The 3.1-Angstrom Cryo-electron Microscopy Structure of the Porcine Epidemic Diarrhea Virus Spike Protein in the Prefusion Conformation

To structurally characterize the prefusion conformation of PEDV CV777 S, we expressed the soluble ectodomain, encompassing residues 1 to 1319. This construct was designed to be truncated just after heptad repeat 2 (HR2) such that the membrane-proximal external region (MPER), transmembrane domain (TM), and intravirion tail (IV) were excluded. To these residues we appended a C-terminal T4 fibritin trimerization motif, an 8× His tag, and a Twin-Strep-tag. Affinity purification from a 500-ml transfection volume yielded approximately 0.5 mg of protein, which was then further purified by size exclusion chromatography (SEC). SDS-PAGE revealed a single band between 160 and 220 kDa, corresponding to the molecular weight of a glycosylated monomer that showed no evidence of cleavage by endogenous proteases. The SEC chromatogram revealed a single, symmetrical peak with an elution volume corresponding to a molecular weight of roughly 600 kDa, suggesting a heavily glycosylated homotrimer. The purified protein was then used to prepare cryo-EM grids. Initially, CF-1.2/1.3 grids were used, but the PEDV S proteins were occluded from the thin ice and exhibited a tendency to cluster around the edge of the holes rather than being uniformly embedded throughout the vitreous ice layer. To mitigate these effects, CF-2/2 grids were prepared using optimized blotting conditions, which allowed collection of full fields of view that contained PEDV S proteins throughout.

Micrograph movies collected on a K2 Summit detector using a Titan Krios operating at 300 kV were first processed in Warp to perform motion correction, contrast transfer function (CTF) estimation, and BoxNet-based nontemplated particle picking (35). The 269,838 Warp-extracted particles were then imported into cryoSPARC v2 for further processing (36). Two-dimensional (2D) classification removed 122,440 “junk” particles, and the remaining 147,398 particles were used to generate three ab initio volumes without imposition of any symmetry restraints. These three volumes were then used as templates to sort all 147,398 particles by 3D classification. This more stringent classification method sorted 34,743 particles into two classes which did not resemble biological macromolecules. The remaining 112,655 particles were sorted into a third class in which secondary structural elements were clearly visible. This final set of 112,655 particles was used to perform nonuniform homogeneous 3D refinement. These data were initially processed without imposing symmetry, resulting in a 3.3-Å map with obvious C3 symmetry. After C3 symmetry was imposed, the resolution improved to 3.1 Å (Fig. 1; see also Fig. S2 in the supplemental material). This map was then sharpened using LocalDeBlur before being used for model building and refinement (37).

The PEDV CV777 S map has a length of ∼150 Å and a radius of ∼85 Å, measuring from the 3-fold axis of symmetry to the tip of D∅. Our final model contains 1,064 amino acids per monomer, spanning Thr43 to Tyr1242. The majority of the residues that are missing from our model are contained within D∅, where a relatively low (∼5-Å) local resolution prohibited unambiguous building of some regions. Similarly, the density began to fade after Tyr1242 and the flexible HR2 domain at the C terminus of S could not be resolved. Our model encompasses 21 of the 29 N-linked glycosylation sites on each protomer of the PEDV spike, with 16 of these contained within the S1 subunit and 5 contained within the S2 subunit. We observed density for and built corresponding glycan molecules at 18 of the 21 sites. For the majority of these sites, we built only a single N-acetylglucosamine, whereas larger glycan chains could be built at 5 sites (Fig. S4D). The PEDV S1 subunit is almost entirely composed of β-sheets, whereas the S2 subunit is made up of a series of discontinuous α-helices that are characteristic of a class I fusion protein in the prefusion conformation (Fig. S3A).

D∅ (Arg34 to Cys231) is the domain of PEDV S located farthest away from the central 3-fold axis of symmetry. Although we were unable to build roughly half of this domain, eight β-strands that are organized into two β-sheets are clearly observable. D∅ contains four N-linked glycosylation sites, two of which could be clearly resolved and one of which contained density corresponding to the glycan itself. The S1-NTD (Thr232 to Ser471) adopts a tertiary structure similar to that of D∅, forming 12 β-strands that are organized into two β-sheets. Overall, the density in this region allowed unambiguous model building, excluding a flexible loop at the apex of the trimer (Leu354 to Ala363) that could not be clearly resolved. The NTD is densely glycosylated, with seven N-linked glycosylation sites, all of which could be resolved and six of which contained corresponding glycan density. The density at Asn261 was particularly prominent and allowed us to build a branched, high-mannose glycan that was packed between the NTD and the α-helices of the S2 fusion machinery. Notably, similar elongated glycans have been observed playing the same structural role at Asn74 and Asn240 in the cryo-EM structures of porcine deltacoronavirus (PDCoV) S and NL63 S, respectively (34, 38).

