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Cryo-electron Microscopy Structure of S-Trimer, a Subunit Vaccine Candidate for COVID-19

An effective vaccine against SARS-CoV-2 is critical to end the COVID-19 pandemic. In this study, using Trimer-Tag technology, we were able to produce stable and large quantities of WT S-Trimer, a subunit vaccine candidate for COVID-19 with high safety and efficacy from animal and phase 1 clinical trial studies.

A successful vaccine that could truly impact the course of the ongoing COVID-19 pandemic has to have four key characteristics: safety, efficacy, scalability (to billions of doses to meet global demand), and speed. Although protein subunit vaccines have excellent track records for the first three requirements, exemplified by the highly successful vaccine Gardasil used to prevent human papillomavirus (HPV) infections (3) and Shingrix vaccine for containing herpes zoster virus infections (4), subunit vaccine development can take years to decades to complete. Many of the difficulties reside in the manufacturing processes that have to ensure that a fully native-like antigen structure is retained, starting from subunit vaccine designs. Similar to other enveloped RNA viruses, such as HIV, respiratory syncytial virus (RSV), and influenza virus, coronaviruses, including SARS-CoV-2, use a ubiquitous trimeric viral surface antigen (spike protein) to gain entry into host cells. In the case of SARS-CoV-2, this occurs via binding to the ACE2 receptor expressed in target cells (5). Approaches for using a noncovalent trimer foldon from T4 bacteriophage fibritin to stabilize the trimer conformation while simultaneously introducing mutations in viral antigens to abolish furin cleavage and stabilize the antigen in prefusion forms (6,7) are common strategies inherited from decades of HIV and RSV vaccine studies, but these have yet to yield a successful vaccine.

RESULTS
Construct design and negative staining. Using Trimer-Tag technology (8), we produced both soluble wild-type (WT) and a furin site mutant (MT) (R685A) forms of S-Trimer from CHO cells in serum-free fed-batch processes in bioreactors, with protein yields ranging from 0.5 to 1 g/liter. These antigen titers are 3 orders of magnitude higher than that reported previously for foldon-derived S proteins with two Pro mutations (S-2P) (6,7), laying a solid foundation for the scalability requirement of a successful COVID-19 vaccine. Both S-Trimers consist of the ectodomain (amino acid residues 1 to 1211) of SARS-CoV-2 spike protein fused in-frame to the C-terminal region of human type I (a) collagen, which spontaneously form a disulfide-bond linked homotrimer, thereby stabilizing the antigens in trimeric forms ( Fig.1A and B). Using a tailored affinity purification scheme that employs a collagen receptor Endo180-Fc fusion protein that binds to the Trimer-Tag with high affinity, the secreted S-Trimers were purified to near homogeneity in a single step (9). Reducing SDS-PAGE analysis of the purified S-Trimers revealed that the WT S-Trimer was metastable and partially cleaved precisely at S1/S2 boundary by furin, while a single point mutation (R685A) in MT S-Trimer fully abolished the protease cleavage (Fig.1C). WT spike proteins from live SARS-CoV-2 (10) or recombinant full-length S (11) were all previously shown to be partially cleaved apparently at the S1/S2 boundary by furin proteases. In contrast, an S-Trimer derived from wild-type SARS-CoV-1 S protein lacking a furin cleavage site produced in the same manner was essentially uncleaved by furin protease, like the MT S-Trimer from SARS-CoV-2 (Fig.1C). Receptor binding studies using ForteBio biolayer interferometry showed that all three S-Trimers had similarly high affinities for ACE2-Fc at 1.2 nM (Fig.  1D), similar to previous studies using purified S-2P protein (5). Negative-staining electron microscopy (EM) analysis of the MT S-Trimer revealed homogeneous particles consistent with trimeric spike proteins connected to the Trimer-Tag (Fig. 1E). Particles with similar structural features have also been observed by negative-staining EM for WT S-Trimer (9). Taken together, these results suggested that both MT and WT S-Trimer proteins produced using Trimer-Tag technology were well folded.
