The capacity of SARS-CoV-2 S protein-mediated membrane fusion

From the GISAID Platform (https://platform.gisaid.org), we obtained the full-length amino-acid sequence of SARS-CoV-2 (BetaCoV 2019–2020) S protein (GenBank: QHD43416). Through alignment with SARS-CoV and SL-CoVs S proteins, we located the functional domains in SARS-CoV-2 S protein, which contains S1 subunit and S2 subunit with the cleavage site at R685/S686.15 S1 subunit is located within the N-terminal 14–685 amino acids of S protein, containing N-terminal domain (NTD), receptor binding domain (RBD), and receptor binding motif (RBM). S2 subunit contains fusion peptide (FP), heptad repeat 1 (HR1), heptad repeat 2 (HR2), transmembrane domain (TM) and cytoplasmic domain (CP) (Fig. 1b).

Recent biophysical and structural evidence showed that SARS-CoV-2 S protein binds hACE2 with 10-fold to 20-fold higher affinity than SARS-CoV S protein, suggesting the higher infectivity of the new virus.12 Unlike other β-B coronaviruses, S protein of SARS-CoV-2 harbors a special S1/S2 furin-recognizable site, indicating that its S protein might possess some unique infectious properties. Indeed, in live SARS-CoV-2 infection, we found a typical syncytium phenomenon naturally formed by infected cells, which is rarely reported in SARS-CoV infection (Fig. 1c). To further explore the special characteristic of SARS-CoV-2 infection, we cloned the S gene into PAAV-IRES-GFP vector and established the S-mediated cell–cell fusion system, using 293T cells that express SARS-CoV-2 S protein and EGFP (293T/SARS-CoV-2/EGFP) as the effector cells, and ACE2/293T cells expressing human ACE2 receptor as the target cells (Fig. 1d and Supplementary information, Fig. S1a). After effector cells and target cells were cocultured at 37 °C for 2 h, the fused cells showed at least 2-fold larger size than normal cells and multiple nuclei, and these cells were observed in the SARS-CoV-2 group, but not the SARS-CoV group. After coincubation for 24 h, hundreds of target cells fused together as one big syncytium, containing multiple nuclei (Fig. 1d). Another 24 h later, the syncytium grew bigger and could be easily observed under both light and fluorescence microscopy (Fig. 1e). Similar results were observed in the fusion between 293T/SARS-CoV-2/EGFP cells and Huh-7 cells, which naturally express human ACE2 receptor on the cell surface. Their syncytium was obviously formed after coincubation for 48 h, similar to the syncytium formed by live SARS-CoV-2-infected Huh-7 cells (Fig. 1c, f). On the contrary, SARS-CoV S protein lacked the ability to mediate the cell–cell fusion under the same conditions (Fig. 1d) based on the required presence of exogenous trypsin to complete membrane fusion in our previous studies. Therefore, compared to SARS-CoV, SARS-CoV-2 S protein showed much more efficiency in mediating viral surface-fusion and entry into target cells.14 Meanwhile, no fusion was observed for 293T/EGFP cells without S-expression or 293T cells without ACE2-expression (Fig. 1d and Supplementary information, Fig. S1b), confirming that S-receptor engagement is necessary for the S-mediated viral fusion and entry.

X-ray crystallographic analysis of the 6-HB fusion core formed by HR1 and HR2 domains in S2 subunit of SARS-CoV-2 S protein

Previously, we identified that the 6-HB formed by HR1 and HR2 domains of the S2 subunit plays a very important role in the membrane fusion process mediated by MERS-CoV or SARS-CoV S protein.16,17 Similarly, our recent study suggested that HR1 and HR2 in subunit S2 of SARS-CoV-2 also interacted to form coiled-coil complex to support membrane fusion and viral infection15 (Fig. 2a and Supplementary information, Fig. S2). However, the specific binding characteristics of SARS-CoV-2 6-HB remained to be explored.