The S1-CTD (Phe504 to Asp637) is composed of 10 β-strands, a single α-helix, and 2 N-linked glycosylation sites, both of which could be observed in our map. Although a majority of the S1-CTD is organized into β-sheets, these secondary structural elements are thought to function as a scaffold for key aromatic residues based on the interactions between closely related alphacoronaviruses and their respective host cell receptors (15, 27). These aromatic residues are packed tightly against the NTD of the same protomer in the prefusion conformation, suggesting that the S1-CTD must undergo a conformational rearrangement in order to productively engage a host cell receptor. Subdomain 1 (SD-1) and subdomain 2 (SD-2) (Gln472 to Ser503 and Val638 to Tyr761, respectively) are composed of 14 β-strands and 5 N-linked glycosylation sites, 4 of which had observable glycan density. The putative S1/S2 cleavage site, between Lys755 and Ser756, is located in a small loop between the final two β-strands of SD-2 (Fig. S4A and B) (11). This cleavage site is positioned at the side of PEDV S, roughly equidistant between the viral membrane and the apex of the spike. Although this site is readily accessible to host trypsin-like proteases and can be cleaved by the addition of exogenous trypsin, our reconstruction showed continuous density for the peptide backbone throughout this region, supporting our SDS-PAGE data that indicated that the spike used in these studies had not been proteolytically processed at the S1/S2 cleavage site (Fig. 2). These data are also consistent with previous reports that PEDV S remains uncleaved during viral packaging and becomes proteolytically activated upon exposure to lysosomal proteases in the host cell (39).

The S2 subunit (Ser756 to Tyr1242) is positioned beneath the fusion-suppressive S1 capping subunit, forming extensive contacts with the internal face of the S1-CTD and subdomains 1 and 2. S2 is organized into 16 α-helices and 12 β-strands, three of which form an elongated, twisting β-sheet that resembles a fold observed previously in the fusion machinery of other class I fusion proteins. The hydrophobic fusion peptide (Ser892 to Val910) forms an α-helix that is directly C terminal to the S2′ protease cleavage site at Arg891 (Fig. S4A and C). Although there is density to show Arg891 packing tightly against S2, the “S2′ loop” spanning residues Ser878 to Gln889 could not be resolved, suggesting that this short stretch of residues does not adopt a single, rigid conformation. Overall, the S2 subunit of PEDV S strongly resembles the fusion machinery of other coronavirus spikes from diverse genera. Structural comparisons between PEDV S residues 756 to 1242 and the corresponding residues in NL63 S2, SARS-CoV S2, PDCoV S2, and infectious bronchitis virus (IBV) S2 yielded root mean square deviation (RMSD) values of 2.3 Å, 5.1 Å, 2.5 Å, and 4.1 Å, respectively (Fig. S3B) (30, 34, 38, 40). A small HR1 insertion (residues Ile1018 to Ala1031) that is present in the alphacoronaviruses and deltacoronaviruses but absent in the betacoronaviruses and gammacoronaviruses contributes to the higher level of divergence between SARS-CoV S2, IBV S2 and PEDV S2.

The most dramatic difference between the structure of PEDV S and that of the previously reported alphacoronavirus NL63 S is the position of their respective D∅ domains. In NL63 S, this domain was observed tucked against S2, pointing down toward the viral membrane. However, our reconstruction of PEDV S clearly shows the corresponding density for D∅ jutting out from the apex of the trimer such that it is perpendicular to the 3-fold axis of symmetry and readily accessible to interact with its host cell attachment factor, sialic acid (Fig. 3A). The positions of D∅ in these two structures are roughly related by a 180° rotation about a horizontal axis (see Movie S1 in the supplemental material). Perhaps due to some intrinsic flexibility of this domain, a complete de novo model for PEDV D∅ could not be unambiguously built, and our final model for PEDV S lacks ∼100 residues in this region. However, even with the relatively poor connectivity of our map in this region, we were able to observe density that corresponds to the predicted β-sheet-rich composition of PEDV S D∅ (Fig. 3B and C).

In an attempt to determine whether the presence of sialic acid could alter the conformation of PEDV S D∅, we incubated PEDV CV777 S with an ∼750-fold molar excess of the sialic acid analog 3′-sialyllactose. Screening this complex under cryogenic conditions yielded a 10-Å reconstruction that was indistinguishable from the PEDV CV777 S apo structure (Fig. 4). These observations are consistent with the theory that host glycans such as sialic acid are likely attachment factors that do not have a dramatic influence on the conformation of the prefusion coronavirus spike (41).