Cryo-EM structure of the MT S-Trimer. To further investigate structural details of these vaccine candidates, we first sought to determine the cryo-EM structure of the MT S-Trimer, which is more tractable for structural determination. While the particles showed preferred top-view orientation when embedded in ice, they preferentially adopted side view orientation on graphene oxide (GO)-coated grids ( Fig. 2A). Combination of the two data sets enabled us to obtain a structure at a 2.6-Å resolution ( Fig. 2B to E and Table 1). Due to the high resolution, the EM density for side chains was clear in the structure (Fig. 3). In addition to all the glycosylation sites and disulfide bonds previously observed in the S-2P structure (PDB code 6VXX), the disulfide bond between Cys15 and Cys136 and the N-linked glycan at Asn17, both in the N-terminal domains (NTDs), were well resolved in our structure ( Table 2). The Trimer-Tag was invisible in our structure due to highly flexible nature of the linker between the soluble S and the C-prodomain of collagen (Fig. 2B). After three-dimensional (3D) classification without imposing 3-fold symmetry (C3), we found that all three receptor binding domains (RBDs) in our map adopt a closed conformation (RBD down) without any open conformation (RBD up) that was previously observed in the S-2P structure ( Fig.  2B) (6). Surprisingly, we observed unaccounted-for EM density in both the NTD and RBD of the S1 domain. The presence of polysorbate 80 (PS80) during the purification process suggested that the bulky EM density in the NTD region could be accounted for by PS80 (Fig. 3A and B). Indeed, PS80 can be well fitted into the density of the NTD. Mass spectrometry analysis further confirmed the presence of PS80 in the MT sample (Fig. 4). PS80 is buried deeply in the hydrophobic pocket residues, with a few hydrophilic residues, including N99, N121, R190, and H207, making hydrogen bonds with the hydroxyl group of PS80 ( Fig. 5A and B). Notably, PS80 engages hydrophobic interactions with F175 and M177, which are invisible in the S-2P structure (Fig. 5C). Since no small molecule was shown to bind to the S-2P structure, PS80 likely stabilizes the disordered loops of the NTD, making them more ordered ( Fig. 5B and C). The EM density in the RBD is elongated and in close proximity to R408 (Fig. 3C); we speculate that it may be oleic acid or linoleic acid, which is present in the culture medium. Although both oleic acid and linoleic acid can be ambiguously fitted into the density of the RBD owing to the excellent quality of EM map, mass spectrometry analysis indicated both MT and WT samples contained a higher level of oleic acid than linoleic acid (Fig 4B and  C). Recent studies revealed the presence of linoleic acid at the same binding site but with a slightly different position (7,12). The oleic acid in our structure, located in the hydrophobic pocket of the RBD, engaged a salt bridge interaction with R408 and a hydrogen bond interaction with Q409 at the adjacent protomer through its carboxylic acid group, bringing the RBD domain in close proximity and resulting in the tightly closed conformation (Fig. 5D).
Cryo-EM structure of the WT S-Trimer. Recent studies have shown that low pH can stabilize the S-Trimer (13). Indeed, negative-staining EM analysis of WT S-Trimer at pH 5.5 revealed more homogenous trimer than that at physiological pH (9). In light of this finding, we were able to determine the cryo-EM structure of the WT S-Trimer at a 3.2-Å resolution at pH 5.5 ( Fig. 6 and Table 1). The structure of the WT S-Trimer resembled that of the MT form, with a root mean square deviation (RMSD) of 0.5 Å over 2,773 Ca atoms (Fig. 7A). Oleic acid was well resolved in the WT structure but the density for the PS80 was weak, likely due to the low resolution or low occupancy. As a result, the NTD of the WT S-Trimer was less well resolved than that of MT (Fig. 7A). It has been shown that a pH-dependent switch domain (residues 824 to 848; pH switch 1) undergoes dramatic conformational changes at different pH values (13). However, this region was nearly identical between our two structures (Fig. 7B). The fatty acid binding in our WT S-Trimer structure may stabilize the conformation of pH switch 1, accounting for conformational difference from the previous study (13). In contrast, a fragment (residues 617 to 639) we named pH switch 2 at the CTD1 region of the S1 domain before the furin cleavage site displays significant structural arrangement. Whereas this region appeared disordered in the MT structure at physiological pH conditions, it was well ordered and formed a helix-turn-helix structural motif in the WT structure at pH 5.5 (Fig. 7C). From a structural perspective, lower pH likely contributes to this structural arrangement. At physiological pH, R319 forms salt bridge interactions with D737 and D745 (Fig. 7D). At lower pH, the protonation of D737 and D745 weakens these interactions. As a result, R319 flips to the other side and makes hydrophobic interactions with W633 and L629 through its aliphatic chain, leading to the ordered helix-turn-helix motif (Fig. 7E). The newly formed structural motif makes direct contact with the previously identified pH switch 1 of the adjacent protomer, accounting for the enhanced stability of the WT S-Trimer at lower pH. The structural arrangement of pH switch 2 at different pHs was also observed in previous studies (13), further supporting the notion that the conformational change between the MT and WT S-Trimer structures was due to the different pH but not to the mutation in the furin site. The biological relevance of the structural arrangement of pH switch 2 may require further investigation.
Conformational change of the S protein before receptor binding. In contrast to the structural differences described above for S-2P protein, both of our WT and MT S-Trimer proteins were nearly identical to the recently published structures of full-length wild-type S (PDB code 6XR8) (11) and 3Q-2P-FL with two proline mutations (PDB code 7JJI) (7,14) purified in detergent from HEK293 and sf9 insect cell membranes, respectively. When revisiting the electron density map for full-length wild-type S protein (Electron Microscopy Data Bank [EMDB] no. 22292), we spotted unassigned density at the same position as oleic acid, which was absent in the S-2P map (EMDB no. 21452). Therefore, fatty acid binding at the RBD stabilizes the tightly closed conformation, accounting for the conformational difference from S-2P. Based on the structural similarity among all published structures of SARS-CoV-2 S protein, we can classify them into three distinct states: a tightly closed state, a loosely closed state, and an open state (Fig. 8A). A conformational switch (residues 835 to 858) previously known as the pH switch or the fusion peptide-proximal region (FPPR) is critical for conformational transition from the tightly closed state to the open state (Fig. 8B). In the tightly closed state, which is less accessible to the receptor, three RBDs are down and in closer prox-  imity to one another. The conformational switch region is well ordered and stabilizes this tightly closed state. Recently, the D614G mutation has become predominant over the ancestral form worldwide and has been shown to increase viral infections (15,16).
In the tightly closed state, D614 makes a salt bridge interaction with K854 at the conformational switch region (Fig. 8C). From the tightly to the loosely closed state, the conformational switch undergoes a large conformational arrangement and becomes disordered (Fig. 8D). K854 flips to the opposite side and interacts with D568 and D574 of the CTD1, causing the S1 to move downwards relative to the S2 (Fig. 8A)

DISCUSSION
To our knowledge, this is the first cryo-EM structure of the wild-type S protein in soluble and cleavable form without the transmembrane domain, confirming the structural integrity of this metastable wild-type form of COVID-19 subunit vaccine candidate. Whereas most vaccine candidates currently in clinical trials incorporated furin site and double proline mutations, our S-Trimer vaccine candidate used wild-type sequence without any mutagenesis, providing an important alternative option. Trimer-Tag technology has proven in this study to be able to rapidly produce large quantities of native-like S-Trimer antigen and may offer a platform technology for subunit vaccine development for enveloped RNA viruses that use ubiquitous trimeric antigens to invade host cells.
Like for the previously reported structure of full-length WT S protein purified in detergent micelles, it is unclear whether the furin cleavage site in our resolved WT S-Trimer structure is cleaved. Moreover, we could not exclude the possibility that other conformational states exist in the WT sample that were not captured in our cryo-EM study, since partial cleavage of the furin site may lead to some S1 dissociation from S-Trimer. Nevertheless, we are certain that the highly purified WT S-Trimer predominately adopts a prefusion state, unlike the full-length wild-type spike protein, which forms both pre-and postfusion states in the presence of detergent (11).
Similar to the subunit vaccine candidate with two proline mutations from Novavax, WT S-Trimer largely adopts tightly closed conformation, which is stabilized by fatty acid binding (14). The biological relevance of the fatty acid binding awaits further investigation. One may argue that the tightly closed conformation of WT S-Trimer prevents the production of antibodies recognizing receptor binding region at the RBD, since they are buried in the closed conformation. The binding affinity of the spike protein in open conformation for the receptor is similar to that in tightly closed state, suggesting energy barrier from tightly closed state to open state is relatively low (5). Therefore, it is likely that WT S-Trimer adopts multiple conformational states when injected into the human body. The phase 3 clinical results for the vaccine candidate from Novavax with fatty acid binding and closed conformation showed promising efficacy, indicating that fatty acid binding has little influence on production of neutralizing antibodies.
Preclinical studies of the WT S-Trimer vaccine candidate revealed that it resulted in rapid and high-level induction of both neutralizing antibodies and Th1-biased cellular immune responses in mice, rats, and nonhuman primates. Nonhuman primates immunized with WT S-Trimer were fully protected from SARS-CoV-2 viral challenge (9). The phase 1 clinical trial studies also showed great immunogenicity and safety (17). Global phase 2/3 clinical trials are currently ongoing.

MATERIALS AND METHODS
Protein expression and purification. Endo180-Fc expression vector was generated by subcloning a PCR-amplified cDNA encoding soluble human Endo180 (amino acid residues 1 to 1394) into the HindIII site of pGH-hFc expression vector (GenHunter, Nashville, TN) to allow in-frame fusion to human IgG Fc. The expression vector was transfected into the GH-CHO (dhfr 2/2 ) cell line (GenHunter, Nashville, TN) using FuGENE 6 (Roche) and grown in Iscove's modified Dulbecco's medium (IMDM) with 10% fetal bovine serum (FBS). After stepwise gene amplification with increasing concentrations (0.0 to 10 nM) of methotrexate (MTX) (Sigma), a high-titer clone was then adapted to SFM-4CHO serum-free medium (GE BioSciences). The secreted End0180-Fc fusion protein was then produced in a 15-liter bioreactor (Applikon) under a fed-batch process with Cell Boost 2 supplement (GE HyClone) as instructed by the manufacturer. Endo180-Fc was purified to homogeneity by protein A affinity chromatography using MabSelect PrismA (GE Healthcare) followed by Capto QXP resins (GE BioSciences) in a flowthrough mode to remove any host cell DNA and residual host cell proteins (HCP).
To produce S-Trimer fusion proteins, cDNA encoding the ectodomain of either wild-type SARS-CoV-2 S protein (amino acid residues 1 to 1211) (GenBank accession number MN908947.3) or a SARS-CoV (amino acid residues 1 to 1193) (GenBank accession number AAS00003.1) were synthesized using -optimized codons for Cricetulus griseus (CHO cells) by GenScript. The cDNAs were subcloned into pTRIMER expression vector (GenHunter, Nashville, TN) at HindIII and BglII sites to allow in-frame fusion of the soluble S protein to Trimer-Tag [residues 1156 to 1406 from human type I(a) collagen] as described previously (8). Furin cleavage site mutant S-Trimer (R685A) was generated by site-directed mutagenesis using the QuikChange kit (Stratagene). The expression vectors were transfected into the GH-CHO (dhfr 2/2 ) cell line (GenHunter) using FuGENE 6 (Roche) and grown in IMDM with 10% FBS. After stepwise gene amplification with increasing concentrations (0.0 to 10 nM) of MTX (Sigma), clones producing the highest S-Trimer titer were adapted to SFM-4CHO serum-free medium (GE BioSciences). The secreted S-Trimer fusion proteins were produced in a 15-liter bioreactor (Applikon) under a fed-batch process with Cell Boost 2 supplement (GE HyClone) as instructed by the manufacturer.
Cell culture medium was clarified by depth filtration (Millipore) to remove cell and debris. S-Trimers were purified to homogeneity by consecutive chromatographic steps, including a protein A affinity column using MabSelect PrismA (GE Healthcare) which was preloaded with Endo180-Fc at 3 mg/ml to capture S-Trimer, based on the high-affinity binding between Endo180 and Trimer-Tag (18). After unbound contaminating proteins were washed off, S-Trimers were purified to near homogeneity in a single step using 0.5 M NaCl in phosphate-buffered saline (PBS). For S MT and SARS-CoV S-Trimer, the proteins were dialyzed against PBS plus 0.02% polysorbate 80 before analysis. After 1 h of low-pH (pH 3.5) viral inactivation (VI) using acetic acid, the pH was adjusted to neutral range, and WT S-Trimer was further purified on a Capto QXP resin (GE BioSciences) in a flowthrough mode to remove any host cell DNA and residual HCP. A final preventative viral removal (VR) step was performed using a nanofiltration cartridge (AsahiKASEI) before final buffer exchange to PBS plus 0.02% polysorbate 80 by ultrafiltration/diafiltration (UF/DF) (Millipore). For EM sample preparation, the protein was further purified by size exclusion chromatography on an Agilent 1260 Infinity high-performance liquid chromatograph (HPLC) with a TSK gel G3000 SWxL column (Tosoh) in PBS.
ACE2-Fc expression vector was generated by subcloning a gene-synthesized cDNA template (GenScript) encoding soluble human ACE2 (amino acid residues 1 to 738; GenBank accession number NM_001371415.1) into HindIII and BglII sites of pGH-hFc expression vector (GenHunter, Nashville, TN) to allow in-frame fusion to human IgG Fc. The expression vector was then stably transfected into the GH-CHO (dhfr 2/2 ) cell line, high-expression clones were selected and adapted to SFM-4-CHO (HyClone) serum-free medium, and ACE2-Fc was produced in a 15-liter bioreactor essentially as described for S-Trimer above. ACE2-Fc was purified to homogeneity from the conditioned medium using a PoRos XQ column (Thermo Fisher) following the manufacturer's instructions.
Studies of binding of S-Trimers to human receptor ACE2-Fc. The avidity of binding of different S-Trimers to the SARS-CoV-2 receptor ACE2 was assessed by biolayer interferometry measurements on a ForteBio (Pall, NY) Octet QKe platform. ACE2-Fc (10 mg/ml) was immobilized on protein A (ProA) biosensors (Pall). Real-time receptor binding curves were obtained by applying the sensor in 2-fold serial dilutions of S-Trimer from 22.5 to 36 mg/ml in PBS. Kinetic parameters (K on and K off ) and affinities (dissociation constants [K D ]) were analyzed using Octet software, version 12.0. Dissociation constants were determined using steady-state analysis, assuming a 1:1 binding model for S-Trimer to ACE2-Fc.
Negative staining. Negative staining was performed as previously described (19). In brief, 3 ml of purified S-trimer at a concentration of about 0.01 mg/ml was deposited on a glow-discharged carboncoated copper grid for 30 s before being blotted with filter paper. Grids were quickly washed with 2 drops of water and 1 drop of 2% (wt/vol) uranium acetate. Grids were kept touching to the last drop of 2% (wt/vol) uranium acetate for 100 s and blotted with filter paper. Data collection was performed on a Talos L120C electron microscope operated at 120 kV equipped with an FEI Ceta 4K detector. Images were collected at a magnification of Â57,000 and a defocus of 1.5 mm.
Cryo-EM sample preparation and data collection. Purified MT S-trimer proteins diluted to 0.2 and 0.5 mg/ml in PBS were applied to glow-discharged gold holey carbon 1.2/1.3 300-mesh grids with In the loosely closed conformation, the switch region undergoes structural arrangement, in which K854 flips and pulls the S1 domain downwards through interacting with D574 and D568 in the CTD1 region. (E) The K854G mutation reduced the virus infectivity. TMPRSS2-293T cells expressing hACE2 on a 96-well plate were infected with lentivirus PV expressing luciferase and pseudotyped with the indicated SARS-CoV-2 spike glycoprotein. Luciferase activity (LUC) was measured at 48 h postinfection. and without graphene oxide (GO), respectively. Grids were blotted for 2 to 4 s at a blotting force of 4 and plunge-frozen in liquid ethane using a MarkIV Vitrobot (Thermo Fisher Scientific). The chamber was maintained at 8°C and 100% humidity during freezing. A sample (0.3 mg/ml) of WT S-trimer in low-pH buffer (100 mM sodium citrate [pH 5.5] and 100 mM NaCl) was deposited on glow-discharged gold holey carbon 1.2/1.3 300-mesh grids with graphene oxide. Grids were blotted and vitrified under the same condition.
All movies were collected using a Titan Krios microscope (Thermo Fisher Scientific) equipped with a BioQuantum GIF/K3 direct electron detector (Gatan). The detector was operated in superresolution mode. A complete description of cryo-EM data collection parameters is summarized in Table 1.
Cryo-EM image processing. For MT S-Trimer protein, motion correction for cryo-EM images and contrast transfer function (CTF) estimation were performed using motioncorr2 (20) and CTFFIND4 (21), respectively. A total of 574,832 particles were automatically picked from 534 images collected on grids without GO using Laplacian-of-Gaussian in Relion 3.0.7 (22), and 1,029,938 particles were automatically picked from 1,584 images collected on grids with GO. Extract particles from two data sets were downsized 2-fold and subjected to 2D classification separately, resulting in 438,205 and 560,545 good particles. Particles from GO grids were recentered using scripts written by Kai Zhang before 3D classification. Good particles from both data sets were combined and subjected to 3D classification using the initial model generated from the model of S protein with C1 symmetry (PDB code 6VXX). Two major classes accounting for 26.7% and 25.8% of the particles showed clear and complete structural features. These particles were autorefined, followed by local 3D classification with C1 symmetry to generate two classes with similar structures. The better class was subjected to 3D refinement with C3 symmetry and postprocessed using mask on the entire molecule to yield a 2.9-Å map. CTF refinement was performed to further increase the resolution to 2.6 Å.
For image processing of WT S-Trimer, the MT S-Trimer map was low-pass-filtered to a 20-Å resolution and used as the 3D reference template for autopicking. A total of 752,204 articles were picked from 1,199 images collected on a GO-coated grid and used for two rounds of 2D classification. A total of 541,528 particles were selected from good 2D classes and used for 3D classification with C1 symmetry. One class accounting for 32.2% showing a well-defined structure was refined with C3 symmetry and postprocessed using mask on the entire molecule to give a map at a 3.4-Å resolution. CTF refinement followed by another round of 3D refinement improved the resolution to 3.2 Å. Reported resolutions were calculated based on the gold standard Fourier shell correlation (FSC) at the 0.143 criterion.
Model building. The soluble ectodomain structure S-2P (PDB code 6VXX) was used as a template for model building. The missing region in the S-2P structure can be de novo modeled in the MT S-Trimer map, owing to its high resolution. The model was manually built in COOT (23), and real space refinement was performed in Phenix (24) using rotamer, Ramachandran, and secondary-structure restraints. The model for WT S-trimer was built in COOT based on the MT structure and refined in Phenix using the same strategy. Refinement statistics are summarized in Table 1.
Small-molecule extraction from protein samples. Twenty-microliter volumes of WT and MT S-Trimer samples at 1.21 mg/ml and 1.02 mg/ml, respectively, were transferred to Eppendorf tubes and placed in a heater block at 100°C for 5 min. Samples were then extracted with 100 ml of methanol. The tubes were vortexed and centrifuged at 15,000 rpm and 4°C for 10 min. The supernatant was collected and transferred to glass vials, and 1 ml was injected for liquid chromatography-mass spectrometry (LC-MS) analysis. For oleic acid and linoleic acid reference samples, 10 mM stock solutions of compounds were prepared in dimethyl sulfoxide (DMSO) and diluted to 1 mM by methanol, and 1 ml was injected for the LC-MS analysis.
LC-MS analysis. LC-MS analyses of PS80 and MT S-Trimer sample were performed by an Agilent 1290 ultrahigh-performance liquid chromatograph (UHPLC) coupled to an Agilent quadrupole-time of flight (QTOF) mass spectrometer via an electrospray ionization source (ESI) with JetStream technology. The separation was performed by flow injection using isocratic flow of a solvent composed of 0.1% formic acid in 60% acetonitrile and 40% water. The flow rate was set at 0.2 ml/min for 0.5 min. Mass spectra were recorded in the positive ionization mode over a mass range from m/z 100 to 1,500. The scan parameters were capillary voltage of 4.0 kV and fragmentor voltage of 135 V. The nitrogen pressure and flow rate on the nebulizer were 40 lb/in 2 and 5 liters/min, respectively. Other ion source parameters included drying gas temperature of 325°C, sheath gas temperature of 350°C, and sheath gas flow rate of 10 liters/min. The relative quantitation for oleic acid and linoleic acid was performed by a Thermo Vanquish UHPLC coupled to a Thermo Q Exactive HF-X hybrid quadrupole-Orbitrap mass spectrometer. The chromatographic separation was performed using a Waters CSH C 18 column (2.1 by 100 mm; 1.7 mm) and maintained at 40°C. The separation was performed using isocratic flow of a solvent composed of 90% acetonitrile, 10% water, and 2 mM ammonium acetate.
SARS-CoV-2 pseudovirus production and infection. SARS-CoV-2 pseudovirus (PV) was produced using the lentiviral system. HEK293T cells were transfected at ;80% confluency in a 6-well plate using Lipofectamine 3000 with 4.8 mg of total DNA. The ratio of 4:3:1 by mass was used for the lentivirus packaging plasmid (pHIV-Luc) encoding luciferase, the pSPAX2 plasmid expressing the lentivirus gag, pol, and rev genes, and a plasmid expressing the full-length spike protein of SARS-CoV-2 or mutated S proteins, including D614G, K854G, or D614G/K854G double mutations. The SARS-CoV-2 S protein gene used in the production of pseudovirus was codon optimized containing a C9 tag at its C terminus. Medium was changed with Dulbecco's modified Eagle's medium (DMEM) containing 2% FBS (D2) at 6 h posttransfection, and pseudovirus-containing culture supernatants were collected at 48 h posttransfection, cleared through 0.45-mm filters, and either purified or aliquoted and frozen at 280°C immediately.
Infection assays were performed using hACE2/TMPRSS2-293T cells seeded on a 96-well plate with equal pseudovirus each well. Inoculated plates were incubated for 24 h in a CO 2 incubator and medium was replaced with DMEM containing 2% FBS. Infection levels were assessed 48 h postinfection by measuring luciferase activity using the Bright-Glo luciferase assay system.
Data availability. Data are available in the Protein Data Bank under accession numbers 7E7B and 7E7D and in EMDB under accession numbers EMD-30998 and EMD-30999.

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, MOV file, 0.4 MB.