Fig. 2: Overall structure of post-fusion 6-HB in SARS-CoV-2. a Sequence alignment of HR1 and HR2 domains in SARS-CoV and SARS-CoV-2. b Structure of SARS-CoV-2 6-HB is shown in cartoon representation with HR1 colored in green and HR2 in cyan. The structural dimensions are indicated in angstroms. c HR1 trimer of SARS-CoV-2 6-HB is shown in electrostatic surface, and HR2 domain is shown in cartoon representation, the important binding residues of which are shown in sticks and labeled. d The superposition of 6-HB structure of SARS-CoV (PDB entry 1WYY), MERS-CoV (PDB entry 4NJL) and SARS-CoV-2 is shown in ribbon. The RMSD between structures is indicated. e The sequence comparison of 6-HB structure of different HCoVs is shown in cartoon representation with different colors for HR1 and HR2. The helical fusion core regions are indicated. Full size image

To understand the structural basis of the interactions between HR1 and HR2 regions of SARS-CoV-2, a fusion protein containing the major parts of HR1 (residues 910–988) and HR2 (residues 1162–1206) with a flexible linker (L6, SGGRGG) in between was constructed for crystallographic study. The crystal structure of HR1-L6-HR2 shows a canonical 6-HB structure with a rod-like shape 115 Å in length and 25 Å in diameter (Fig. 2b). The three HR1 domains form a parallel trimeric coiled-coil center, around which three HR2 domains are entwined in an antiparallel manner. The interaction between these two domains is predominantly a hydrophobic force. Each pair of two adjacent HR1 helices forms a deep hydrophobic groove, providing the binding site for hydrophobic residues of the HR2 domain, including V1164, L1166, I1169, I1172, A1174, V1176, V1177, I1179, I1183, L1186, V1189, L1193, L1197 and I1198 (Fig. 2c). The hydrophobic interactions between HR1 and HR2 are mainly located in the helical fusion core region, which will be discussed later.

The overall 6-HB structure of SARS-CoV-2 is similar to that of other HCoVs with root-mean-square deviation (RMSD) of 0.36 Å to SARS-CoV 6-HB and 0.66 Å to MERS-CoV 6-HB for all the Cα atoms (Fig. 2d). This finding suggested that the overall 6-HB conformation is an important and highly conserved component for these dangerous coronaviruses. When comparing with the 6-HB of other common coronaviruses causing mild respiratory disease, such as 229E and NL63, the SARS-CoV-2 6-HB has a similar overall structure, except for the different length of HR2 helix in the 6-HB. The HR2 domain of 229E or NL63 forms a longer and bending helix to interact with trimeric HR1 core (Fig. 2e). The relationship between the structural difference and the pathogenicity of these HCoVs remains to be elucidated.

According to sequence alignment, the S2 subunits of SARS-CoV-2 and SARS-CoV are highly conserved, with 92.6% and 100% overall homology in HR1 and HR2 domains, respectively. Inside the fusion core region of HR1 domain, there are 8 different residues (Fig. 3a), which may contribute the enhanced interactions between HR1 and HR2 and stabilize 6-HB conformation of SARS-CoV-2 as revealed by crystallographic analysis, compared with those of SARS-CoV. This significant difference has not been observed in other SARS-like viruses, such as WIV1, Rs3367, and RsSHC014. As shown in Fig. 3b, the K911 in SARS-CoV HR1 could bind to E1176 in HR2 through a salt bridge 2.9 Å in distance. However, with the Lys-Ser replacement, S929 in SARS-CoV-2 binds to S1196 through a strong hydrogen bond 2.4 Å in distance. In SARS-CoV, Q915 in the HR1 domain does not bind to the HR2 domain. However, with Q-K replacement in the new virus, K933 in the HR1 domain binds to carbonyl oxygen of N1172 in HR2 through a salt bridge 2.7 Å in distance (Fig. 3b). In SARS-CoV, E918 in the HR1 domain binds to R1166 in the HR2 domain through a weak salt bridge 3.7 Å in distance. In SARS-CoV-2, E918 is mutated to D936 and binds to R1185 in the HR2 domain through a salt bridge 2.7 Å in distance (Fig. 3c). In SARS-CoV, K929 in HR1 binds to E1163 in HR2 through a salt bridge 3.2 Å in distance, while T925 is not involved in the interaction. However, when T925 was mutated to S943, it could bind to E1182 in the HR2 domain with a hydrogen bond 2.6 Å in distance, and K947 could also bind to E1182 through a salt bridge 3.0 Å in distance (Fig. 3d). These results suggested that the multiple replacements in the HR1 domain of emerging SARS-CoV-2 virus could enhance the interactions between HR1 and HR2 domain to further stabilize the 6-HB structure, which may lead to increased infectivity of the virus.

