Expression of SARS-CoV-2 S protein

To enhance expression of the S protein of SARS-CoV-2 in mammalian cells, a codon-optimized cDNA encoding the S protein and 3xFLAG tag was synthesized, and to facilitate incorporation of S protein into lentiviral pseudovirons, the last 19 amino acids containing an endoplasmic reticulum (ER)-retention signal from the cytoplasmic tail of the S protein was removed (Fig. 1a). The construct was named SARS-CoV-2 S. HEK293T cells were transfected with SARS-CoV-2 S plasmid and expression of SARS-CoV-2 S protein was determined by western blot. There were two major bands, 180 kDa, and 90 kDa, detected by mouse anti-FLAG M2 antibody (Fig. 1b, lane 2), reflecting the full-length and cleaved S proteins, respectively. The band above 250 kDa likely results from dimeric or trimeric S proteins. Consistent with our previous report29, MERS-CoV S protein was detected by polyclonal goat anti-MHV S antibodies AO4 (Fig. 1c). AO4 also detected SARS-CoV-2 and SARS-CoV S proteins, suggesting the presence of a conserved immunogenic epitope among all four different CoVs. This presumably linear epitope is likely in S229. S1 subunits of SARS-CoV-2 and SARS-CoV share almost 64% in amino acid identities. Nevertheless, SARS-CoV-2 S protein was barely detected by rabbit polyclonal anti-SARS S1 antibodies T62 (Fig. 1d), suggesting that the major epitope(s) for T62 antibodies include non-conserved regions of S1. The SARS-CoV-2 S protein was not detected by either a monoclonal anti-SARS S1 antibody (Fig. 1e) or anti-MERS S2 antibody (Fig. 1f).

Pseudovirion incorporation of SARS-CoV-2 S protein

The efficiency of SARS-CoV-2 S protein incorporation into pseudovirions was evaluated using monoclonal mouse anti-FLAG M2 antibody and polyclonal goat anti-MHV S antibody AO4. While majority of SARS-CoV S proteins incorporated into pseudovirons were full-length, at 180 kDa (Fig. 1h, i), most of SARS-CoV-2 S proteins on lentiviral pseudovirions were cleaved, about 90 kDa (Fig. 1g, h), likely reflecting presence of extra furin site (R682-R683-A684-R685, Fig. 1a) between S1 and S2 in SARS-CoV-2 S protein. Consistent with the results in cell lysate (Fig. 1d), SARS-CoV S protein, but not SARS-CoV-2 S protein, in pseudovirions was readily detected by using polyclonal rabbit anti-SARS S1 antibodies T62 (Fig. 1i).

Human ACE2 is a receptor for SARS-CoV-2

Next, we determined whether SARS-CoV-2 S pseudovirions were able to transduce human, monkey, and bat cells. VSV-G pseudovirons were used as a positive control, whereas bald particles with no spike proteins (mock) served as a negative control. As expected, all cell types were effectively transduced by VSV-G pseudovirons (Fig. 2a). Compared to mock control, SARS-CoV-2 S pseudovirions showed an over 500-fold increase in luciferase activities in Calu3 cells, at a level similar to SARS-CoV S pseudovirions (Fig. 2a). Huh7 and Vero 81 cells also gave about 10-fold increase in luciferase activities when transduced by SARS-CoV-2. Transduction of LLCMK2 cells was higher with SARS-CoV S pseudovirions than with SARS-CoV-2 S pseudovirions (Fig. 2a), suggesting that there might be some differences on virus entry on LLCMK2 cells mediated by S proteins between SARS-CoV-2 and SARS-CoV. We then investigated whether any known CoV receptors might be used by SARS-CoV-2 S protein as entry receptor. The SARS-CoV-2 S pseudovirons were used to transduce BHK cells stably expressing human aminopeptidase N (hAPN), the receptor for human CoV 229E, 293 cells stably expressing hACE2 (293/hACE2), the receptor for SARS-CoV, and HeLa cells stably expressing hDPP4 (HeLa/hDPP4), the receptor for MERS-CoV. While BHK/hAPN and HeLa/hDPP4 cells were not susceptible for the transduction of SARS-CoV-2 S pseudovirions, 293/hACE2 cells were highly transduced by SARS-CoV-2 S pseudovirions, consistent with hACE2 as the receptor for SARS-CoV-213. We then determined whether SARS-CoV-2 S protein could directly bind to hACE2 protein. HEK 293T cells transiently expressing SARS-CoV-2 S protein were incubated with soluble hACE2 and analyzed by flow cytometry. As shown in Fig. 2c, SARS-CoV-2 S protein bound to soluble hACE2 at a level similar to SARS-CoV S protein, although the mean fluorescence intensity (MFI) for SARS-CoV-2 S protein was slightly lower than SARS-CoV S protein. To further investigate if hACE2 is the receptor for SARS-CoV-2, we performed inhibition experiments using soluble hACE2. Soluble hACE2 proteins were pre-incubated with SARS-CoV-2 S pseudovirons on ice for 1 h, then virus-protein mixture was added onto 293/hACE2 cells. Entry of SARS-CoV-2 S pseudovirions was significantly prevented by pre-incubation of soluble hACE2 at both 10 μg/ml and 50 μg/ml (Fig. 2d), further supporting the notion that hACE2 is the receptor and soluble hACE2 might be used as a therapeutic inhibitor against SARS-CoV-2 infection.

