Sigma) with 1.5 M ammonium sulfate, 0.1 M NaCl, and 0.1 M Bis-Tris (pH 6.5) as the cryoprotectant and then flash-frozen in liquid nitrogen. X-ray diffraction data for the Ad4 E3-19K/HLA-A2 complex was collected to 2.64 Å resolution with a MAR-225 CCD detector at LS-CAT beamline 21-ID-F of the Advanced Photon Source (Argonne National Laboratory, Argonne, IL). The space group was determined to be P3 (see Table I). Data were processed and scaled with the HKL-2000 program package (41).

Table I.

X-ray data processing and refinement statistics

Data collection
Wavelength (Å) 1.00
Space group P3
Unit cell parameters
 a, b, c (Å) 165.73, 165.73, 122.86
 α, β, γ(°) 90.0, 90.0, 120.0
Resolution (Å) 100-2.64 (2.69-2.64)a
Unique reflections 110878
Rmerge 0.172 (0.619)
Redundancy 7.9 (6.7)
Completeness (%) 100 (100)
<I/σI> 23.4 (2.5)
Refinement
Resolution (Å) 49.62-2.64
Rwork/Rfreeb 0.266/0.285
Twin law (-h, -k, l) 0.47
Number of protein atoms 22994
Number of water molecules 40
Rms deviations
 Bond lengths (Å) 0.006
 Bond angles (°) 1.2
B-factors (Å2)
 Protein 57.3
 Water 20.7
Ramachandran plotc
 Most favored regions (%) 82.1
 Allowed regions (%) 16.8
 Generously allowed regions (%) 1.1
 Disallowed regions (%) 0
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a

Values in parentheses are for highest-resolution shell.

b

Rwork and Rfree are defined as Σ||Fobs|-|Fcalc||/Σ|Fobs| for the reflections in the working or the test set, respectively.

c

As defined by PROCHECK (46).

Structure determination and refinement

The structure of Ad4 E3-19K/HLA-A2 complex was determined by molecular replacement in AMoRe (42) using free HLA-A2/Tax (PDB 1HHK) as a search model. Six solutions were identified for both the rotation and translation functions, corresponding to six molecules of Ad4 E3-19K/HLA-A2 in the asymmetric unit. After molecular replacement and an initial refinement with REFMAC (CCP4) (43), the electron density maps were calculated and examined manually using Coot (44), and residues with a poor fit to the electron density map were omitted from the model. Subsequent model rebuilding and refinement was carried out in Coot and phenix.refine (45). Phenix.refine was then used for several rounds of refinement with the imposition of non-crystallographic symmetry restraints and application of the appropriate twin law (-h,-k, l) with a twin fraction of 0.470 as suggested from Xtriage (45). In the final rounds of refinement the non-crystallographic symmetry restraints were released, and a composite omit map was calculated to reduce model bias. Water molecules were added and checked with the FO-FC electron density map (>3σ) at 3.8 Å, or less, from hydrogen bond donors or acceptors. A total of 40 water molecules were added to the model. Throughout refinement, agreement between the model and the observed data was monitored by calculating Rfree based on 2% of the reflections. The final Rwork and Rfree values (with a bulk solvent correction) are 25.7% and 28.8%, respectively, for all reflections between 49.62-2.64 Å (Table I). In the final model, the structural geometry was checked using PROCHECK (46). All backbone ϕ–ψ torsion angles of the model were within allowed regions of the Ramachandran plot. Because of poor quality or missing electron density, the final model does not include residues 273-275 of chains A,E,I,M,Q,U, residues 97-100 of chains C,G,K,O,S,W, residues 1-6 of chain D, and residues 1-5 of chains H,L,P,T,X. Buried surface areas were calculated with the program Areaimol in the CCP4 package, using a probe radius of 1.4 Å. All structural figures were prepared using PyMol (47).

Results

Structure of the Ad4 E3-19K/HLA-A2 complex

The Ad4 E3-19K/HLA-A2 complex was assembled using an in vitro rescue-refolding strategy that we described previously (31). Briefly, the inclusion bodies of Ad4 E3-19K were injected into a refolding buffer containing a submolar amount of HLA-A2/LLFGYPVYV complex; the presence of folded HLA-A2 in the buffer was absolutely necessary to promote formation of Ad4 E3-19K/HLA-A2 complex (see Materials and Methods). The structure of Ad4 E3-19K/HLA-A2 was determined to 2.64 Å by molecular replacement from twinned crystals (see Materials and Methods, and Table I). Only minor differences were observed between the six molecules of E3-19K in the asymmetric unit (rms deviation of 0.31–0.39 Å for 103 equivalent Cα atoms) such that only chain B will be discussed. We observed clear electron density for most residues of the structure: residues 6-108 of Ad4 E3-19K, residues 1-271 of HLA-A2 heavy chain, residues 0–97 of β2m, and HIV-1 Tax LLFGYPVYV peptide.

The overall structure of the Ad4 E3-19K/HLA-A2 complex is shown in Fig. 1A. Ad4 E3-19K binds at the N-terminal end of the HLA-A2 groove contacting the N-terminus of the α1-helix, C-terminus of the α2-helix, a loop of the α3-domain, and β2m subunit. This mode of binding is reminiscent of the way Ad2 E3-19K associates with HLA-A2/LLFGYPVYV (31), with unique features (see below).

FIGURE 1.