Fig. 3: Interaction between HR1 and HR2 of SARS-CoV-2 and SARS-CoV. a–d The 6-HB structure of SARS-CoV-2 and SARS-CoV is shown in cartoon representation. The HR1 domain is shown in green for SARS-CoV-2 and forest for SARS-CoV, while the HR2 domain is shown in cyan for SARS-CoV-2 and orange for SARS-CoV. Important residues are shown in sticks and labeled. Full size image

Design and structure-activity relationship (SAR) analysis of lipopeptides with remarkably improved fusion inhibitory activity

Previously, we found that peptide EK1 could disturb viral 6-HB formation and effectively inhibit SARS-CoV-2 PsV infection. However, the potent stability of SARS-CoV-2 6-HB structure might reduce the antiviral efficacy of EK1. Recently, numerous reports have shown that the lipidation strategy can effectively improve the antiviral activity of fusion inhibitory peptides, such as the ant-HIV-1 peptide LP-19,18 and the anti-Nipah virus lipopeptides.19 In order to improve the inhibitory activity of EK1, cholesterol (Chol) and palmitic acid (Palm) were covalently attached to the C-terminus of EK1 sequence under the help of a flexible polyethylene glycol (PEG) spacer, and the corresponding lipopeptides EK1C and EK1P were constructed, respectively (Fig. 4a). Both of them could completely inhibit SARS-CoV-2 mediated cell–cell fusion at the concentration of 2.5 μM (Fig. 4b). The inhibitory activity with mean 50% inhibitory concentration (IC 50 ) values is 48.1 nM for EK1C and 69.2 nM for EK1P, respectively (Fig. 4c). Meanwhile, the EK1-scrambled peptide showed no inhibitory activity with the concentration up to 5 μM (Fig. 4c). These results strongly suggest that lipidation of EK1 is a promising strategy to improve its fusion-inhibitory activity against SARS-CoV-2 infection, especially, cholesterol-modification.

Fig. 4: EK1-Lipopeptides showed potent inhibitory activity against SARS-CoV-2 infection. a Amino acid sequences of the designed peptides EK1, EK1P and EK1C. The dotted lines represent E–K salt-bridge with i to i + 3, or i + 4 arrangement. b SARS-CoV-2 S protein-mediated cell–cell fusion in the presence of EK1-scramble (I), EK1 (II), EK1C (III), and EK1P (IV) at 2.5 μM (scale bar: 400 µm). c Inhibitory activity of EK1-scramble, EK1, EK1C and EK1P against SARS-CoV-2 S-mediated cell–cell fusion. d Design diagram of EK1-lipopeptides with cholesterol modification, including EK1C1-EK1C7. e Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 S-mediated cell–cell fusion. f Inhibitory activity of EK1-lipopeptides on SARS-CoV-2 PsV infection. Experiments were repeated twice, and the data are expressed as means ± SD (error bar). Full size image

On the basis of the structure of EK1C, series of cholesteryl EK1 with multiple linkers were constructed, where the glycine/serine-based linker, i.e., GSG, or PEG-based spacer was employed between EK1 and the cholesterol moiety (Fig. 4d). Compared with EK1C1, EK1C2 and EK1C showed similar inhibitory activities. Strikingly, EK1C3 peptide with both the 3-amino acid linker “GSG” and the PEG4-based spacer, exhibited 4-fold more potency than EK1C1. It is noteworthy that changing “GSG” in EK1C3 to a longer 5-amino acid linker “GSGSG” significantly increased the inhibitory potency of the hybrid molecule, and EK1C4 had IC 50 value of 1.3 nM, which was 43-fold more potent than EK1C1. These findings indicate that the linker length has a significant effect on the overall activity of lipopeptides. Comparison of increasing PEG-based arm lengths in EK1C4 shows that inhibitors potency slightly decreased in the cell–cell fusion assay (Fig. 4e). The data suggest that “GSGSG-PEG4” linker was optimal to bridge both parts of the conjugates. Similarly, EK1C4 showed the most potent inhibitory activity against SARS-CoV-2 PsV infection, with IC 50 value of 15.8 nM, providing 149-fold stronger anti-SARS-CoV-2 activity than that of EK1 (IC 50 = 2,375 nM) (Fig. 4f).

