Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV

Abstract

Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002-2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients' sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.

Fig. 1. Incorporation of SARS-CoV-2 S protein into pseudovirions.

a Diagram of full-length SARS-CoV-2 S protein with a 3xFLAG tag. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane domain; NTD, N-terminal domain; pFP, potential fusion peptide; HR-N, heptad repeat-N; HR-C, heptad repeat-C; b–f Detection of CoVs S protein in cells lysate by western blot. Mock, 293T cells transfected with empty vector. b Mouse monoclonal anti-FLAG M2 antibody; c Polyclonal goat anti-MHV-A59 S protein antibody AO4. d Polyclonal rabbit anti-SARS S1 antibodies T62. e Mouse monoclonal anti-SARS S1 antibody. f Mouse monoclonal anti-MERS-CoV S2 antibody. g–j Detection of CoVs S protein in pseudovirions by western blot.Gag-p24 served as a loading control. g Anti-FLAG M2. h Polyclonal goat anti-MHV-A59 S protein antibody AO4. i Polyclonal rabbit anti-SARS S1 antibodies T62. j Polyclonal anti-Gag-p24 antibodies. uncleaved S protein, about 180 kDa; cleaved S protein, about 90 kDa. Experiments were done twice and one is shown. Source data are provided as a Source Data file.

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.

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 NH₄Cl 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.

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.

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.

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.