FIGURE 1

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The structure of Ad4 E3-19K/HLA-A2. (A) Ribbon representation of the Ad4 E3-19K/HLA-A2 structure: E3-19K, slate; HLA-A2 heavy chain (α1-, α2-, and α3-domains), yellow; β2m, green; HIV-1 Tax peptide (LLFGYPVYV), orange; and disulfide bonds, magenta. The N- and C-termini of Ad4 E3-19K are labeled. (B) Ribbon representation of the complex structure of Ad4 E3-19K (top) and topology diagram (bottom). The N-terminal domain (residues 6-86), α-helix (residues 87-101), and tail (residue 102-108) are indicated. The β-strands are represented with arrows, and α-helix as a cylinder. The β-strands and N6- and C108-termini are labeled. (C) Ribbon representation of the complex structure of Ad4 E3-19K showing the side chains of hydrophobic core-forming residues (slate) (only some side chains were labeled for clarity). Core-forming residues (>90% buried) were determined by SPDBV (48). The β-strands and N- and C-termini are labeled. The regions of major conformational differences (RMS deviation greater than 2.5 Å) between Ad4 E3-19K and Ad2 E3-19K (superimposition of Ad4 E3-19K/HLA-A2 and Ad2 E3-19K/HLA-A2) are colored in green and labeled I-VI (see also legend of Fig. 3A).

The Ad4 E3-19K structure (Fig. 1B) consists of a long N-terminus segment followed by a large domain formed by two antiparallel β-sheets defined by β-strands A, B, and D and β-strands C, E, and F. The two β-sheets overlap only slightly with each other and adopt a V-shaped arrangement. The N-domain is followed by a short α-helix and then a tail segment. There are a number of critical interactions along the entire length of the Ad4 E3-19K structure. First, the three disulfide bonds are strategically positioned (Fig. 1B and 1C): Cys10/Cys39 links the long N-terminus segment (Lys6 to Lys17) to the BC-loop (loop connecting β-strands B and C), Cys18/Cys35 connects β-strands A and B, and Cys29/Cys91 keeps the α-helix packed against the large N-domain. Second, a number of residues form a well-defined hydrophobic core (Fig. 1C). These residues are: for the ABD β-sheet, Val31 and Ile33 (β-strand B) and Leu55 (β-strand D), and for the CEF β-sheet, Val42, Ile44, and Tyr46 (β-strand C), Tyr67 and Val69 (β-strand E), and Asn83 (β-strand F). Other residues contributing to the hydrophobic core are Asn53, Trp59, and Asp63 of the large N-terminus domain, Phe87 and Met95 of the α-helix, and Trp104 of the tail. Finally, several hydrogen bonds contribute to the well-ordered electron density of the long N-terminus segment (Lys6 to Lys17): the main chain of Pro9 forms a hydrogen bond with the side chain of Thr12, and the main chain of Asp16 forms a hydrogen bond with the side chain of Asn83. A hydrogen bond between the main chain atoms of Ser98 and Met103 is also critical for stabilizing the α-helix against the N-terminus domain. Overall, Ad4 E3-19K adopts a similar tertiary fold as Ad2 E3-19K (31), a fold unique to this family of proteins, but also shows distinct structural features (see below).

The Ad4 E3-19K/HLA-A2 binding interface

The Ad4 E3-19K/HLA-A2 binding interface shields a molecular surface area of 2112 Å2 from solvent. The shape complementarity coefficient, Sc, of the complex was determined to be 0.66 (Sc = 1 for perfect geometrical fits) (49). We divided the overall binding interface into four sites (sites 1–4) (Fig. 2). Site 1, located at the N-terminus of the α1-helix on HLA-A2, is defined by numerous hydrophobic contacts involving Gln23 (β-strand A), Trp37 (BC-loop), and both Thr54 and Ala56 (β-strand D) of E3-19K and a number of HLA-A2 heavy chain residues. Site 2, located at the C-terminus of the α2-helix on HLA-A2, is characterized by a few hydrogen bonds mediated by Lys49 and Arg51 (CD-loop) and Ala56 (β-strand D) of E3-19K and Asn174 and Glu177 of HLA-A2 heavy chain. A number of hydrophobic contacts also contribute to site 2. Site 3 is more localized and comprises a hydrogen bond between Gly26 (AB-loop) of E3-19K and Asp238 of the α3-domain of HLA-A2 heavy chain. Finally, site 4 is defined by hydrogen bonds involving Gln100 and Tyr101 (α-helix) of E3-19K and lys19 and Ser20 of β2m. This region is also characterized by numerous hydrophobic contacts.

FIGURE 2.

FIGURE 2

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Interaction surface between Ad4 E3-19K and HLA-A2. Interactions at sites 1–4 of Ad4 E3-19K/HLA-A2 are shown in an overview panel and in three close-up view panels (same color code as in Fig. 1A). Nitrogen and oxygen atoms are colored blue and red, respectively. Hydrogen bonds (distance ≤ 3.5 Å) are indicated by dashed green lines, and hydrophobic contacts (distance < 4.0 Å) are represented by dashed magenta lines. The β-strands of Ad4 E3-19K, α1-, α2-, and α3-domains of HLA-A2 heavy chain, and β2m are labeled.

The Ad4 E3-19K versus Ad2 E3-19K structures

An alignment of the Ad4 and Ad2 E3-19K amino acid sequences (Fig. 3A) shows that although the ER-lumenal domains of Ad4 and Ad2 E3-19K proteins share only 36% sequence identity (residue marked in red in Fig. 3A), their overall complex structures are very similar (Fig. 3B, Ad4 E3-19K structure serves as the reference). There are 36 strictly (red) and 9 highly (pink) conserved residues (Fig. 3A) which are distributed along the entire length of the structures (Fig. 3B), clustering especially on β-strand B and DE-loop of the large domain, α-helix, and tail. Remarkably, the majority (approximately 70%) of residues that make up the hydrophobic core in Ad4 and Ad2 structures (see Fig. 1C, and residues marked with a star in Fig. 3A) are strictly and highly conserved residues. The hydrophobic core is therefore critical for conservation of the E3-19K tertiary fold, and accordingly, regions with the largest differences between the two structures (defined as regions I–VI in Fig. 1C and 3A) are devoid of core-forming residues, except for Asn53 and Val69. There are four strictly conserved Cys residues in Ad4 and Ad2 E3-19K (Fig. 3A) that form two disulfide bonds (Cys18/Cys35 and Cys29/Cys91) along β-strand B (Fig. 3B), tethering it to the long N-terminus segment and α-helix, respectively. These conserved disulfide bonds are critical to the E3-19K fold, as demonstrated previously (50), and their strategic positioning on the ABD β-sheet likely provides conformational rigidity to this face of the proteins. The long N-terminus segment of Ad4 E3-19K is tethered to the BC-loop by an additional disulfide bond (Cys10/Cys39) (Fig. 3B); this disulfide bond is conspicuously absent in Ad2 E3-19K owing to a shorter N-terminus. Overall, the two E3-19K proteins have similar complex structures owing to the conservation of key structural residues and determinants, and also because of their shared MHC I-binding function.

