key: cord-0957361-v530u7h0
authors: Walls, Alexandra C.; Tortorici, M. Alejandra; Frenz, Brandon; Snijder, Joost; Li, Wentao; Rey, Félix A.; DiMaio, Frank; Bosch, Berend-Jan; Veesler, David
title: Glycan shield and epitope masking of a coronavirus spike protein observed by cryo-electron microscopy
date: 2016-09-12
journal: Nature Structural & Molecular Biology
DOI: 10.1038/nsmb.3293
sha: f75fbbb1502a00fdc8d63ab5d380faba55c24b37
doc_id: 957361
cord_uid: v530u7h0

The threat of a major coronavirus pandemic urges the development of suitable strategies to combat these pathogens. HCoV-NL63 is an α-coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. We report here the 3.4 Å resolution cryo-electron microscopy reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass-spectrometry, provides a structural framework to understand accessibility to antibodies. The structure also reveals a remarkable modular architecture of the receptor-binding subunit and the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on masking of epitopes with glycans and activating conformational changes, to evade the immune system of infected hosts.

a r t i c l e s Coronaviruses are enveloped viruses with large single-stranded positive-sense RNA genomes, classified in four genera (α, β, γ and δ). In humans, coronaviruses are responsible for 30% of respiratorytract infections 1 . In addition, coronaviruses have received substantial attention in the past decade, owing to the emergence of two deadly viruses with tremendous pandemic potential: severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) 2 . To date, there are no approved antiviral treatments or vaccines for any human coronavirus.

Coronaviruses are zoonotic viruses, and surveillance studies have suggested that both SARS-CoV and MERS-CoV originated from bats and that camels are also likely hosts for MERS-CoV 3, 4 . Moreover, sequencing data have demonstrated that bats serve as a reservoir of coronaviruses that have the potential to cross the species barrier and infect humans. This phenomenon is illustrated by the observation that substitution of three amino acid residues in the spike (S) glycoprotein receptor-binding domain of the bat-infecting HKU4-CoV enhances its affinity for human DPP4 (the MERS-CoV receptor) by two orders of magnitude 5, 6 . In addition, substitution of two other residues enables processing by human proteases and allows the HKU4-CoV S protein to mediate entry into human cells 7 . As a result, cross-species transmission of coronaviruses poses an imminent and long-term threat to human health. Recombination with coronaviruses frequently involved in mild respiratory infections may potentially lead to the emergence of highly pathogenic viruses 4 . Understanding the pathogenesis, cross-species transmission and recombination of coronaviruses is crucial to prevent or control their spread in humans and to evaluate the potential for long-term emerging diseases.

To date, αand β-coronavirus genera have been implicated in human diseases and zoonoses. The human coronavirus NL63 (HCoV-NL63) is an α-coronavirus that is genetically distinct from the β-coronaviruses mouse hepatitis virus (MHV, the prototypical coronavirus), MERS-CoV and SARS-CoV, and was first isolated from a 7-month-old patient with a respiratory-tract infection 8,9 . Further studies have revealed that HCoV-NL63 infections appear to be common in childhood, and most adult sera contain antibodies that neutralize the virus 8, 10 . HCoV-NL63 is a major cause of bronchiolitis and pneumonia in newborns worldwide and can cause severe lower-respiratory-tract infections that require hospitalization, especially among young children, the elderly and immunocompromised adults 11 . HCoV-NL63 infections have been reported in countries across Europe, Asia and North America, thus indicating its circulation among the human population worldwide. Other α-coronaviruses related to the human respiratory pathogen HCoV-229E have recently been identified in camels co-infected with MERS-CoV 4 , an observation further underscoring the importance of characterizing this coronavirus genus. Additionally, the emergence of the highly lethal porcine epidemic diarrhea coronavirus (PEDV, α-genus) has recently had devastating consequences for the US swine industry 12 .