Fig. 2: Entry and receptor of SARS-CoV-2 S pseudovirons. a, b Entry of SARS-CoV-2 S pseudovirions on indicated cell lines. Cells from human and animal origin were inoculated with SARS-CoV-2 S (red), SARS-CoV S (blue), or VSV-G (gray) pseudovirions. At 48 h post inoculation, transduction efficiency was measured according to luciferase activities. RS, Rhinolophus sinicus bat embryonic fibroblast; BHK/hAPN, BHK cells stably expressing hAPN, the hCoV-229E receptor; 293/hACE2, 293 cells stably expressing hACE2, the SARS-CoV receptor; HeLa/hDPP4, HeLa cells stably expressing hDPP4, the MERS-CoV receptor. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Binding of SARS-CoV S and SARS-CoV-2 S proteins to soluble hACE2. HEK293T cells transiently expressing SARS-CoV and SARS-CoV-2 S proteins were incubated with the soluble hACE2 on ice, followed by polyclonal goat anti-hACE2 antibody. Cells were analyzed by flow cytometry. The experiments were repeated at least three times. d Inhibition of SARS-CoV-2 S pseudovirion entry by soluble hACE2. SARS-CoV S, SARS-CoV-2 S, or VSV-G pseudovirions were pre-incubated with soluble hACE2, then mixture were added to 293/hACE2 cells. Cells were lysed 40 h later and pseudoviral transduction was measured. Experiments were done twice and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file. Full size image

The SARS-CoV-2 enters 293/hACE2 cells through endocytosis

The majority of S proteins on SARS-CoV-2 S pseudovirions are cleaved (Fig. 1g, h). We next determined whether SARS-CoV-2 S pseudovirons entered cells through endocytosis or cell surface. HEK 293/hACE2 cells were treated with lysosomotropic agents, ammonia chloride and bafilomycin A, and their effect on virus entry was evaluated. Consistent with previous reports, 20 mM NH 4 Cl and 100 nM bafilomycin A decreased entry of SARS-CoV S and VSV-G pseudovirions by over 99%, compared to no treatment control. More than 98% reduction in transduction on 293/hACE2 cells by SARS-CoV-2 S pseudovirions was also shown when the cells were incubated with either NH 4 Cl or bafilomycin A (Fig. 3a), indicating that SARS-CoV-2 S pseudovirions enter 293/hACE2 cells mainly through endocytosis, despite that its spike proteins were cleaved.

Fig. 3: Endocytosis of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH 4 Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod. HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively. The luciferase activity was measured 40 h post transduction. VSV-G pseudovirions were used as a control. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod. The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01. Viral infection and cell viability were determined by using qPCR and MTT assay, respectively. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. d, e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine. HEK 293/hACE2 cells were pretreated with either apilimod (d), YM201636 (e), or tetrandrine (f), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug. The luciferase activity were measured 40 h post transduction. YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor. The experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM of technical triplicates. Source data are provided as a Source Data file. Full size image