FIGURE 3.

FIGURE 3

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Conserved residues in Ad4 and Ad2 E3-19K. (A) Amino acid sequence alignment of Ad4 and Ad2 E3-19K. Strictly (red) and highly (pink) conserved residues are indicated: residues in Ad4, and Ad2 E3-19K at interaction sites 1–4 are indicated by numbers, and core-forming residues (>90% buried) are indicated by a star. Regions of major conformational differences between Ad4 and Ad2 E3-19K are indicated by green boxes labeled I–VI (see also legend of Fig. 1C). (B) Strictly (red) and highly (pink) conserved residues are mapped in the Ad4 and Ad2 E3-19K (31) structures. The β-strands and N- and C-termini are labeled.

Comparison of the Ad4 E3-19K/HLA-A2 and Ad2 E3-19K/HLA-A2 structures

A comparative analysis of the Ad4 E3-19K/HLA-A2 and Ad2 E3-19K/HLA-A2 structures reveals similarities and differences in the binding mode of E3-19K proteins onto HLA-A2. Fig. 4A (left top panel) shows a front view of the residues that Ad4 E3-19K (yellow balls) and Ad2 E3-19K (orange balls) interact with on HLA-A2, highlighting how both proteins contact generally the same four regions of HLA-A2. Notably, although a number of HLA-A2 residues that the E3-19K proteins bind to are the same at sites 1, 2, and 4, each protein also has unique contact residues at sites 1 and 2 and in particular at site 3 (Fig. 4A). A side view in Fig. 4A (right top panel) shows how sites 1, 2, and 4 are spatially organized along the interaction surface, with contact residues Gln54 (site 1) and Glu177 (site 2) on HLA-A2 heavy chain, and both Lys19 and Ser20 on β2m (site 4) occupying prominent positions (see also Fig. 4C). Fig. 4A (bottom panel) shows an amino acid sequence alignment of consensus HLA-A, -B, and -C heavy chains at interaction sites 1–4. The information shows that sites 1 and 2 are comprised mostly of conserved MHC I residues, while site 3 is comprised largely of polymorphic residues. Site 4 involves β2m residues and is therefore strictly conserved in all MHC I molecules. Thus, overall, Fig. 4A shows that Ad4 and Ad2 E3-19K proteins contact largely conserved residues on HLA-A2, though not necessarily the same conserved residues.

FIGURE 4.

FIGURE 4

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Contact residues in Ad4 E3-19K/HLA-A2 and Ad2 E3-19K/HLA-A2 structures. (A) Superimposition of Ad4 E3-19K/HLA-A2 and Ad2 E3-19K/HLA-A2 (31) structures (top), in front (left panel) and side (right panel) views, showing contact residues on HLA-A2 (sites 1–4) for Ad4 E3-19K (yellow balls) and Ad2 E3-19K (orange balls). The side view representation shows that residues Gln54, Glu177, Lys19, and Ser20 occupy prominent positions along the binding interface. The α1-, α2-, and α3-domains of HLA-A2 heavy chain and β2m are labeled. Amino acid sequence alignment of HLA-A2 and the consensus HLA-A, -B, and -C heavy chain sequences (consensus sequences were obtained from the IMGT-HLA Sequence Database (www.ebi.ac.uk/imgt/hla/align.html)) at sites 1–3 is shown at the bottom. Capital letters designate contact residues at each site. (B) Residues in Ad4 E3-19K (yellow balls) and Ad2 E3-19K (orange balls) that contact HLA-A2 at sites 1–4 are indicated with the site numbers. Non-conserved residues are shown in black, strictly conserved residues in red, and highly conserved residue in pink. Residues in both Ad4 and Ad2 E3-19K interacting with HLA-A2 residue Gln54 are shown as solid black underlines; similarly, residues interacting with HLA-A2 residue Glu177 are shown as dashed black underlines. Strictly conserved residues in both Ad4 and Ad2 E3-19K interacting with β2m residues Ser19 and Lys20 are shown as solid red underlines. Regions of major conformational differences between Ad4 and Ad2 E3-19K are colored in green (see also legend of Fig. 1C). (C) A close-up view of the superimposed Ad4 E3-19K/HLA-A2 (slate) and Ad2 E3-19K/HLA-A2 (grey) (31) structures showing interactions at residues Gln54 (left panel) and Glu177 (right panel). All interactions are shown as dashed red lines for Ad4 E3-19K/HLA-A2 and dashed blue lines for Ad2 E3-19K/HLA-A2. The α1- and α2-domains of HLA-A2 heavy chain and β-strands of Ad4 E3-19K (only) are labeled. (D) Native gel band-shift assay showing how a single-point mutation at Gln54 and Glu177 in HLA-A2 heavy chain impairs interaction with Ad4 E3-19K (left) and Ad2 E3-19K (right). See Materials and Methods for conditions.