The lipopeptide EK1C4 exhibits the most potent inhibitory activity against membrane fusion mediated by S proteins and entry of pseudotyped coronaviruses

We have previously demonstrated that EK1 could effectively inhibit divergent HCoV infection by targeting the HR1 domains, including α-HCoV and β-HCoV. Here, we further systematically evaluated the broad-spectrum surface-fusion inhibitory activity of EK1C4 on cell–cell fusion mediated by S proteins of divergent coronaviruses, including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-NL63 and HCoV-229E. Among them, SARS-CoV has the closest relative to SARS-CoV-2, and its S protein-mediated cell–cell fusion could be effectively inhibited by EK1C4 with IC 50 of 4.3 nM, which is about 94-fold more active than that of EK1 (IC 50 = 409.3 nM) (Fig. 5a). Similarly, EK1C4 showed extremely potent fusion-inhibitory activity on MERS-S- and OC43-S-mediated cell–cell fusion with IC 50 of 2.5 nM and 7.7 nM, which were 95- and 101-fold more potent when compared to EK1, respectively, indicating that EK1C4 could potently and broadly inhibit S protein-mediated cell–cell fusion of various β-HCoVs (Fig. 5b, c). For α-HCoVs, EK1C4 also effectively blocked the fusion process mediated by the S protein of HCoV-229E and HCoV-NL63 with IC 50 of 5.2 nM and 21.4 nM, respectively, while EK1 showed inhibitory activity of IC 50 ranging from 207.4 to 751.0 nM (Fig. 5d, e). Moreover, with their potential for human infection, SL-CoVs, including WIV1, Rs3367 and RsSHC014, the fusion process of which is mediated by S protein, could also be significantly prevented by EK1C4 with IC 50 ranging from 4.3 to 8.1 nM, as well as EK1 with IC 50 ranging from 237.0 to 279.6 nM (Fig. 5f–h). As control, the EK1-scrambled peptide showed no inhibitory activity with concentration up to 5 μM in all those coronavirus cell–cell fusion assays (Fig. 5a–h).

Fig. 5: EK1C4 broadly and potently inhibited cell–cell fusion and PsV infection mediated by S protein of divergent HCoVs. a–h Inhibitory activity of EK1C4 in cell–cell fusion mediated by the S proteins of SARS-CoV (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), HCoV-NL63 (e), WIV1 (f), Rs3367 (g) and SHC014 (h). i–o Inhibitory activity of EK1C4 in PsV infection assays against SARS-CoV (i), MERS-CoV (j), HCoV-OC43 (k), HCoV-229E (l), NL63 (m), WIV1 (n) and Rs3367 (o). Experiments were repeated twice, and the data are expressed as means ± SD. Full size image

We also assessed the antiviral activity of EK1C4 on PsV infection by divergent coronaviruses. As expected, EK1C4 showed much more potent activity than EK1 (IC 50 ranging from 631.8 to 3,237 nM) against SARS-CoV, MERS-CoV, and HCoV-OC43 infection with IC 50 of 11.7 nM, 11.1 nM and 37.7 nM, respectively (Fig. 5i-k). EK1C4 also effectively blocked PsV infection of α-HCoVs, including HCoV-229E and HCoV-NL63, with IC 50 of 12.4 nM and 76.6 nM, respectively, which was about 319- and 99-fold more active than EK1 (IC 50 ranging from 3,963 to 7,666 nM) (Fig. 5l, m). Similarly, by cholesteryl modification with “GSGSG-PEG4” linker, the inhibitory activity of EK1 could be significantly increased on PsV infection from SL-CoVs, including WIV1 and Rs3367, where EK1C4 showed potent inhibitory activity with IC 50 of 30.8 nM and 66.9 nM, respectively, which is 175-fold to 89-fold more potent than that of EK1 (Fig. 5n, o).

EK1C4 possesses the most potent inhibitory activity against in vitro infection by live coronaviruses