Coronaviruses use S homotrimers to promote cell attachment and fusion of the viral and host membranes. Because it is virtually the only antigen present at the virus surface, S is the main target of neutralizing antibodies during infection and a focus of vaccine design 13 . S is a class I viral fusion protein that is synthesized as a single-chain precursor of ~1,300 amino acids and trimerizes after folding 14 . It is composed of a r t i c l e s an N-terminal S 1 subunit, containing the receptor-binding domain, and a C-terminal S 2 subunit, driving membrane fusion. After virion uptake by target host cells, cleavage at the S 2 ′ site (next to the putative fusion peptide) is required for fusion activation of all coronavirus S proteins, so that they can subsequently transition to the postfusion conformation [15] [16] [17] .

Our previously reported cryo-EM reconstruction of the MHV S glycoprotein at 4.0-Å resolution reveals the prefusion architecture of the machinery mediating entry of β-coronaviruses into cells 18 . It also demonstrates that coronavirus S and paramyxovirus F proteins share a common evolutionary origin. Here, we set out to characterize the conservation of the 3D organization of spike proteins among coronaviruses belonging to different genera. We report the atomic-resolution structure of the pathogenic HCoV-NL63 S-glycoprotein trimer, which belongs to the α-coronavirus genus. The substantial resolution improvement as compared with earlier studies allows visualization of the S glycoprotein at an unprecedented level of detail, which is a prerequisite for guiding drug and vaccine design, and reveals both shared and unique features of the α-genus of human pathogens. Our results suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on epitope masking with glycans and activating conformational changes, to evade the immune system of infected hosts, in a manner similar to that described for HIV-1.

We used Drosophila S2 cells to produce the HCoV-NL63 S ectodomain N-terminally fused to a GCN4 trimerization motif downstream from the heptad-repeat 2 (HR2) helix. We imaged frozen-hydrated HCoV-NL63 S ectodomain particles with an FEI Titan Krios electron microscope equipped with a Gatan Quantum GIF energy filter operated in zero-loss mode, with a slit width of 20 eV, and a Gatan K2 Summit electron-counting camera 19 (Online Methods).

We determined a 3D reconstruction of the HCoV-NL63 S at 3.4-Å resolution, using the gold-standard Fourier shell correlation (FSC) criterion of 0.143 (refs. 20,21) ( Fig. 1 and Supplementary  Fig. 1 ). The final model, which we built and refined with Coot 22 and Rosetta [23] [24] [25] , includes residues 23 to 1224, with internal breaks between residues 110-121, 882-890 and 992-1001 (Supplementary Fig. 1 and Table 1 ). The HCoV-NL63 S ectodomain is a 160-Å-long trimer with a triangular cross-section.

A notable feature of this structure is the extraordinary number of N-linked oligosaccharides that cover the spike trimer. In the cryo-EM npg a r t i c l e s reconstruction, we observed density for 31 N-linked glycans extending tangentially relative to the protein surface ( Fig. 2a, Using on-line reversed-phase liquid chromatography with electron transfer/high-energy collision-dissociation tandem MS 26 , we detected 25 N-linked glycosylation sites overlapping with those observed in the cryo-EM map and identified three additional sites (Fig. 2c, Supplementary Fig. 2 and Supplementary Table 1) . We identified these sites from both intact glycopeptides and peptides with the glycan trimmed down to the N-linked core N-acetylglucosamine moiety. The cryo-EM and MS data together provide evidence for glycosylation at 34 out of 39 possible NXS/T glycosylation sequons. The intact glycopeptides detected by MS/MS for HCoV-NL63 S expressed in Drosophila S2 cells corresponded to either paucimannosidic glycans containing three mannose residues (with or without core fucosylation) or high-mannose glycans containing four to nine mannose residues. Although glycan processing differences exist between insect and mammalian cell expression systems, the same glycosylation sequons are expected to be recognized and glycosylated in both cases. Previous reports have suggested that several coronavirus S glycans are of the high-mannose type, as a result of direct budding from the endoplasmic reticulum-Golgi intermediate compartment 27, 28 , thus supporting the biological relevance of the potential glycan structures identified.