The residues in E3-19K protein that bind at sites 1–4 on HLA-A2 are shown in Fig. 4B. A number of observations can be made (Ad4 E3-19K/HLA-A2 structure serves as the reference). First, all contact residues are found in regions of highest structural similarities between the two structures, except for Trp37 in Ad4 and Tyr46 in Ad2. Second, in both structures, the contact residues are located on the ABD β-sheet, in particular along β-strand D, and on the α-helix; there are no contact residues on the CEF β-sheet. Third, both E3-19K proteins use mostly non-conserved residues of the large N-domain and loops to associate with HLA-A2 heavy chain at sites 1–3 (Fig. 3A and 4B). On the other hand, both E3-19K proteins use strictly conserved residues of the α-helix to interact with β2m at site 4 (Fig. 3A and 4B); there are only 4 strictly conserved residues (Lys49, Met97, Gln100, and Tyr101) that are used functionally by both E3-19K proteins, further underlining that conservation of residues in E3-19K is primarily to preserve the tertiary fold. Finally, Ala56 in Ad4 and Tyr49 in Ad2, which occupy equivalent positions structurally on β-strand D (Fig. 4B), are the only residues in each E3-19K protein that mediate interactions with HLA-A2 heavy chain at two different sites, sites 1 and 2, bridging the α1- and α2-helices. This further underlines the critical functional role of β-strand D.

It is also noteworthy that both E3-19K proteins make extensive contacts with HLA-A2 residues Gln54 and Glu177 (Fig. 4C), residues that occupy striking positions along the binding interface (see Fig. 4A, right panel). Specifically, Ad4 E3-19K uses Trp37, Thr54, and Ala56 to interact with Gln54 while Ad2 E3-19K uses Ala47 and Tyr49 (left panel, see also Fig. 4B as solid, black underline); and Ad4 E3-19K uses Ala56, Ser57, and Thr58 to interact with Glu177 while Ad2 E3-19K uses Lys42 and Ile51 (right panel, see also Fig. 4B as dashed, black underline). Overall, this is consistent with that HLA-A2 residues Gln54 and Glu177 play critical roles at the E3-19K/HLA-A2 interfaces.

MHC I residues 54 and 177 modulate interactions with E3-19K proteins

The above analysis provides a strong rationale for examining the roles of HLA-A2 heavy chain residues 54 and 177 in modulating interaction with Ad2 and Ad4 E3-19K proteins. We made the single-point mutations Gln54-to-Gly54 (Q54G) and Glu177-to-Lys177 (E177K) in HLA-A2 heavy chain and probed association with E3-19K using a native gel band-shift assay (Fig. 4D). The results show that Ad4 and Ad2 E3-19K interact more weakly with HLA-A2(Q54G), relative to the control HLA-A2, as evidenced by the presence of residual uncomplexed HLA-A2(Q54G), the reduced intensities of the bands corresponding to complexes (more evident for Ad2 E3-19K), and the protein smear (more evident for Ad4 E3-19K). Furthermore, interaction of Ad4 and Ad2 E3-19K with HLA-A2(E177K) failed to generate bands at the positions expected for complexes. This result is noteworthy given that Ad4 and Ad2 E3-19K mediate different types of interactions with E177; hydrophobic interactions for Ad4 E3-19K (Fig. 2) and salt bridge for Ad2 E3-19K (31). Together, results from native gel analysis provide evidence that residue 54 and especially residue 177 in HLA-A2 heavy chain play important roles at the binding interface, consistent with our structural analysis.

Ad4 E3-19K/HLA-A2 and Ad2 E3-19K/HLA-A2 structures; insights into locus-specific interactions

The HCMV-encoded US2 protein, which also binds to HLA-A2 at the N-terminal end of the groove (51), does not interact with HLA-C molecules (35). To gain insights into the structural basis underlying the MHC I-locus specificity of US2 and E3-19K, we mapped the binding surface of Ad4 E3-19K (yellow), Ad2 E3-19K (orange), and US2 (green) on HLA-A2 (Fig. 5A). The analysis shows that Ad4 E3-19K and US2 (left panel) share contact residue Glu177 (cyan), while Ad2 E3-19K and US2 (right panel) share residues Glu177 and Arg181 (cyan). As discussed above, Glu177 is critical for Ad4 and Ad2 E3-19K interaction with HLA-A2 (Fig. 4D), as well as HLA-A11 (33). Similarly, Arg181 was shown to be critical for Ad2 E3-19K and US2 function (19, 51). Remarkably, Fig. 5B shows that the side chain of Glu177 in HLA-Cw4 (PDB 1QQD) (blue) points in a different orientation relative to the orientations this residue adopts in its free and liganded HLA-A2 forms; compare free HLA-A2/Tax (PDB 1HHK) (magenta) with Ad4 E3-19K/HLA-A2 (yellow) and Ad2 E3-19K/HLA-A2 (orange). Taken together, this analysis points to the region of HLA-A2 in the vicinity of site 2 as playing a potential role in the MHC I-locus specificity of these viral immunoevasins.

FIGURE 5.

FIGURE 5

Open in a new tab

MHC I-locus specificity of viral immunoevasins. (A) The binding surface of Ad4 E3-19K (yellow), Ad2 E3-19K (31) (orange), and US2 (51) (green) at sites 1–4 on HLA-A2 showing overlapping contact residues Glu177 and Arg181 in cyan. The α1-, α2, and α3-domains of HLA-A2 heavy chain and β2m are labeled. (B) The side chain orientation of Glu177 in the superimposed structures of free HLA-Cw4 (PDB 1QQD) (blue), free HLA-A2/Tax (PDB 1HHQ) (magenta), Ad4 E3-19K/HLA-A2 (yellow), and Ad2 E3-19K/HLA-A2 (orange).

Discussion

Viruses have evolved specific mechanisms to interfere with the MHC class I pathway and suppress antigen presentation. Ads encode the E3-19K protein that targets MHC class I molecules for retention within the ER of infected cells. Here, we determined the x-ray crystal structure of species E Ad4 E3-19K bound to HLA-A2, defined the mode of binding and contact sites, and compared the findings with those of our species C Ad2 E3-19K/HLA-A2 structure (31).