Several previous studies have described the isolation of PEDV S D∅-deletion variants that have arisen spontaneously in animals as well as in cell culture (23, 24). One such strain, PEDV PC177, was recently used to inoculate piglets in a PEDV challenge model, and it was shown that not only was the primary infection with PEDV PC177 significantly attenuated compared to infection with PEDV CV777, but also the piglets inoculated with the D∅-deletion variant were significantly more susceptible to subsequent reinfection with PEDV CV777 than those that were originally inoculated with PEDV CV777 (25). In an attempt to discover a structural explanation for these observations, we expressed and purified residues 1 to 1125 of PEDV PC177 S using the same C-terminal T4 fibritin motif and affinity tags that were used to express and purify PEDV CV777 S. Excluding the D∅ deletion at the N terminus of the spike, this variant shares 100% sequence identity with PEDV CV777 S. Because of this deletion, we were able to detect a slight but measurable shift of the PEDV PC177 S SEC elution volume relative to the elution volume of PEDV CV777 S (Fig. 5A). The purified PEDV PC177 S was then observed by negative-stain EM (Fig. 5B). The final 3D reconstruction of this S variant has a resolution of ∼20 Å, which was sufficient to show that, other than the absence of D∅, the structure of PC177 S does not dramatically differ from that of CV777 S (Fig. 5C). Because the previously reported differences in susceptibility to reinfection cannot be ascribed to any large-scale conformational differences between the spikes, these data suggest that the enhanced protection against reinfection in piglets inoculated with PEDV CV777 is due to a porcine immune response against D∅.

The S1-CTDs of PEDV S adopt the same arrangement that has been described in the alphacoronaviruses and deltacoronaviruses, wherein the putative receptor-binding loops are tucked against the NTD of the same protomer (34, 38, 42). Although these loops are present at the apex of the trimer (Fig. 6, top panel; see also Fig. S5), it is unlikely that they would be able to engage a host cell protein receptor without the receptor sterically clashing with both the NTD of the same protomer and the S1-CTD of the neighboring protomer (Fig. 6, bottom panel). For the betacoronaviruses SARS-CoV and MERS-CoV, this clash is avoided when the S1-CTDs undergo a hinge-like movement that repositions the domains above the rest of S1 (31, 32). This conformational change is thought to allow the SARS-CoV and MERS-CoV S proteins to mask their determinants of receptor binding from antibodies in one conformation while making them readily accessible for host cell receptor engagement in another conformation. However, after extensive 3D classification of our PEDV CV777 data set, we were unable to observe any such conformational dynamism of the PEDV S1-CTDs in the intact PEDV S trimer. Despite this, the inaccessibility of the putative receptor-binding loops in their observed conformation suggests that there is likely to be an equivalent phenomenon in the alphacoronaviruses.

During 2D classification of the PEDV CV777 S cryo-EM data set, roughly 10% of particles were consistently assigned into classes that resembled a trimeric S1 ring, although these classes lacked the interior density that would correspond to the S2 fusion machinery (Fig. 7A). The presence of these dissociated rings suggests that some small proportion of the purified spike was processed by endogenous trypsin-like proteases, even though SDS-PAGE analysis revealed only a single band corresponding to uncleaved S (Fig. 2). Similar dissociated S1 rings have been reported during cryo-EM analysis of MERS-CoV S (31). Despite the relatively few dissociated PEDV S1 caps that were observed, a 3D reconstruction generated from these particles resulted in a 9.4-Å map that showed evidence of the conformational rearrangement for two of the three S1-CTDs (Fig. 7B and C). These domains appear to have rotated ∼30° away from the 3-fold axis of symmetry, presumably undergoing a hinge-like motion similar to what has been observed in the betacoronaviruses (Fig. 7D). The two S1-CTDs that are observed in the “out” conformation are stacked directly on their respective SD-1s such that their receptor-binding loops are now pointed away from the NTD and are presumably readily accessible to engage with a putative host cell receptor without forming any steric clashes. Although further experimentation will be required to obtain high-resolution structures of these transient conformations, these observations provide some of the first insights into the process of alphacoronavirus receptor engagement and triggering.