In the refined model, N-linked glycans cover a substantial amount of the accessible surface of the trimer (Fig. 2a,b) . The higher glycan density per accessible surface area detected for the S 2 subunits (819 Å 2 /glycan) compared with the S 1 subunits (1,393 Å 2 /glycan) may explain why most coronavirus neutralizing antibodies isolated to date target the latter region. Because many of the observed glycosylation sites are topologically conserved among coronavirus S proteins, we suggest that the glycan footprint observed here may be representative of those of other S proteins. Besides potentially contributing to immune evasion, as discussed below, S glycans have been proposed to play a role in host-cell entry 29 via L-SIGN lectin, which is an alternative receptor for SARS-CoV 30 and HCoV-229E 27 .

The HCoV-NL63 and MHV S 2 fusion machineries are structurally similar and can be superimposed with excellent agreement (Fig. 3a and Supplementary Fig. 3 ; DALI 31 Z score 29.7, r.m.s. deviation 2.2 Å over 312 residues). In contrast to our previous MHV S structure 18 , most of the HCoV-NL63 S 2 ′ trigger loop, which connects the upstream helix to the fusion peptide and participates in fusion activation, is resolved in the reconstruction (Fig. 3b) . The trigger loop runs almost perpendicularly to the long axis of the S 2 subunit and forms three helical segments before looping back to connect to the fusion peptide. Multiple arginine residues, forming two putative furin-cleavage sites, are present in the C-terminal region of the S 2 ′ loop (863-RNIRSSR-870), which is characterized by weaker density, as would be expected from a proteasesensitive polypeptide segment. These observations are consistent with results of previous studies suggesting that fusion activation of the HCoV-NL63 S glycoprotein occurs after S 2 ′ proteolytic processing at the plasma membrane (by trypsin-like proteases such as TMPRS2) or in the endosomal pathway (by furin or cysteine proteases) 15, 32 .

The lack of strict amino acid sequence conservation at the S 2 ′ cleavage site among coronavirus S proteins reflects the usage of different proteases found in distinct cellular compartments for fusion activation 15, 17 . Similarly to the additional cleavage site present between the S 1 and S 2 subunits of MERS-CoV 7 , the multiple glycans present in the vicinity of the S 2 ′ loop probably further influence protease sensitivity (Fig. 3b) . However, we emphasize that S 2 ′ processing occurs at topologically equivalent positions for HCoV-NL63 S, MERS-CoV S, MHV S and probably most coronavirus S glycoproteins.

The HCoV-NL63 S reconstruction (Fig. 3a) resolves a large part of the S 2 C-terminal region that has not been observed in previous studies 18, 33 . We were able to build an atomic model for the connector domain and the stem helix, which connect to the HR2 region. The connector folds as a β-rich domain decorated with one short α-helix. At its C-terminal end, the polypeptide chain folds as an α-helix (stem helix, Fig. 3a ,c,d) aligned along the three-fold molecular axis, which turns into the HR2 domain, corresponding to 71 additional residues not resolved in our map. In the trimer, the connector domains The coronavirus S connector domain and the equivalent paramyxovirus F domain share a related topology, although their tertiary structures are different, and several structural motifs have been added to the latter domain throughout evolution 34, 35 (Fig. 3e,f) . Moreover, the trimer of stem helices assembles as a helical bundle, which initiates the HR2 domain in a manner reminiscent of the heptad repeat B (HRB) region of paramyxovirus prefusion F structures 34, 35 . These observations lend further support to the evolutionary connection that we have previously proposed for the fusion machineries of these two viral families 18 .

Comparison of the prefusion HCoV-NL63 S 2 subunit with the structure of the postfusion core suggests that the C-terminal region of the connector domain and the stem helix must refold and/or change conformation to yield the canonical 'trimer of hairpin' conformation that mediates fusion of the host and viral membrane in all class I fusion proteins 18, 36, 37 .