PIKfyve and TPC2 critical for SARS-CoV-2 entry

Phosphoinositides play many essential roles in endocytosis. Among them, one is phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ), which regulates early endosome to late endosome dynamics33,34. Phosphatidylinositol 3-phosphate 5-kinase (PIKfyve) is the main enzyme synthesizing PI(3,5)P 2 in early endosome. HEK 293/hACE2 cells were treated with apilimod, a potent inhibitor for PIKfyve35. Inhibition of PIKfyve by apilimod significantly reduced entry of SARS-CoV S pseudovirions on 293/hACE2 cells in a dose dependent manner (Fig. 3b), whereas it had no effect on entry of VSV-G pseudovirions, which occurs in early endosomes. Similar inhibitory effects were observed when HeLa/hDPP4 cells and HeLa cells stably expressing mouse carcinoembryonic antigen related cell adhesion molecule 1a (mCEACAM1a) (HeLa/mCEACAM1a) were treated with apilimod and transduced with MERS-CoV and MHV S pseudovirions (Fig. 3b), respectively. Moreover, infection of live MHV on 17Cl.1 cells was also strongly inhibited by apilimod treatment (Fig. 3c). No significant cell toxicity was observed on apilimod at any concentration tested (Fig. 3c). We then determined whether apilimod could inhibit entry of SARS-CoV-2 S pseudovirions on 293/hACE2. As expected, apilimod treatment inhibited entry of SARS-CoV-2 S pseudovirions in a dose dependent manner (Fig. 3d). Similar effects were shown when 293/hACE2 cells were treated with YM201636, another PIKfyve inhibitor (Fig. 3e). These results suggested that PIKfyve might be a potential general drug target for viruses that enter cells through endocytosis. Two pore channel subtype 2 (TPC2) and TRPML1 in lysosome are two major downstream effectors of PI(3,5)P 2 36. While blocking TPC2 activity by tetrandrine, an inhibitor for TPC237, decreased entry of SARS-CoV-2 S pseudovirions (Fig. 3f), treatment of cells with 130, a TRPML1 inhibitor, had no effect (Supplementary Fig. 1), indicating that TPC2, not TRPML1, is important for SARS-CoV-2 entry.

Effect of cathepsin inhibitors on SARS-CoV-2 entry

Protease activation on S protein is an important step for coronavirus entry, and cathepsins in lysosome have been shown to be critical for SARS-CoV and MERS-CoV entry through endocytosis. To investigate whether cathepsins are required for SARS-CoV-2 entry, HEK 293/hACE2 cells were treated with either a broad inhibitor for cathepsin B, H, L, and calpain (E64D), cathepsin L inhibitor (SID 26681509), or cathepsin B inhibitor (CA-074). VSV-G pseudovirions were used as a negative control, since virus entry mediated by VSV-G does not require protease activation. E64D treatment of 293/hACE2 cells reduced entry of SARS-CoV-2 S pseudovirions by 92.5%, indicating that at least one of cathepsins or calpain might be required for SARS-CoV-2 entry (Fig. 4a). While cathepsin B inhibitor treatment did not show any marked effect on virus entry, cathepsin L inhibition treatment decreased entry of SARS-CoV-2 S pseudovirions into 293/hACE2 by over 76% (Fig. 4a), suggesting that cathepsin L should be essential for priming of SARS-CoV-2 S protein in lysosome for entry into 293/hACE2 cells.

Fig. 4: Activation of SARS-CoV-2 S protein by cathepsin and trypsin. a Effects of cathepsin inhibitors on entry of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. HEK 293/hACE2 cells were pretreated with broad-spectrum cathepsin inhibitor E64D, cathepsin L-specific inhibitor (SID 26681509), or cathepsin B-specific inhibitor (CA-074) and then transduced with SARS-CoV-2 S and VSV-G pseudovirions. Pseudoviral transduction was measured at 40 h post inoculation. Experiments were done in triplicates and repeated at least three times. One representative is shown. Error bars indicate SEM of technical triplicates. b Cell–cell fusion mediated by SARS-CoV-2 S protein. HEK 293T cells were transiently expressing eGFP and either SARS-CoV-2 or SARS-CoV S protein were detached with either trypsin or EDTA, and co-cultured with 293/hACE2 or 293 cells for 4 h at 37 °C. The scale bar indicates 250 µm. c Quantitative analysis of syncytia in panel b. d, e Thermostability analysis of SARS-CoV-2 S protein. d SARS-CoV and SARS-CoV-2 S pseudovirons were incubated at 37 °C for the specified times (0 to 4 h) in the absence of serum, and then assayed on 293/hACE2 cells. The results from infection at 0 h were set as 100%, and the experiments were repeated four times, and means with standard deviations are shown. e SARS-CoV and SARS-CoV-2 S pseudovirions without serum were incubated at the indicated temperature (37 to 51 °C) for 2 h and then assayed on 293/hACE2 cells. The results are reported as the percentage of transduction at 37 °C. The experiments were repeated four times, and means with standard deviations are shown. Source data are provided as a Source Data file. Full size image