A human-codon-optimized gene fragment encoding residues 1 to 1319 of PEDV CV777 S was generously provided by Kizzmekia Corbett and Barney Graham of the Vaccine Research Center. This gene was subcloned into a pαH eukaryotic expression plasmid with a C-terminal T4 fibritin motif, an HRV3C protease cleavage site, an 8× His tag, and a Twin-Strep-tag. Further MegaPrimer subcloning was performed to remove the 194 amino acids corresponding to D∅, thereby generating a gene encoding residues 1 to 1125 of PEDV PC177 S. These plasmids were transiently transfected into FreeStyle 293-F cells (GibCo) using polyethylenimine. Transfected FreeStyle 293-F cells were treated with 5 μM kifunensine to ensure uniform high-mannose glycosylation. PEDV S CV777 and PEDV S PC177 were purified over Strep-Tactin resin (IBA Lifesciences) before being run over a Superose6 10/300 column (GE Healthcare Biosciences) in 2 mM Tris (pH 8.0), 200 mM NaCl, 0.02% NaN₃.

SEC-purified PEDV CV777 S was mixed with tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin from bovine pancreas (Sigma-Aldrich) at a ratio of 10:1 (wt/wt). This mixture was incubated at 37°C, and samples from various time points were collected, reduced by the addition of lithium dodecyl sulfate (LDS) loading dye containing 5% 2-mercaptoethanol, and analyzed by SDS-PAGE after being heated for 5 min at 98°C.

CF-2/2 grids (Electron Microscopy Sciences) were plasma cleaned for 30 s in a Gatan Solarus 950 plasma cleaner with a 4:1 O₂/H₂ ratio. PEDV S CV777 at a concentration of 0.4 mg/ml was mixed with 2 mM Tris (pH 8.0), 200 mM NaCl, 0.02% NaN_3, and 0.01% Amphipol A8-35 and was deposited onto the grids before being blotted for 6 s and plunge-frozen in liquid ethane using a Vitrobot Mark IV instrument (Thermo Scientific). The frozen grid was imaged using an FEI Titan Krios transmission electron microscope (Thermo Scientific) equipped with a K2 direct electron detector (Gatan) at a nominal magnification of ×22,500, corresponding to a calibrated pixel size of 1.075 Å/pixel. A total of 3,186 movies were collected using Leginon (45). A full description of the cryo-EM data collection parameters can be found in Table 1.

Motion correction and CTF estimation were performed for PEDV S CV777 movies in Warp (35). Unbiased, nontemplated particle picking was also performed in Warp using BoxNet. BoxNet-selected particles were imported into cryoSPARC v2.4.6 (Structura Biotechnology) (36) for 2D classification, ab initio reconstruction, 3D classification, and nonuniform 3D refinement. The resulting 3.1-Å map was then subjected to local-resolution-guided sharpening in LocalDeblur (37). A detailed description of the cryo-EM data processing workflow can also be found in Fig. S1. Model-building was performed by fitting a small, homologous portion of the NL63 S2 fusion machinery (PDB identifier [ID] 5SZS) into the PEDV S CV777 map. The rest of the model was built manually in Coot (46) and refined in both PHENIX (47) and ISOLDE (48) (Table 2).

PEDV CV777 S at a concentration of 0.4 mg/ml was incubated with 2 mM 3′-sialyllactose (Sigma-Aldrich) in 2 mM Tris (pH 8.0), 200 mM NaCl, 0.02% NaN₃, 0.01% Amphipol A8-35 for 3 h at 4 °C. A 3-μl volume of the spike and 3′-sialyllactose mixture was deposited on a plasma-cleaned CF-2/2 grid before being blotted for 6 s and plunge-frozen in liquid ethane using a Vitrobot Mark IV instrument. The frozen grid was imaged using an FEI Talos transmission electron microscope (Thermo Scientific) equipped with a Ceta 16M detector. Micrographs were collected manually using TIA v4.14 (Thermo Scientific) at a nominal magnification of ×92,000, corresponding to a calibrated pixel size of 1.63 Å/pixel. A full description of the EM data collection parameters can be found in Table 1. CTF estimation, particle picking, 2D classification, ab initio 3D reconstruction, and 3D refinement were performed in cisTEM (49).

PEDV S PC177 was deposited onto a plasma-cleaned CF400-Cu grid (Electron Microscopy Sciences) at a concentration of 0.035 mg/ml before being stained with methylamine tungstate (Nanoprobes). The stained grid was imaged using an FEI Talos transmission electron microscope equipped with a Ceta 16M detector. Micrographs were collected manually using TIA v4.14 at a nominal magnification of ×92,000, corresponding to a calibrated pixel size of 1.63 Å/pixel. A full description of the negative-stain EM data collection parameters can be found in Table 1. CTF estimation, particle picking, 2D classification, ab initio 3D reconstruction, and 3D refinement and sharpening were all performed in cisTEM.

Cryo-EM maps have been deposited in the Electron Microscopy Data Bank (EMDB) under accession codes EMD-20671 and EMD-20672. The atomic model has been deposited in the Protein Data Bank (PDB) under accession code 6U7K.