To function effectively as an immunomodulatory protein, the ER-lumenal domain of E3-19K must establish “cross-reactive” and high-affinity interactions with the ER-lumenal domain of MHC I molecules. First, Ad4 and Ad2 E3-19K proteins achieve functional promiscuity by contacting mostly conserved residues on HLA-A2, albeit not necessarily the same conserved residues, and by avoiding contacts with the bound antigenic peptide. Second, we showed that Ad4 and Ad2 E3-19K indeed bind tightly to HLA-A2 (see Fig. 4D); we also previously determined a Kd of 12 nM for Ad2 E3-19K/HLA-A2 complex (32). Our structures show that although both E3-19K proteins bind to the same hot spots on HLA-A2 (sites 1–4), overall, they mediate different types of interactions at sites 1–3 and more similar interactions at site 4. Indeed, relative to Ad2 E3-19K, Ad4 E3-19K establishes fewer hydrogen bonds and no salt bridges at sites 1–3 and instead relies extensively on hydrophobic contacts. These differences are not surprising given that Ad4 and Ad2 E3-19K use almost exclusively non-conserved residues to bind to HLA-A2 heavy chain at sites 1–3. On the other hand, Ad4 and Ad2 E3-19K proteins use three strictly conserved residues (Met97, Gln100, and Tyr101) to contact the same β2m residues at site 4. Therefore, taken together, Ad4 and Ad2 E3-19K proteins achieve high-affinity binding from the energetic contribution of all interactions along their respective interfaces, including the conservation of extensive hydrophobic contacts at site 4. In addition, we suggest that shape complementarity plays a role in the favorable recognition of HLA-A2 by E3-19K proteins.

A role for shape complementarity at the interface of E3-19K/HLA-A2 complexes is supported by a number of observations. First, the shape complementarity coefficients are reasonably high at all sites except site 3, and also remarkably similar between the two E3-19K/HLA-A2 structures: site 1, Sc = 0.709 (Ad4) and 0.693 (Ad2) (31); site 2, Sc = 0.605 (Ad4) and 0.658 (Ad2); site 3, Sc = 0.333 (Ad4) and 0.341 (Ad2); and site 4, Sc = 0.799 (Ad4) and 0.801 (Ad2). Second, the binding interfaces of both E3-19K/HLA-A2 structures are characterized by relatively large buried surface area: 2,112 Å2 for Ad4 E3-19K/HLA-A2 and 1,929 Å2 for Ad2 E3-19K/HLA-A2 (31). Finally, we and others showed that the mutation of even single contact residues in either E3-19K or MHC I can have global inhibitory effects on complex formation (Fig. 4D and (32–34)). Together, these observations are consistent with favorable shape complementarity at the E3-19K/HLA-A2 interfaces.

It is interesting to note that in both Ad4 and Ad2 E3-19K/HLA-A2 structures, the Sc values are highest at site 4 (see Sc values above). Site 4 is particularly striking in that it involves an extended network of hydrophobic contacts between a few strictly conserved residues of the E3-19K α-helix, one of the most conserved regions of E3-19K proteins across Ad species (Supplementary Fig. 1), and residues of β2m (Fig. 2 and (31)). Another noticeable feature of both structures is the pronounced backbone curvatures of the E3-19K C-terminus tails. The curvatures arise from internal packing interactions involving the strictly conserved WPP motif (Supplementary Fig. 1). As we discussed previously (31), the proline residues of the WPP motif likely provide conformational rigidity to the tail, reducing conformational freedom of the membrane-anchored ER-lumenal domain of E3-19K. The strictly conserved WPP motif may therefore serve to correctly orient the ER-lumenal domain of E3-19K and initiate some favorable recognition event with HLA-A2, perhaps between the membrane-proximal E3-19K α-helix and β2m (site 4), as a starting point that drives interactions at the other three sites.

It is also interesting to note that in both Ad4 and Ad2 E3-19K/HLA-A2 structures, the Sc values are lowest at site 3 (see Sc values above); this may be because interactions at site 3 involve residues on flexible loops of E3-19K and HLA-A2 α3-domain (Fig. 2 and (31)). Another distinct feature of site 3 is that it involves a region of HLA-A2 where the consensus sequences of HLA heavy chains are most divergent (Fig. 3A). Together, these characteristics likely contribute to why interactions at site 3, more than at any other sites, are distinctively different between the two structures. It is likely that these differences contribute to the differential binding affinities that E3-19K proteins of different Ad species have for a given MHC I molecule.

E3-19K proteins selectively associate with MHC I molecules, recognizing HLA-A and -B molecules but not HLA-C molecules. No studies so far have explained at the molecular level how viral immunomodulatory proteins discriminate between seemingly similar MHC I molecules. In our analysis of Fig. 5A, we identified that Glu177 on HLA-A2 heavy chain is a common contact for Ad4 E3-19K, Ad2 E3-19K, and HCMV US2, which all discriminate against HLA-C, while Arg181 is common to Ad2 E3-19K and HCMV US2. We also showed that the side chain of Glu177 is oriented differently in free HLA-Cw4 in comparison to free HLA-A2/Tax, and that there is minimal movement of this side chain upon formation of E3-19K/HLA-A2 complexes. Therefore, the region surrounding residues 177 and 181 on HLA heavy chains can potentially have a role in defining the binding specificity of E3-19K proteins for MHC I molecules.

A comparative analysis of Ad4 and Ad2 E3-19K/HLA-A2 structures reveals other notable features. At site 1, while A4 E3-19K uses Trp37 to make numerous hydrophobic contacts with the backbone of Gly56 at the tip of α1-helix (Fig. 2A), Ad2 E3-19K uses instead Tyr46 on a different loop to mediate similar interactions with Gly56 (31). Because we showed previously that residue 56 is critically important for the recognition of MHC I by Ad2 E3-19K (32), it is clear that different E3-19K proteins find different ways of maintaining interactions with MHC residue 56. An examination of the amino acid sequences of E3-19K proteins of species B and D at position 37 (Supplementary Fig. 1) shows that they carry Trp and His, respectively, while in contrast those of species C carry Thr. Therefore, this together with other structural points discussed above, makes the Ad4 E3-19K/HLA-A2 structure, rather than the Ad2 E3-19K/HLA-A2 structure, a more universal structure to make predictions on how E3-19K proteins of species B and D interact with HLA-A2. In this regards, however, it is worth mentioning that E3-19K proteins of species D are distinctively different from all other species in that they have several divergent amino acids in the α-helix (Supplementary Fig. 1), including non-conserved substitutions at the three contact residues Met97, Gln100, and Tyr101. These differences in species D E3-19K proteins are likely to impair interactions between α-helix residues and β2m residues, as seen at site 4 in our structures (Fig. 2 and (31)). Consistent with this, Ad37 E3-19K of species D has a very low affinity for HLA-A molecules, and no measurable affinity for HLA-B molecules, relative to E3-19K proteins of species B, C, and E (33). Determining the x-ray structure of a species D E3-19K protein bound to HLA-A2 would be highly valuable in revealing interaction in a low-affinity complex (see also below).