The HCoV-NL63 S structure shows the presence of an additional N-terminal domain not present in β-coronaviruses. Phylogenetic analyses suggest that this is a canonical feature of most α-coronavirus S glycoproteins (Fig. 4a-c) . This domain, which we named domain 0, adopts a galectin-like β-sandwich fold supplemented with a three-stranded β-sheet, similarly to domain A ( Fig. 4d-f , DALI Z score 6.7, r.m.s. deviation 3.8 Å over 147 residues), thus suggesting a gene-duplication event. Domain 0 interacts with the viral-membraneproximal side of domain A and with domain D. We determined that domain 0 is also structurally similar to the VP8* sialic acid-binding domain of the rotavirus VP4 spike protein 38 ( Fig. 4g ; PDB 1KQR, DALI Z score 8.9, r.m.s. deviation 3.0 Å over 109 residues). In line with this finding, domain 0 of transmissible gastroenteritis coronavirus (TGEV) and of PEDV bind to sialic acid, and deletion of this domain in α-coronavirus S appears to correlate with a loss of enteric tropism 39 . We detected no sialic acid binding activity for the HCoV-NL63 S 1 subunit (Supplementary Fig. 4) , thus possibly explaining the strict respiratory tropism of this virus. Instead, host-cell heparan sulfate proteoglycans have been shown to participate in HCoV-NL63 anchoring and infection 40 , and we detected binding of heparan sulfate to the HCoV-NL63 S protein by using surface plasmon resonance (SPR) (Supplementary Fig. 5a) . We hypothesize that these interactions may be mediated either by domain 0, which exhibits several positively charged patches on its surface ( Supplementary  Fig. 5b) , or domain A, which has been reported to bind carbohydrates in the case of a bovine coronavirus 41 .

A putative immune-evasion strategy Domain B, which is the HCoV-NL63 receptor-binding domain, exhibits a structure distinct from those of β-coronavirus B domains, although a topological relatedness has been detected among these β-rich domains 42 . Superimposition of the HCoV-NL63 and MHV S 1 subunits highlights that their B domains feature opposite orientations related by an ~180° rotation (Fig. 5a,b) . As a result, many of the HCoV-NL63 receptor-binding residues are buried through interaction with domain A of the same protomer, are masked by the glycan at residue Asn358 and are not available to engage the host-cell receptor (human angiotensin-converting enzyme 2, ACE2). Comparison of the HCoV-NL63 domain-B structure in our cryo-EM-derived model with the crystal structure of the same domain in complex with ACE2 (ref. 43 ) revealed that the receptor-binding loop containing residues 531-539 undergoes substantial conformational changes after binding (and is defined by weak density ; Fig. 5c ). These findings explain the markedly higher ACE2 binding affinity of HCoV-NL63 domain B, compared with that of the full-length S 1 domain (Fig. 5d) .

Because the receptor-binding loops elicit potent neutralizing antibodies in the case of TGEV 44 , MERS-CoV 45 and SARS-CoV 46-49 , we speculate that HCoV-NL63 has evolved to limit exposure of this vulnerable site to B-cell receptors via protein-protein interactions and glycan masking. This mechanism is reminiscent of the HIV-1 immune evasion strategy, which relies on a glycan shield and conformational changes that are triggered by binding of CD4 and expose the chemokine-receptor-interacting motifs 50, 51 .