Cell–cell fusion mediated by SARS-CoV-2 S protein

Type II membrane serine proteases (TMPRSS)-mediated cleavage can activate the fusion potential of SARS-CoV and MERS-CoV S protein and induce receptor-dependent syncytium formation28,29,38,39. To test whether SARS-CoV-2 S protein cleaved by these cellular proteases could trigger syncytium formation, we overlaid HEK 293T cells expressing SARS-CoV-2 S or control proteins on 293/hACE2 cells in presence or absence of proteases. Expression of TMPRSS 2, 4, 11 A, 11D, and 11E on 293/hACE2 cells enhanced SARS-CoV-2 S protein-mediated cell–cell fusion similarly to SASR-CoV S (Supplementary Fig. 2). Next we evaluated the role of trypsin. Consistent to our previous report23, addition of trypsin triggered syncytium formation on 293/hACE2 cells by SARS-CoV S protein after 4 h incubation (Fig. 4b, c). Large syncytia were formed at a level similar to SARS-CoV S proteins when SARS-CoV-2 S protein expressing 293T cells were added onto 293/hACE2 cells with trypsin. Of note, SARS-CoV-2, but not SARS-CoV, S proteins induced syncytia formation on 293/hACE2 cells even in the absence of trypsin (Fig. 4c), suggesting that SARS-CoV-2 S proteins could be triggered upon the receptor binding without exogenous protease priming or activation. During 2013–2016 Ebola virus outbreak, A82V and T544I mutations in glycoprotein (gp) of Ebola virus were linked to increase of virus transmissibility40,41,42. Further study showed that A82V and T544I mutation decreases the thermostability of gp while they increases virus infectivity in cell culture42. We then investigated the thermostability of SARS-CoV-2 S protein. Compared to SARS-CoV S protein, SARS-CoV-2 S protein was less stable, requiring significant shorter time and lower temperature to be inactivated (Fig. 4d, e). The native S protein is metastable, and there is an energy barrier that prevents it from undergoing conformational change before triggering, SARS-CoV-2 S protein might decrease its energy barrier by reducing its thermostability. This might contribute to high-transmission efficiency of SARS-CoV-2.

Effect of anti-SARS S1 antibodies T62 on SARS-CoV-2 S protein

Since SARS-CoV-2 S reacted weakly with polyclonal rabbit anti-SARS S1 antibodies T62 in western blot (Fig. 1d), we investigated whether SARS-CoV-2 S protein in native conformation could be recognized by anti-SARS S1 antibodies T62. HEK293T cells transiently expressing SARS-CoV-2 S proteins were incubated with polyclonal anti-SARS S1 antibodies T62 and analyzed by flow cytometry. SARS-CoV S was used as a positive control. As expected, expression of SARS-CoV S proteins on 293T cell surface were readily detected by polyclonal anti-SARS S1 antibodies T62 (Fig. 5a). In contrast, only low level of binding of SARS-CoV-2 S protein to polyclonal rabbit anti-SARS S1 antibodies T62 was detected (Fig. 5a). To further delineate where the conformational epitopes for this polyclonal antibodies are located, two new constructs were generated, a SARS-CoV S protein backbone with RBD from SARS-CoV-2 (SARS-CoV S/nRBD) and a SARS-CoV-2 S protein backbone with RBD from SARS-CoV (SARS-CoV-2 S/sRBD). Replacement of SARS-CoV RBD with SARS-CoV-2 RBD (SARS-CoV S/nRBD) decreased binding of polyclonal antibodies T62 to S protein, whereas substitution of SARS-CoV-2 RBD with SARS-CoV RBD (SARS-CoV-2 S/sRBD) increased the affinity of S protein to polyclonal antibodies T62 (Fig. 5a). All chimera proteins were expressed at levels similar to SARS-CoV-2 S protein (Fig. 5b, top panel). These results suggest that at least one major conformational epitope for polyclonal antibodies T62 might be located in RBD where the sequences differ between SARS-CoV and SARS-CoV-2 (Fig. 5c). Of note, while SARS-CoV-2 S/sRBD bound to polyclonal antibodies T62 better than SARS-CoV S/nRBD in flow cytometry, the signal for SARS-CoV S/nRBD was stronger than SARS-CoV-2 S/sRBD in western blot by polyclonal antibodies T62, indicating that major linear epitopes of polyclonal antibodies T62 were located on SARS-CoV NTD. Among residues in SARS-CoV RBD critical for receptor binding and virus entry43,44, seven of them are different between SARS-CoV and SARS-CoV-2 RBDs (Fig. 5c). We then mutated each of them in SARS-CoV S protein to the corresponding residues in SARS-CoV-2 S. With exception of SARS-CoV mutant Y442L, which had reduced protein expression (Supplementary Fig. 3), binding of SARS-CoV S mutants to polyclonal rabbit anti-SARS S1 antibodies T62 was not affected (Fig. 5d). This suggests that these residues might not be direct binding sites for polyclonal antibodies T62. Next, we tested whether polyclonal rabbit anti-SARS S1 antibodies T62 could inhibit entry of SARS-CoV-2 S pseudovirions. CoV S protein pseudovirions were incubated with polyclonal antibodies T62 on ice for 1 h, and their transduction was measured according to luciferase activities. As expected, polyclonal antibodies T62 neutralized SARS-CoV S pseudovirion effectively in a dose dependent manner (Fig. 5e). In contrast, even at a concentration of 50 μg/ml, polyclonal antibodies T62 did not have marked effect on transduction by SARS-CoV-2 S proteins (Fig. 5e).