Overall, our study provides a structural understanding of how two E3-19K proteins of different Ad species, Ad4 E3-19K of species E and Ad2 E3-19K of species C, with low with levels of amino acid sequence homologies nonetheless share the same tertiary fold and MHC I-binding function. The conservation of these features underscores a critical role for E3-19K proteins in the pathogenesis of Ads. This is corroborated by the fact that species D Ads have evolved a unique immunomodulatory protein, E3/49K protein, that binds to CD45 in order to suppress the functions of T-cells and NK cells (52, 53). This evasion mechanism may have been evolved by species D partly because of the low binding affinity that their E3-19K proteins have for MHC I molecules, as discussed above. Overall, our structural findings help better understand the molecular basis by which Ad counteracts cellular immune defenses for survival inside host cells.

Supplementary Material

1

Acknowledgments

This work was supported, in whole or in part, by the US National Institute of Allergy and Infectious Diseases grants R01AI108546 and R03AI114611 (to MB), and US National National Center for Complementary and Alternative Medicine 1U41AT008706 (to BDS).

The authors thank Yasameen Muzahim for technical help and Dr. Karl Volz for discussion. We also thank Argonne National Laboratory for beam time and staff support.

Abbreviations used in this article

Ad

Adenovirus

β2m

β2-microglobulin

ER

endoplasmic reticulum

Footnotes

Disclosures

The authors have no financial conflicts of interest.

The atomic coordinates and structure factors of the Ad4 E3-19K/HLA-A2 complex have been deposited in the PDB (ID code 5IRO; http://www.rcsb.org/pdb/home/home.do) and will be released immediately upon publication.