Viruses have evolved several immune-evasion strategies including rapid antigenic evolution, masking of epitopes and exposure of nonneutralizing immune-dominant 'decoy' epitopes. For example, HIV-1 (ref. 52), Lassa virus 53 , hepatitis C virus 54 and Epstein-Barr virus 55 exhibit extensive N-linked glycosylation, covering exposed protein surfaces, with glycan masses that may exceed that of the protein component. The HCoV-NL63 S trimer is covered by an extensive glycan shield consisting of 102 N-linked oligosaccharides obstructing the protein surface. This observation is reminiscent of descriptions of the HIV-1 envelope trimer 52 , although the glycan density is 30% higher in the latter case. Furthermore, our data suggest that, similarly to HIV-1, coronavirus S glycans mask the protein surface and consequently limit access to neutralizing antibodies and thwart the humoral immune response. This strategy is illustrated by the presence of a glycan linked to Asn358 in the HCoV-NL63 structure reported here. This glycan, along with the proteinaceous moiety of domain A, contributes to masking the receptor-binding loops, which have been shown to elicit potent neutralizing antibodies for other coronaviruses [44] [45] [46] [47] [48] [49] and appear to represent a potential ' Achilles' heel' of these viruses. This hypothesis is further supported by the observation of three additional glycans directly protruding from the viral-membranedistal side of domain B. As a result, conformational changes are npg a r t i c l e s required for the HCoV-NL63 S glycoprotein to be able to interact with ACE2 (ref. 43 ). These rearrangements and/or receptor binding are likely to participate in initiating the fusion reaction by disrupting the interactions formed between domain B and the HR1 C-terminal region. Interactions with heparan sulfate proteoglycans present at the host-cell surface might potentially contribute to activating HCoV-NL63 S and promote subsequent interactions with ACE2. A common theme arising from the analysis of αand β-coronavirus S-glycoprotein structures is that domain-B-mediated host anchoring involves major structural rearrangements that expose the binding motifs 18, 33 . Visualization of the glycan shield obstructing access to the S surface and deciphering the molecular trickery used by some coronaviruses provide a rational basis for understanding the accessibility to neutralizing antibodies and may pave the way for guiding future design of immunogens or therapeutics. We have previously suggested that targeting the fusion machinery bears the promise of finding broadly neutralizing inhibitors of coronavirus infection 18 , and the high density of glycans decorating this region will need to be taken into consideration to increase the likelihood of success.

Methods and any associated references are available in the online version of the paper. 

The authors declare no competing financial interests.

Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 

A gene fragment encoding the HCoV-NL63 S ectodomain (residues 16-1291, UniProt Q6Q1S2) was PCR-amplified from a plasmid containing the fulllength S gene. The PCR product was ligated to a gene fragment encoding a GCN4 trimerization motif (LIKRMKQIEDKIEEIESKQKKIENEIARIKKIK) 18 Cryo-EM data processing. Whole-frame alignment was carried out with DOSEFGPU DRIFTCORR 19 . The parameters of the microscope contrast-transfer function were initially estimated with CTFFIND4 (ref. 58 ) and then with GCTF 59 . Micrographs were manually masked with Appion 60 to exclude the visible carbon edge from images. Particles were automatically picked with DoGPicker 61 . Particle images were extracted and processed with Relion 1.4 (ref. 62) with a box size of 320 pixels 2 and a pixel size of 1.36 Å. After reference-free 2D classification, we retained 180,000 out of 474,000 particles to run 3D classification with C1 symmetry 62 . We used the initial model previously generated for MHV 18 with Optimod 63 and low-pass-filtered the data to 60 Å as a starting reference for 3D classification. 118,000 particles were selected and used to run gold-standard 3D refinement with Relion 20 , thus yielding a map at 3.95-Å resolution. After particle-motion and radiation-damage correction with Relion particle polishing 64 , another round of 3D classification with C3 symmetry was performed to select 79,667 particles. After gold-standard 3D refinement with this subset of particles, we obtained a reconstruction at 3.76-Å resolution. Per-particle defocus parameters were estimated with GCTF and used to run an identical round of 3D refinement that yielded the final 3.4-Å-resolution map. Post processing was performed with Relion to apply an automatically generated B factor of −129 Å 2 . Reported resolutions were based on the gold-standard FSC = 0.143 criterion 20, 21 , and FSC curves were corrected for the effects of soft masking by high-resolution noise substitution 65 . The soft mask used for FSC calculation had a 10-pixel cosine-edge fall-off.