Fig. 5: Characterization of polyclonal rabbit anti-SARS S1 antibodies T62. a Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to SARS-CoV-2, SARS-CoV S, and chimeric S proteins. HEK293T cells transiently expressing either SARS-CoV-2 S, SARS-CoV S, SARS-CoV S/nRBD, or SARS-CoV-2 S/sRBD proteins were incubated with polyclonal rabbit anti-SARS-CoV S1 antibody T62 for 1 h on ice, followed by a FITC-conjugated secondary antibody, then cells were analyzed by flow cytometry. Experiments were done three times and one representative is shown. b Expression of SARS-CoV-2 S, SARS-CoV S, or chimeric S proteins on 293T cells. Cells from panel A were lyzed and blotted with anti-FLAG M2 antibody and polyclonal anti-SARS S1 antibody T62. c Amino acid sequence alignment of SARS-CoV and SARS-CoV-2 S RBDs. Stars (*) indicate the seven critical residues different between SARS-CoV-2 and SARS-CoV RBDs. d Binding of polyclonal rabbit anti-SARS S1 antibodies T62 to mutant SARS-CoV S proteins. e Neutralization of SARS-CoV-2 S and SARS-CoV S pseudovirions by polyclonal rabbit anti-SARS S1 antibody T62. Pseudovirons were pre-incubated with serially diluted polyclonal rabbit anti-SARS S1 antibodies T62 on ice, then virus-antibody mixture was added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file. Full size image

Cross-neutralization of SARS and COVID-19 patient sera

As rabbit polyclonal anti-SARS S1 antibodies did not show significant neutralization activity against SARS-CoV-2 S pseudovirions, we then asked whether sera from recovered SARS and COVID-19 patients could neutralize SARS-CoV S and SARS-CoV-2 S pseudovirions. We obtained sera from one recovered SARS patient and five recovered COVID-19 patients and tested their neutralization activities against transduction on 293/hACE2 cells by SARS-CoV and SARS-CoV-2 S pseudovirions. Serum from recovered SARS patient showed strong inhibition on transduction by SARS-CoV S pseudovirions, and it had modest neutralization activity against SARS-CoV-2 S pseudovirons (Fig. 6a and Table 1). In contrast, sera from all five COVID-19 patients neutralized SARS-CoV-2 S pseudovirions effectively, and none of them had an effect on transduction by SARS-CoV S pseudovirions (Fig. 6b and Table 1).

Fig. 6: Limited cross-neutralization of SARS and COVID-19 sera. All sera were incubated on 56 °C for 30 min to eliminate complement. SARS-CoV S and SARS-CoV-2 S pseudovirons were pre-incubated with serially diluted SARS patient serum (a) or COVID-19 patient sera (b) for 1 h on ice and then added on 293/hACE2 cells. Pseudoviral transduction was measured 40 h later. Experiments were done in triplicates and repeated twice, and one representative is shown. Error bars indicate SEM of technical triplicates. Source data are provided as a Source Data file. Full size image