References

  • 1.Wadell G. Molecular epidemiology of human adenoviruses. Curr Top Microbiol Immunol. 1984;110:191–220. doi: 10.1007/978-3-642-46494-2_7. [DOI] [PubMed] [Google Scholar]
  • 2.Berk AJ. Adenoviridae: the viruses and their replication. In: Knipe DM, Howley PM, editors. Fields Virology. 5. Lippincott Williams & Wilkins; Philadelphia, PA: 2007. pp. 2355–2394. [Google Scholar]
  • 3.Hage E, Liebert UG, Bergs S, Ganzenmueller T, Heim A. Human mastadenovirus type 70: a novel, multiple recombinant species d adenovirus isolated from diarrheal faeces of a haematopoietic stem cell transplantation recipient. J Gen Virol. 2015;96:2733–2742. doi: 10.1099/vir.0.000196. [DOI] [PubMed] [Google Scholar]
  • 4.Horwitz MS. Adenoviridae and their replication. In: Fields BN, Knipe DM, editors. Virology. 2. Raven Press; New York, NY: 1990. pp. 1723–1740. [Google Scholar]
  • 5.Hierholzer JC. Adenovirus in the immunocompromised host. Host Clin Microbiol Rev. 1992;5:262–274. doi: 10.1128/cmr.5.3.262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Flomenberg P, Babbitt J, Drobyski WR, Ash RC, Carrigan DR, Sedmak GV, McAuliffe T, Camitta B, Horowitz MM, Bunin N, Casper JT. Increasing incidence of adenovirus disease in bone-marrow transplant recipients. J Infect Dis. 1994;169:775–781. doi: 10.1093/infdis/169.4.775. [DOI] [PubMed] [Google Scholar]
  • 7.Zahradnik JM, Spencer MJ, Porter DD. Adenovirus infection in the immunocompromised patient. Am J Med. 1980;68:725–732. doi: 10.1016/0002-9343(80)90262-4. [DOI] [PubMed] [Google Scholar]
  • 8.Krilov LR, Rubin LG, Frogel M, Gloster E, Ni K, Kaplan M, Lipson SM. Disseminated adenovirus infection with hepatic necrosis in patients with human immunodeficiency virus infection and other immunodeficiency states. Rev Infect Dis. 1990;12:303–307. doi: 10.1093/clinids/12.2.303. [DOI] [PubMed] [Google Scholar]
  • 9.Kosulin K, Geiger E, Vecsei A, Huber WD, Rauch M, Brenner E, Wrba F, Hammer K, Innerhofer A, Potschger U, Lawitschka A, Matthes-Leodolter S, Fritsch G, Lion T. Persistence and reactivation of human adenoviruses in the gastrointestinal tract. Clin Microbio Infect. 2016 Dec 19; doi: 10.1016/j.cmi.2015.12.013. pii: S1198-743X(15)01039-3. [DOI] [PubMed] [Google Scholar]
  • 10.Kvist S, Ostberg L, Persson H, Philipson L, Peterson PA. Molecular association between transplantation antigens and cell surface antigen in adenovirus-transformed cell line. Proc Natl Acad Sci USA. 1978;75:5674–5678. doi: 10.1073/pnas.75.11.5674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Burgert HG, Kvist S. An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens. Cell. 1985;41:987–997. doi: 10.1016/s0092-8674(85)80079-9. [DOI] [PubMed] [Google Scholar]
  • 12.Paabo S, Bhat BM, Wold WSM, Peterson PA. A short sequence in the COOH-terminus makes an adenovirus membrane glycoprotein a resident of the endoplasmic reticulum. Cell. 1987;50:311–317. doi: 10.1016/0092-8674(87)90226-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Cox JH, Yewdell JW, Eisenlohr LC, Johnson PR, Bennink JR. Antigen presentation requires transport of MHC class I molecules from the endoplasmic reticulum. Science. 1990;247:715–718. doi: 10.1126/science.2137259. [DOI] [PubMed] [Google Scholar]
  • 14.Cox JH, Bennink JR, Yewdell JW. Retention of adenovirus E19 glycoprotein in the endoplasmic reticulum is essential to its ability to block antigen presentation. J Exp Med. 1991;174:1629–1637. doi: 10.1084/jem.174.6.1629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Burgert HG, Kvist S. The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition. EMBO J. 1987;6:2019–2026. doi: 10.1002/j.1460-2075.1987.tb02466.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Liu H, Stafford WF, Bouvier M. The endoplasmic reticulum lumenal domain of the adenovirus type 2 E3-19K binds to peptide-filled and peptide-deficient HLA-A*1101 molecules. J Virol. 2005;79:13317–13325. doi: 10.1128/JVI.79.21.13317-13325.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Beier DC, Cox JH, Vining DR, Cresswell P, Engelhard VH. Association of human class I MHC alleles with the adenovirus E3/19K protein. J Immunol. 1994;152:3862–3872. [PubMed] [Google Scholar]
  • 18.Feuerbach D, Etteldorf S, Ebenau-Jehle C, Abastado JP, Madden D, Burgert HG. Identification of amino acids within the MHC molecule important for the interaction with the adenovirus protein E3/19K. J Immunol. 1994;153:1626–1636. [PubMed] [Google Scholar]
  • 19.Flomenberg P, Gutierrez E, Hogan KT. Identification of class I MHC regions which bind to the adenovirus E3-19K protein. Molec Immunol. 1994;31:1277–1284. doi: 10.1016/0161-5890(94)90078-7. [DOI] [PubMed] [Google Scholar]
  • 20.Gabathuler R, Levy F, Kvist S. Requirement for the association of adenovirus type 2 E3-19K wild-type and mutant proteins with HLA antigens. J Virol. 1990;64:3679–3685. doi: 10.1128/jvi.64.8.3679-3685.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Hermiston TW, Tripp RA, Sparer RA, Gooding LR, Wold WSM. Deletion mutation analysis of the adenovirus type 2 E3-gp19K protein: identification of sequences within the endoplasmic reticulum lumenal domain that are required for class I antigen binding and protection from adenovirus-specific cytotoxic T lymphocytes. J Virol. 1993;67:5289–5298. doi: 10.1128/jvi.67.9.5289-5298.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Flomenberg P, Szmulewicz J, Gutierrez E, Lupatkin H. Role of the adenovirus E3-19K conserved region in binding major histocompatibility complex class I molecules. J Virol. 1992;66:4778–4783. doi: 10.1128/jvi.66.8.4778-4783.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sester M, Ruszics Z, Mackley E, Burgert HG. The transmembrane domain of the adenovirus E3/19K protein acts as an endoplasmic reticulum retention signal and contribute to intracellular sequestration of major histocompatibility complex class I molecules. J Virol. 2013;87:6104–616. doi: 10.1128/JVI.03391-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Flomenberg P, Piaskowski V, Truitt RL, Casper JT. Human adenovirus-specific CD8+ T-cell responses are not inhibited by E3-19K in the presence of gamma interferon. J Virol. 1996;70:6314–6322. doi: 10.1128/jvi.70.9.6314-6322.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Andersson M, McMichael A, Peterson PA. Reduced allorecognition of adenovirus-2 infected cells. J Immunol. 1987;138:3960–3966. [PubMed] [Google Scholar]
  • 26.Burgert HG, Maryanski JL, Kvist S. “E3/19K” protein of adenovirus type 2 inhibits lysis of cytolytic T lymphocytes by blocking cell surface expression of histocompatibility class I antigens. Proc Natl Acad Sci USA. 1987;84:1356–1360. doi: 10.1073/pnas.84.5.1356. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Tanaka Y, Tevethia SS. Differential effect of adenovirus 2 E3/19K glycoprotein on the expression of H-2Kb and H-2Db class I antigens and H-2Kb- and H-2Db-restricted SV40-specific CTL-mediated lysis. Virol. 1988;165:357–366. doi: 10.1016/0042-6822(88)90580-6. [DOI] [PubMed] [Google Scholar]