We further assessed the inhibitory activity of EK1C4 against live HCoVs infection, including SARS-CoV-2, MERS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63. Importantly, EK1C4 effectively blocked SARS-CoV-2 infection at the cellular level in a dose-dependent manner with IC 50 of 36.5 nM, being 67-fold more active than that of EK1 (IC 50 = 2,468 nM) (Fig. 6a), which is consistent to the results of cell–cell fusion assay and PsV infection assay mediated by SARS-CoV-2 S protein. Similarly, EK1C4 also showed more potent antiviral activity than EK1 against MERS-CoV, HCoV-OC43, HCoV-229E, and HCoV-NL63 infection with IC 50 of 4.2 nM, 24.8 nM, 101.5 nM and 187.6 nM, respectively, which are 190-, 62-, 42- and 19-fold more potent than those of EK1, respectively (Fig. 6b–e). We next assessed the cytotoxicity of EK1C4 on various target cells and found that the half cytotoxic concentration (CC 50 ) was beyond 5 μM, which is the highest detection concentration of EK1C4 (Supplementary information, Fig. S3). Therefore, the selectivity index (SI = CC 50 /IC 50 ) of EK1C4 is >136, suggesting that EK1C4 is a promising SARS-CoV-2 fusion inhibitor with little, or even no, toxic effect in vitro. Further, we explored the potent antiviral mechanism of EK1C4 and found that the complexes of EK1C4/SARS-HR1, EK1C4/MERS-HR1, and EK1C4/SARS-2-HR1 harbor higher stability and increased Tm values than those of the complexes formed by EK1 and HR1s (Supplementary information, Fig. S4). These results suggested that increased antiviral activity of EK1C4 should be related to its increased binding affinity with HR1, but their detailed interactions require further studies.

Fig. 6: EK1C4 effectively inhibited live-CoVs infection in vitro and in vivo. a–e Inhibitory activity of EK1C4 on live HCoV replication for SARS-CoV-2 (a), MERS-CoV (b), HCoV-OC43 (c), HCoV-229E (d), and HCoV-NL63 (e). f–g In vivo prophylactic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (f) and survival curves (g) of mice challenged with HCoV-OC43. h–i In vivo therapeutic efficacy of EK1C4 against HCoV-OC43 infection in mice. Body weight change (h) and survival curves (i) of mice challenged with HCoV-OC43. Experiments were repeated twice, and the data are expressed as means ± SD. Full size image

Intranasally applied EK1C4 showed strong protection of mice against HCoV-OC43 infection

Recently, SARS-CoV-2 rapidly spread in humans by transmitting through the respiratory tract. Here, we used an HCoV-OC43 infection mouse model to further investigate the potential prophylactic effect of EK1C4 in clinical applications via the intranasal administration route (Fig. 6f, g). In the OC43-infected mouse model, we treated newborn mice with EK1C4 at a single dose of 0.5 mg/kg 0.5 h (Pre-0.5), 2 h (Pre-2), 4 h (Pre-4), 12 (Pre-12) and 24 h (Pre-24) before challenging with HCoV-OC43 at 100 TCID 50 (50% tissue culture infectious dose). Starting from 4 days’ post-infection (dpi), the body weight of mice in the viral control group decreased significantly along with 100% mortality (Fig. 6f, g). The final survival rates of mice in Pre-0.5, Pre-2, Pre-4, Pre-12 and Pre-24 groups were 100%, 100%, 100%, 83 and 0%, respectively (Fig. 6f, g). In contrast, EK1 with a single dose of 20 mg/kg via nasal administration exhibited very promising prophylactic effect in the Pre-0.5 h and Pre-1 h groups, whereas all mice in the EK1-Pre-2 h group eventually died similarly to the mice in the viral control group (Supplementary information, Fig. S5). These results suggested that EK1C4 has better stability, antiviral activity, and prolonged half-life in the airway environment when compared with EK1.

We then tested the therapeutic effect of EK1C4 0.5 h (Post-0.5 group) and 2 h (Post-2 group) after HCoV-OC43 infection (Fig. 6h, i). The Post-0.5 group and Post-2 group mice showed 100% and 16.7% survival rate, respectively, suggesting that EK1C4 harbors good therapeutic effect after a short period of HCoV-OC43 infection, possibly resulting from the establishment of HCoV-OC43 infection in mouse brain where EK1C4 cannot get through the blood brain barrier via nasal administration.14 As shown in Supplementary information, Fig. S6, high viral titer was detected in brains of all 5 mice in Pre-24 group and 4 out of 5 mice in Post-2 group, but was not detected in brain tissues of all mice in Pre-0.5, Pre-2, Pre-4, and Post-0.5 groups, while only moderate level of viral titer was detected in brain tissue in one of the 5 mice in Pre-12 group (Supplementary information, Fig. S6a, b). Similar to those in the viral control mice, mice in Pre-24 and Post-2 groups exhibited similar histopathological changes in brain tissues, including vacuolation, degeneration, and infiltration. However, the brain tissues of mice in Pre-0.5, Pre-2, Pre-4, Pre-12 and Post-0.5 group as well as the normal control group showed no apparent histopathological changes (Supplementary information, Fig. S6c).