ABSTRACT
Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2 causes worldwide COVID-19 pandemic and poses a great threat to global public health. Due to its high pathogenicity and infectivity, live SARS-CoV-2 is classified as a BSL-3 agent and has to be handled in BSL-3 condition. Nevertheless, entry of SARS-CoV-2 is mediated by viral spike (S) glycoprotein, and pseudovirus with SARS-CoV-2 S protein can mimic every entry step of SARS-CoV-2 virus and be studied in BSL-2 settings. In this chapter, we describe a detailed protocol of production of lentivirus-based SARS-CoV-2 S pseudovirus and its application in study of virus entry and determination of neutralizing antibody titer of human sera against SARS-CoV-2.
Subject(s)
COVID-19 , SARS-CoV-2 , Humans , SARS-CoV-2/metabolism , Antibodies, Viral , Neutralization Tests/methods , Pandemics , Antibodies, Neutralizing , Spike Glycoprotein, Coronavirus , GlycoproteinsABSTRACT
Bat coronavirus (CoV) RaTG13 shares the highest genome sequence identity with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among all known coronaviruses, and also uses human angiotensin converting enzyme 2 (hACE2) for virus entry. Thus, SARS-CoV-2 is thought to have originated from bat. However, whether SARS-CoV-2 emerged from bats directly or through an intermediate host remains elusive. Here, we found that Rhinolophus affinis bat ACE2 (RaACE2) is an entry receptor for both SARS-CoV-2 and RaTG13, although the binding of RaACE2 to the receptor-binding domain (RBD) of SARS-CoV-2 is markedly weaker than that of hACE2. We further evaluated the receptor activities of ACE2s from additional 16 diverse animal species for RaTG13, SARS-CoV, and SARS-CoV-2 in terms of S protein binding, membrane fusion, and pseudovirus entry. We found that the RaTG13 spike (S) protein is significantly less fusogenic than SARS-CoV and SARS-CoV-2, and seven out of sixteen different ACE2s function as entry receptors for all three viruses, indicating that all three viruses might have broad host rages. Of note, RaTG13 S pseudovirions can use mouse, but not pangolin ACE2, for virus entry, whereas SARS-CoV-2 S pseudovirions can use pangolin, but not mouse, ACE2 enter cells efficiently. Mutagenesis analysis revealed that residues 484 and 498 in RaTG13 and SARS-CoV-2 S proteins play critical roles in recognition of mouse and human ACE2s. Finally, two polymorphous Rhinolophous sinicus bat ACE2s showed different susceptibilities to virus entry by RaTG13 and SARS-CoV-2 S pseudovirions, suggesting possible coevolution. Our results offer better understanding of the mechanism of coronavirus entry, host range, and virus-host coevolution.
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.