Model building and analysis. UCSF Chimera 66 and Coot 22,67 were used to fit atomic models into the cryo-EM map. The MHV S 2 subunit was fit into the density and rebuilt manually in Coot. The crystal structure of HCoV-NL63 domain B was then fit into the density, and the rest of the S 1 subunit was built with a combination of manual building in Coot and de novo building with Rosetta 23-25 . Glycan density coming after an NXS/T motif was initially manually built into the density, and glycan geometry was then refined with Rosetta, optimizing the fit-to-density as well as the energetics of protein-glycan contacts. The glycans were not as well defined as the protein region in the reconstruction, owing to flexibility and compositional heterogeneity. The final model was refined by application of strict noncrystallographic symmetry constraints with Rosetta, with a training map corresponding to one of the two maps generated by the goldstandard refinement procedure in Relion. The second map (testing map) was used only for calculation of the FSC compared with the atomic model and preventing overfitting 68 26 was used with calibrated charge-dependent ETD parameters and a supplemental higher-energy collision dissociation energy of 0.15 for the samples with intact glycopeptides and 0.2 for the samples treated with endoglycosidases. A resolution setting of 120,000 with an AGC target of 2 × 10 5 was used for MS1, and a resolution setting of 30,000 with an AGC target of 1 × 10 5 was used for MS2. The data were searched against a custom database including recombinant coronavirus S-glycoprotein sequences, a list of common contaminant proteins including trypsin, chymotrypsin and the endoglycosidases, as well as 998 decoy reverse yeast sequences, with trypsin or chymotrypsin as the protease, allowing up to two missed cleavages. All searches included carbamidomethylation of cysteine as a fixed modification and oxidation of methionine as a variable modification. An initial comprehensive search for glycosylation revealed that (core-fucosylated) paucimannose and high-mannose structures were the only identified glycan species in the samples. On the basis of these findings, a final search was performed with COMET 75 on the same data with the following list of variable modifications of asparagine residues: +HexNAc(2)Hex(3), +HexNAc(2)Hex(3)dHex(1), +HexNAc(2)Hex(3)dHex(2), +HexNAc(2)Hex(4), +HexNAc(2)Hex(5), +HexNAc(2)Hex(6), +HexNAc(2)Hex(7), +HexNAc(2)Hex(8) and +HexNAc(2)Hex (9) . The samples treated with endoglycosidases were searched with +HexNAc, +HexNAc(1)dHex(1) and +HexNAc(1)dHex(2) as variable modifications of asparagine. We used a precursor mass tolerance of 20 p.p.m., 0.02 fragment bin size, including b/c/y/z fragments, with monoisotopic masses for both precursor and fragment ions. The search results were filtered for modification of asparagine residues and the presence of an NX(S/T) sequon at the protein level. All appropriate peptide spectrum matches (PSMs) were manually inspected, and only those with reasonable peptide sequence coverage were kept. In addition, the spectra were inspected for the presence of glycan fragment ions. All glycosylation sites identified by MS listed in Supplementary Table 1 are based on multiple PSMs, often with multiple different glycans and additional confirmation from overlap between the trypsin-and chymotrypsin-treated samples. The greatest number of glycopeptide identifications was made in the chymotrypsin-digested samples.

Hemagglutination assay. The S 1 subunit of HCoV-NL63 C-terminally tagged with the Fc portion of human IgG (S 1 -Fc) was tested alone or premixed with 1 µl of Protein A-coupled, 200-nm-sized nanoparticles (nano-screenMAG-Protein A beads; Chemicell, cat.no. 4503-1) to increase the avidity of S 1 -Fc proteins for sialic acids on the erythrocyte surface. The sialic acid-binding S 1 subunit of PEDV (strain GDU, GenBank AFP81695.1) C-terminally fused to the human Fc portion was used as a positive control. 'Mock' indicates the conditions in which no S 1 subunit was used (negative control). The initial concentration of S 1 -Fc was 5 µg, and two-fold serial dilutions of S 1 -Fc-nanoparticle mixtures were made in 50 µl phosphate-buffered saline supplemented with 0.1% bovine serum albumin. 50 µl erythrocyte suspension (0.5%) was mixed with 50 µl of S 1 -Fc-nanoparticle dilution in V-shaped 96-well plates and incubated for 2 h on ice, after which the wells were photographed.

