Introduction
Coronaviruses (CoVs) infect human and animals and cause varieties of diseases, including respiratory, enteric, renal, and neurological diseases1. They are classified into four genera, alpha-CoV, beta-CoV, gamma-CoV, and delta-CoV2. Since beginning of this century, there have already been three zoonotic outbreaks of beta-CoVs. In 2002–2003, severe acute respiratory syndrome coronavirus (SARS-CoV)3,4, a lineage B beta-CoV, emerged from bat and palm civet5,6, and infected over 8000 people and caused about 800 deaths7. In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV), a lineage C beta-CoV, was discovered as the causative agent of a severe respiratory syndrome in Saudi Arabia8, currently with 2494 confirmed cases and 858 deaths9, it remains endemic in Middle East, and dromedary camel is considered as the zoonotic reservoir host of MERS-CoV. At the end of 2019, a novel coronavirus, named SARS-CoV-2, was found in patients with severe pneumonia in Wuhan, China10–12. Viruses were isolated from patients and sequenced. Phylogenetical analysis revealed that it is a lineage B beta-CoV and closely related to a SARS-like (SL) CoV, RaTG13, discovered in a cave of Yunnan, China, in 201313. They share about 96% nucleotide sequence identities, suggesting that SARS-CoV-2 might have emerged from a Bat SL-CoV. However, the intermediate host or whether there is an intermediate host remains to be determined.
CoV uses its spike glycoprotein (S), a main target for neutralization antibody, to bind its receptor, and mediate membrane fusion and virus entry. Each monomer of trimeric S protein is about 180 kDa, and contains two subunits, S1 and S2, mediating attachment and membrane fusion, respectively. In the structure, N- and C- terminal portions of S1 fold as two independent domains, N-terminal domain (NTD) and C-terminal domain (C-domain) (Fig. 1a). Depending on the virus, either NTD or C-domain can serve as the receptor-binding domain (RBD). While RBD of mouse hepatitis virus (MHV) is located at the NTD14, most of other CoVs, including SARS-CoV and MERS-CoV use C-domain to bind their receptors15–19. MHV uses mouse carcinoembryonic antigen related cell adhesion molecule 1a (mCEACAM1a) as the receptor20, and the receptors for SARS-CoV and MERS-CoV are human angiotensin-converting enzyme 2 (hACE2)21 and human dipeptidyl peptidase 4 (hDPP4)22, respectively. While S proteins of SARS-CoV-2 share about 76% and 97% of amino acid identities with SARS-CoV and RaTG13, respectively, the amino acid sequence of potential RBD of SARS-CoV-2 is only about 74% and 90.1% homologous to that of SARS-CoV and RaTG13, respectively. Recently, Zhou et al.13 reported that SARS-CoV-2 uses hACE2 as the receptor.
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.
CoV S proteins are typical class I viral fusion proteins, and protease cleavage is required for activation of the fusion potential of S protein23. A two-step sequential protease cleavage model has been proposed for activation of S proteins of SARS-CoV and MERS-CoV24,25, priming cleavage between S1 and S2 and activating cleavage on S2’ site. Depending on virus strains and cell types, CoV S proteins may be cleaved by one or several host proteases, including furin, trypsin, cathepsins, transmembrane protease serine protease-2 (TMPRSS-2), TMPRSS-4, or human airway trypsin-like protease (HAT)25–32. Availability of these proteases on target cells largely determines whether CoVs enter cells through plasma membrane or endocytosis. However, whether any of these proteases could promote virus entry of SARS-CoV-2 remains elusive.
In this study, using a lentiviral pseudotype system, we determine cell type susceptibility, virus receptor, entry pathway, and protease priming for SARS-CoV-2, and identify several potential drug targets for SARS-CoV-2. We demonstrate limited cross-neutralization between convalescent sera from SARS and COVID-19 patients.