key: cord-0309071-i9r77o70 authors: Li, Tingting; Zheng, Qingbing; Yu, Hai; Wu, Dinghui; Xue, Wenhui; Zhang, Yuyun; Huang, Xiaofen; Zhou, Lizhi; Zhang, Zhigang; Zha, Zhenghui; Chen, Tingting; Wang, Zhiping; Chen, Jie; Sun, Hui; Deng, Tingting; Wang, Yingbin; Chen, Yixin; Zhao, Qinjian; Zhang, Jun; Gu, Ying; Li, Shaowei; Xia, Ningshao title: Characterization of the SARS-CoV-2 Spike in an Early Prefusion Conformation date: 2020-03-18 journal: bioRxiv DOI: 10.1101/2020.03.16.994152 sha: 1ccdb6a71a89b14490f0f1d533f3f4ec8afde0e8 doc_id: 309071 cord_uid: i9r77o70 Pandemic coronavirus disease 2019 (COVID-19) is caused by the emerging severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), for which there are no efficacious vaccines or therapeutics that are urgently needed. We expressed three versions of spike (S) proteins—receptor binding domain (RBD), S1 subunit and S ectodomain—in insect cells. RBD appears monomer in solutions, whereas S1 and S associate into homotrimer with substantial glycosylation. The three proteins confer excellent antigenicity with six convalescent COVID-19 patient sera. Cryo-electron microscopy (cryo-EM) analyses indicate that the SARS-CoV-2 S trimer dominate in a unique conformation distinguished from the classic prefusion conformation of coronaviruses by the upper S1 region at lower position ~15 Å proximal to viral membrane. Such conformation is proposed as an early prefusion state for the SARS-CoV-2 spike that may broaden the knowledge of coronavirus and facilitate vaccine development. The novel coronavirus grouped in betacoronavirus genus has become the third serious 41 virus intruder to human in the coronaviridae, after sever acute respiratory syndrome Organization (WHO) has declared the SARS-CoV-2 epidemic as a pandemic of 53 international concern and updates the COVID-19 situation every day. Like other coronaviruses, S is responsible for initiating the engagement to a specific 58 cellular receptor angiotensin-converting enzyme 2 (ACE2) and mediating the cell-virus 59 membrane fusion by the class I fusion mechanism 4, 5 . Thus, S is the main target for 60 neutralizing antibodies against viral infection and the core immunogen constituent of 61 vaccine design. S is consisted of S1 and S2 subunits and the cleavage on S1/S2 62 boundary by protease during biosynthesis is prerequisite for coronaviruses cellular during RBDs "down" to "up" was associated with the binding of receptor ACE2 as well 79 as the recognition of neutralizing monoclonal antibodies 11 . A safe and efficacious vaccine is urgently needed to control and eliminate the 81 SARS-CoV-2 infection. Various forms of vaccine candidates, mostly aiming to elicit 82 neutralizing antibodies against S proteins, are under preclinical research or even 83 subjected to clinical trials 12 . Here, we cloned S ectodomain and its fragments RBD and 84 S1 into recombinant baculovirus and expressed the proteins in insect cells. We found 85 that S and S1 formed homotrimer in solutions and the three proteins reacted well with 96 To screen a potent immunogen for COVID-19 vaccine development, we designed three 97 constructs-S ectodomain, S1 and RBD-for the SARS-CoV-2 Spike (S) protein 98 expression by aligning the SARS-CoV-2 S gene (Genbank accession no. NC_045512.2) 99 to a SARS-CoV strain (Genbank accession no. NC_004718) S gene sequence in terms 100 of structure-defined domain profile of the SARS-CoV S protein (Fig. 1A) . The gene of 101 SARS-CoV-2 S ectodomain encoding amino acids (aa) 15-1,213 with removal of its 102 original signal sequence was cloned to the downstream of the gp67 signal sequence in 103 pAcgp67B plasmid vector (Fig.1B) . and with its C-terminal addition of a thrombin 104 cleavage site, a T4 trimerization foldon motif and his tag. The segments S1 (aa 15-105 680) and RBD (aa 319-541) were cloned similar to S ectodomain, keeping gp67 signal 106 peptide and his tag to facilitate secretory outside cell and affinity purification, 107 respectively, but without the thrombin site and T4 foldon (Fig. 1A) . The three 108 constructed plasmids were respectively co-transfected into Sf9 insect cells with v-109 cath/chiA gene deficient baculovirus DNA for the generation and amplification of 110 recombinant baculovirus, which were then harnessed to infect Hive Five insect cells to 111 eventually produce recombinant proteins, named S, S1 and RBD respectively. as detection antibody (Fig. 1C) . Interestingly, about one half S proteins were cleaved 119 into S1 (identical migration site to S1 lane in Fig. 1C ) and S2 (about 80kDa developed 120 in anti-His WB) possibly by innate furin of insect cell that was also found in other cases (Fig. 1D ). These peaks fractionated at retention volume 28mL, 36mL, 48mL, 124 and 65mL, were further harvested and subjected to SDS-PAGE analysis. The results 125 indicated that S proteins together with cleaved S1/S2 were resolved at peak 1 in size-126 exclusion chromatography (Fig. 1D ) and showed a high purity of over 95% total 127 S/S1/S2 in gel (Fig. 1E ). Overall, one-step Ni-NTA affinity chromatography produced 128 RBD with 95% purity and a yield of 30 mg per L cell culture, S1 with about 90% purity 129 and 10 mg per L yield, while further purification through a size-exclusion 130 chromatography (SEC), the resultant S sample had over 95% purity regarding intact S 131 and cleaved S1/S2, and was harvested in a yield of 1 mg per L cell culture. These data Physiochemical properties of SARS-CoV-2 S-RBD, S1 and S proteins 137 We next investigated the physiochemical properties of the recombinant S protein and 138 its fragments purified from insect cells, including association potential, thermal 139 stability