Protein expression of S 1 variants and ACE2. Different S 1 variants of HCoV-NL63 S protein, including S 1 (residues 1-718), S 1 domain 0 (S 1 -0, residues 1-209) and S 1 domain B (S 1 -B, residues 481-616), were C-terminally fused to the Fc region of mouse IgG (mFc), expressed in HEK-293T cells and affinity purified as previously described 76 . Likewise, an S 1 -mFc expression plasmid was made for the SARS-CoV S 1 domain (isolate CUHK-W1, residues 1-676) and the PEDV S 1 domain (strain GDU; residues 1-728). Expression of the human angiotensinconverting enzyme ectodomain (ACE2; residues 1-614) fused to the Fc portion of human IgG (hFc) was performed as previously described 76 .

Coronaviruses: drug discovery and therapeutic options

Middle East respiratory syndrome and severe acute respiratory syndrome

Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor

Co-circulation of three camel coronavirus species and recombination of MERS-CoVs in Saudi Arabia

Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus

Bat origins of MERS-CoV supported by bat coronavirus HKU4 usage of human receptor CD26

Two mutations were critical for bat-to-human transmission of Middle East respiratory syndrome coronavirus

Identification of a new human coronavirus

A previously undescribed coronavirus associated with respiratory disease in humans

Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry

Human coronavirus NL63 infection and other coronavirus infections in children hospitalized with acute respiratory disease in Hong Kong

Deadly pig virus slips through US borders

The spike protein of SARS-CoV: a target for vaccine and therapeutic development

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner

Host cell entry of Middle East respiratory syndrome coronavirus after two-step, furin-mediated activation of the spike protein

Host cell proteases: critical determinants of coronavirus tropism and pathogenesis

Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer

Electron counting and beam-induced motion correction enable nearatomic-resolution single-particle cryo-EM

Prevention of overfitting in cryo-EM structure determination

Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy

Tools for macromolecular model building and refinement into electron cryo-microscopy reconstructions

Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement

De novo protein structure determination from near-atomicresolution cryo-EM maps

High-resolution comparative modeling with RosettaCM

Unambiguous phosphosite localization using electron-transfer/ higher-energy collision dissociation (EThcD)

Human coronavirus 229E can use CD209L (L-SIGN) to enter cells

Identification of N-linked carbohydrates from severe acute respiratory syndrome (SARS) spike glycoprotein

A single asparagine-linked glycosylation site of the severe acute respiratory syndrome coronavirus spike glycoprotein facilitates inhibition by mannosebinding lectin through multiple mechanisms

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

Dali server: conservation mapping in 3D

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Pre-fusion structure of a human coronavirus spike protein

Structure of RSV fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody

Structure of the parainfluenza virus 5 F protein in its metastable, prefusion conformation

Viral membrane fusion

Core structure of S2 from the human coronavirus NL63 spike glycoprotein

The rhesus rotavirus VP4 sialic acid binding domain has a galectin fold with a novel carbohydrate binding site

Point mutations in the S protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus

Human coronavirus NL63 utilizes heparan sulfate proteoglycans for attachment to target cells

Crystal structure of bovine coronavirus spike protein lectin domain

Evidence for a common evolutionary origin of coronavirus spike protein receptor-binding subunits

Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

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Structure of severe acute respiratory syndrome coronavirus receptor-binding domain complexed with neutralizing antibody