  • 28.Rawle FC, Tollefson AE, Wold WSM, Gooding LR. Mouse anti-adenovirus cytoxic T lymphocytes. J Immunol. 1989;138:3960–3966. [PubMed] [Google Scholar]
  • 29.Ginsberg HS, Lundholm-Beauchamp U, Horswood RL, Pernis B, Wold WS, Chanock RM, Prince GA. Role of early region 3 (E3) in pathogenesis of adenovirus disease. Proc Natl Acad Sci USA. 1989;86:3823–3827. doi: 10.1073/pnas.86.10.3823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Ginsberg HS, Moldawer LL, Sehgal PB, Redington M, Kilian PL, Chanock RM, Prince GA. A mouse model for investigating the molecular pathogenesis of adenovirus pneumonia. Proc Natl Acad Sci USA. 1990;88:1651–1655. doi: 10.1073/pnas.88.5.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Li L, Muzahim Y, Bouvier M. Crystal structure of adenovirus E3-19K bound to HLA-A2 reveals mechanism for immunomodulation. Nat Struc Molec Biol. 2012;19:1176–1181. doi: 10.1038/nsmb.2396. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Liu H, Fu J, Bouvier M. Allele- and locus-specific recognition of class I MHC molecules by the immunomodulatory E3-19K protein from adenovirus. J Immunol. 2007;178:4567–4575. doi: 10.4049/jimmunol.178.7.4567. [DOI] [PubMed] [Google Scholar]
  • 33.Fu J, Li L, Bouvier M. Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions towards major histocompatibility class I molecules. J Biol Chem. 2011;286:17631–17639. doi: 10.1074/jbc.M110.212050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Fu J, Bouvier M. Determinants of the endoplasmic reticulum (ER) lumenal-domain of the Adenovirus serotype 2 E3-19K protein for association with and ER-retention of major histocompatibility complex class I molecules. Molec Immunol. 2011;48:532–538. doi: 10.1016/j.molimm.2010.10.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Gewurz BE, Wang EW, Tortorella D, Schust DJ, Ploegh HL. Human cytomegalovirus US2 endoplasmic reticulum-lumenal domain dictates association with major histocompatibility complex class I in a locus-specific manner. J Virol. 2001;75:5197–5204. doi: 10.1128/JVI.75.11.5197-5204.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Machold RP, Wiertz EJ, Jones TR, Ploegh HL. The HCMV gene products US11 and US2 differ in their ability to attack allelic forms of murine major histocompatibility complex (MHC) class I heavy chains. J Exp Med. 1997;185:363–366. doi: 10.1084/jem.185.2.363. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Cohen GB, Gandhi RT, Davis DM, Mandelboim O, Chen BK, Strominger JL, Baltimore D. The selective downregulation of class I major histocompatibility complex proteins by HIV-1 protects HIV-infected cells from NK cells. Immunity. 1999;10:661–671. doi: 10.1016/s1074-7613(00)80065-5. [DOI] [PubMed] [Google Scholar]
  • 38.Le Gall S, Erdtmann L, Benichou S, Berlioz-Torrent C, Liu L, Benarous R, Heard JM, Schwartz O. Nef interacts with the mu subunit of clathrin adaptor complexes and reveals a cryptic sorting signal in MHC I molecules. Immunity. 1998;8:483–495. doi: 10.1016/s1074-7613(00)80553-1. [DOI] [PubMed] [Google Scholar]
  • 39.Ishido S, Choi JK, Lee BS, Wang C, DeMaria M, Johnson RP, Cohen GB, Jung JU. Inhibition of natural killer cell-mediated cytotoxicity by Kaposi’s sarcoma-associated herpesvirus K5 protein. Immunity. 2000;13:365–374. doi: 10.1016/s1074-7613(00)00036-4. [DOI] [PubMed] [Google Scholar]
  • 40.Garboczi DN, Hung DT, Wiley DC. HLA-A2-peptide complexes: Refolding and crystallization of molecules expressed in. Escherichia coli and complexed with single antigenic peptides. Proc Natl Acad Sci USA. 1992;89:3429–3433. doi: 10.1073/pnas.89.8.3429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Otwinowski Z, Minor W. Processing of X-ray Diffraction Data Collected in Oscillation Mode. In: Carter CW Jr, Sweet RM, editors. Methods in Enzymology: Macromolecular Crystallography, Part A. Vol. 276. Academic Press; New York, NY: 1997. pp. 307–326. [DOI] [PubMed] [Google Scholar]
  • 42.Navaza J. AMoRe: an automated package for molecular replacement. Acta Cryst. 1994;A50:157–16. [Google Scholar]
  • 43.Murshudov GN, Vagin AA, Dodson EJ. Refinement of Macromolecular Structures by the Maximum-Likelihood Method. Acta Cryst. 1997;D53:240–255. doi: 10.1107/S0907444996012255. [DOI] [PubMed] [Google Scholar]
  • 44.Emsley P, Cowtan K. Coot: model-building tools for molecular graphics. Acta Cryst. 2004;D60:2126–2132. doi: 10.1107/S0907444904019158. [DOI] [PubMed] [Google Scholar]
  • 45.Afonine PV, Grosse-Kunstleve RW, Echols N, Headd JJ, Moriarty NW, Mustyakimov M, Terwilliger TC, Urzhumtsev A, Zwart PH, Adams PD. Towards automated crystallographic structure refinement with phenix.refine. Acta Cryst. 2012;D68:352–367. doi: 10.1107/S0907444912001308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK: A program to check the stereochemical quality of protein structures. J Appl Cryst. 1993;26:283–291. [Google Scholar]
  • 47.Delano WL. The PyMOL Molecular Graphics System. 2002. [Google Scholar]
  • 48.Guex N, Peitsch MC. SWISS-MODEL and the Swiss-Pdb Viewer: An environment for comparative protein modeling. Electrophoresis. 1997;18:2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  • 49.Lawrence MC, Colman PM. Shape complementarity at protein/protein interfaces. J Mol Biol. 1993;234:946–950. doi: 10.1006/jmbi.1993.1648. [DOI] [PubMed] [Google Scholar]
  • 50.Sester M, Burgert HG. Conserved cysteine residues within the E3/19K protein of adenovirus type 2 are essential for binding to major histocompatibility complex antigens. J Virol. 1994;68:5423–5432. doi: 10.1128/jvi.68.9.5423-5432.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Gewurz BE, Gaudet R, Tortorella D, Wang EW, Ploegh HL. Antigen presentation subverted: Structure of the human cytomegalovirus protein US2 bound to the class I molecule HLA-A2. Proc Natl Acad Sci USA. 2001;98:6794–6799. doi: 10.1073/pnas.121172898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Windheim M, Shouthcombe JH, Kremmer E, Chaplin L, Urlaub D, Falk CS, Claus M, Mihm J, Braithwaite M, Dennehy MK, Renz H, Sester M, Watzl C, Burgert H-G. A unique secreted adenovirus E3 protein binds to the leukocyte common antigen CD45 and modulates leukocyte functions. Proc Natl Acad Sci USA. 2013;110:E4884–4893. doi: 10.1073/pnas.1312420110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Windheim M, Honing S, Leppard KN, Butler L, Seed C, Ponnambalam S, Burgert HG. Sorting motifs in the cytoplamisc tail of the immunomodulatory E3/49K protein of species D adenoviruses modulate cell surface expression and ectodomain shedding. J Biol Chem. 2016;25:6796–6812. doi: 10.1074/jbc.M115.684787. [DOI] [PMC free article] [PubMed] [Google Scholar]

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Supplementary Materials

1
NIHMS793624-supplement-1.pdf (3.9MB, pdf)

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