and glycosylation situation. Firstly, high pressure size-exclusion 140 chromatography (HPSEC) and sedimentation velocity analytical ultracentrifugation 141 (SV-AUC) analyses were carried out to measure the oligomerization potential of the 142 three proteins in solution. RBD, S1 and S all showed single major peak in HPSEC 143 profiles at elution volume of 9.0 mL, 5.5 and 5.3 mL, respectively ( Fig. 2A and Fig. 144 2B). RBD, S1 and S were further verified by SV-AUC, where RBD sedimented as 145 single species of 3.1S in c(s) profile, corresponding to apparent molecular weight 22 146 kDa (Fig. 2D) ; S1 existed as a dominant species of 11.3 S (estimated as 277 kDa 147 corresponding to S1 trimer) and a minor aggregate form of 20 S (Fig. 2E ); S and cleaved 148 S1/S2 resolved as 15.2 S, equivalent to 577 kDa, approximately as the theoretical 149 molecular weight of intact S trimer. The three proteins were further analyzed by 150 differential scanning calorimetry (DSC) that was usually used to investigate the inner 151 thermostability of macromolecules or their complexes 14 . RBD and S1 showed one 152 major peak at comparable thermal denaturation midpoints (Tm) of 46.0 °C and 45.5 °C, 153 respectively ( Fig. 3G and 3H ), whereas S sample showed two major peaks at Tm of 154 45.5°C (identical to Tm of S1) and 64.5°C (Fig. 3I) , which might reflect the coexistence 155 of intact S and cleaved S1/S2. On the other hand, we investigated the glycosylation extent of the three protein by of about ~20 kDa decrease (Fig. 2J ). The analyses conclude that the glycosylation 169 extent within S glycoprotein is RBD < S1 ~ S2, consistent to the predicted glycosylation 170 profile of S polypeptide (Fig. 1A) . Reactivity of SARS-CoV-2 RBD, S1 and S proteins against convalescent COVID-173 19 human sera 174 We next evaluated the antigenicity of the three versions of S proteins by WB and ELISA 175 using a panel of six COVID-19 convalescent human sera, which was collected from Taken together, RBD, S1 and S proteins from insect cells maintain the native-like Structurally, three S monomers intertwine around each other and associate to 211 homotrimers with 145 Å height seen from side-view and 160 Å diameter in top-view 212 ( Fig. 4C and 4D ). We then recruited the recently reported cryo-EM map of S prefusion 213 trimer (EMD-21374, at resolution of 3.17 Å, low pass to 5.43 Å prior to structural 214 comparison) and compared our cryo-EM map at same resolution (Fig. 4D) . It was 215 worthy noted that the compared prefusion SARS-CoV-2 S trimer was engineered with 216 site-directed mutations to stabilize prefusion conformation and expressed in 239F cells. The mutant included two stabilizing proline mutations at residues 986, 987 and a 218 "GSAS" substitution at the furin cleavage site 7 . Surprisingly, the alignment 219 demonstrated that the two cryo-EM structures share similar mushroom-shaped 220 architecture in particular nearly identical at stalk moiety (S2 region), but our S-pre 221 shows the cap part (S1 region) at ~15Å lower position than the reported S trimer in 222 RBD-down prefusion conformation (Fig. 4D ). Regarding to substantial mismatch at the 223 density of 3 S1 subunits, we respectively fitted 5 individual domains (NTD, RBD, SD1, 224 SD2 and S2) of the SARS-CoV-2 S structure (PDB code 6VSB) to our S-pre map. In 225 the fitting map, NTD, RBD, SD2 and S2 could be well placed in the S-pre map, 226 especially for the latter two, which reflects the aforementioned good match at the stalk 227 of the mushroom-shape ( Supplementary Fig. 2) . However, there is no observable 228 density between RBD and SD2 to accommodate an SD1 model ( Supplementary Fig. 2 CoV-2 spike may retain at more precedent state than the classic prefusion conformation 294 that has been determined for other coronaviruses. This early prefusion conformation 295 features that the cap of the mushroom-shaped spike constituted by three S1 subunits is 296 more proximal to viral membrane by 15 Å than in the classic prefusion conformation. The SARS-CoV-2 spike expressed in insect cells predominantly retains a unique 298 early prefusion conformation, which was repeatable in at least three batches of samples 299 and is ascribed to two possible reasons -native aa sequence used in the S ectodomain 300 construct and over-expression in insect cells. There is about a half of S proteins 301 undergoing cleavage on the S1/S2 boundary site in the purified samples both after the 302 first Ni-NAT and the second SEC purification (Fig. 1C and 1E ). Further analyses 303 suggest the split between S1 and S2 likely takes no effect on the trimerization of S Various versions of S proteins are the major targets for vaccine immunogen candidate. In addition to potent neutralizing antibody elicitation upon immunization, potential 315 antibody-dependent disease enhancement (ADE) is the major concern for an efficacious The funders had no role in study design, data collection and analysis, decision to publish, 454 or preparation of the manuscript. Anti-His antibody was used as detection antibody in western blotting. 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The model fitted in our S-605 pre map is a combined model comprised of individually fitted NTD, RBD, SD2 and S2 606 domain, which were evicted from the model of prefusion S trimer (PDB no. 6VSB). A 607 deposited postfusion core of SARS-CoV-2 S2 subunit Domains are designated by different colors, red: NTD, orange: RBD, 609 yellow: SD1, green: SD2 and cyan: S2. (B) Simplified schematic diagram of the S 610 monomer interpreting the conformation change across different states The authors have declared that no competing interests exist.