Structural basis of neutralization by a human anti-severe acute respiratory syndrome spike protein antibody, 80R

Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association

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Structure of a V3-containing HIV-1 gp120 core

Trimeric HIV-1-Env structures define glycan shields from clades A, B, and G

Arenavirus glycan shield promotes neutralizing antibody evasion and protracted infection

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Structure of the Epstein-Barr virus major envelope glycoprotein

After immobilization quenching, running buffer was flowed for 10 min to ensure a steady baseline before experimental binding. Heparan sulfate (Sigma Aldrich) was reconstituted in running buffer at 5.0 mg/mL. Two concentrations of heparan sulfate, 5.0 mg/mL and 2.5 mg/mL, were injected for 80 s with a dissociation time of 400 s. All data were subtracted from the blank flow cell

Crystal structure of GCN4-pIQI, a trimeric coiled coil with buried polar residues

Automated molecular microscopy: the new Leginon system

CTFFIND4: fast and accurate defocus estimation from electron micrographs

Real-time CTF determination and correction

Appion: an integrated, database-driven pipeline to facilitate EM image processing

DoG Picker and TiltPicker: software tools to facilitate particle selection in single particle electron microscopy

RELION: implementation of a Bayesian approach to cryo-EM structure determination

Optimod: an automated approach for constructing and optimizing initial models for single-particle electron microscopy

Beam-induced motion correction for sub-megadalton cryo-EM particles

High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy

Visualizing density maps with UCSF Chimera

Features and development of Coot

Cryo-EM model validation using independent map reconstructions

MolProbity: all-atom structure validation for macromolecular crystallography

Privateer: software for the conformational validation of carbohydrate structures

The interpretation of protein structures: estimation of static accessibility

PDB2PQR: an automated pipeline for the setup of Poisson-Boltzmann electrostatics calculations

Electrostatics of nanosystems: application to microtubules and the ribosome

Quantifying the local resolution of cryo-EM density maps

Comet: an open-source MS/MS sequence database search tool

Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC

ACE2 binding ELISA. The ability of the HCoV-NL63 S 1 -mFc and S 1 -B-mFc chimeric proteins to bind the ACE2-hFc receptor was evaluated with an ELISA-based assay. 100 µl of hACE2-hFc (20 µg/ml, diluted in PBS) was coated on a 96-well MaxiSorb plate overnight at 4 °C. Nonspecific binding sites were subsequently blocked with a 3% (w/v) solution of bovine serum albumin in PBS. Plates were washed with washing buffer (PBS with 0.05% Tween 20) and subsequently incubated with serially diluted S 1 -mFc proteins (starting with equimolar concentrations) for 1 h at room temperature, after which plates were washed three times with washing buffer. mFc-tagged S 1 proteins were detected with HRP-conjugated polyclonal rabbit-anti-mouse immunoglobulins (1:2,000 dilution in PBS with 0.1% BSA; DAKO, P0260; validation on manufacturer's website), and a colorimetric reaction was produced after incubation with tetramethylbenzidine substrate (BioFX). The optical density (OD) was subsequently measured at 450 nm with an ELISA reader (EL-808, BioTEK). Background (signal from HRP-conjugated anti-mFc antibody alone) was subtracted from the OD 450nm values. The mFc-tagged SARS-CoV S 1 subunit was used as a positive control, whereas the mFc-tagged HCoV-NL63 S 1 domain 0 (HCoV-NL63 S 1 -0-mFc) and PEDV S 1 subunit (PEDV S 1 -mFc), both of which do not bind ACE2, were used as negative controls.Surface plasmon resonance (SPR). SPR was performed on a GE Healthcare Biacore T200 with a running buffer containing 20 mM HEPES, pH 7.5, 100 mM NaCl and 0.5% Tween-20, with a flow rate of 30 µL/min at 25 °C. A carboxymethylated dextran (CM5) chip (GE Healthcare) was activated with N-hydroxysulfosuccinimide (NHS) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide