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.
About 70% of the emerging pathogens infecting humans originate from animals, and CoVs are among the forefronts of these pathogens45. The newly emerged SARS-CoV-2 infects human and causes severe pneumonia, and as of 10 February, 2020, the current outbreak has spread to 25 countries with over 40,000 confirmed cases and 900 deaths46. However, little is known about its biology. Since the virus is categorized as a biosafety level 3 (BSL3) agent, according to WHO guideline, we developed a pseudotype system with S protein of SARS-CoV-2 to study virus entry in BSL2 settings. Understanding how SARS-CoV-2 enters cell will provide valuable information for virus pathogenesis, vaccine design, and drug target.
Utilizing this pseudotype system, we screened a panel of human and monkey cell lines for their susceptibility of SARS-CoV-2 S-mediated transduction. In line with SASR-CoV-2 causing respiratory infections in humans, we found that human lung cancer cell line Calu3 is highly susceptible to SARS-CoV-2 S-mediated entry. LLCMK2 cells, a rhesus monkey kidney epithelium cell line, exhibited different susceptibility to SARS-CoV S and SARS-CoV-2 S transduction, but the reasons for this are currently not known and require further investigation. Both S proteins use hACE2 as the receptor for binding and entry13,21, which we further confirmed by flow cytometry analysis and competitive inhibition experiment using soluble hACE2 in this study (Fig. 3b, c). While full-length S proteins of SARS-CoV-2 and SARS-CoV S share almost 76% identities in amino acid sequences, the NTDs show only 53.5% of homology. Of note, NTDs of different CoVs S proteins have been shown to bind different sugar. While NTD of MERS-CoV prefers α2,3-linked sialic acid over α2,6-linked sialic acid47, NTDs of human CoVs OC43 and HKU1 bind to 9-O-acetylated sialic acids48,49. No sugar binding has been reported for NTD of SARS-CoV. Whether or not NTD of SARS-CoV-2 binds to sugar and whether sugar binding of NTD affects virus entry remains to be determined.
Successful conformational changes of S proteins, leading to membrane fusion, not only require receptor binding, but also need appropriate protease activation. There is a furin site between S1 and S2 (amino acids 682-685, RRAR) subunits in SARS-CoV-2 S protein, similar to what happens in high-pathogenic influenza viruses. Whether the presence of this furin site has any effect on viral pathogenesis and virus spreading among humans remains to be determined. Although majority of SARS-CoV-2 S proteins in pseuodvirions were cleaved, endocytosis is the main entry pathway on 293/hACE2 cell. In our experiments, lysosomal cathepsin L, not B, appeared critical for SARS-CoV-2 S protein activation, similar to what was reported for SARS-CoV and MERS-CoV28,29,50. Trypsin could also activate SARS-CoV-2 S protein efficiently and induce large syncytium formation. To our surprise, we found that, even without trypsin, SARS-CoV-2 S protein could trigger syncytium formation on 293/hACE2 cells, a phenomena similar to what was observed in MHV S protein in certain aspect51. This led us to speculate that SARS-CoV-2 S protein is capable of triggering protease-independent and receptor-dependent syncytium formation. Such a mechanism might enhance virus spreading through cell–cell fusion and this might partially explain rapid progress of the diseases10,11. However, we have not excluded a potential role for other endogenous proteases in HEK293T cells for syncytium formation and we have also not evaluated entry into other cell types. This requires further research.
Recent studies showed that early to late endosome maturation is regulated by PI(3,5)P2, and inhibition of PIKfyve, the key enzyme synthesizing PI(3,5)P2, and TPC2, a downstream effector in lysosome, significantly reduced virus entry of MERS-CoV52. We confirmed this in this study, and further showed that blocking PIKfyve and TPC2 also strongly inhibited entry mediated by SARS-CoV-2 S protein, indicating that PI(3,5)P2 pathway might be considered as potential general drug target for CoV infection.
CoV S protein is one of the key components determining virus virulence, tissue tropism and host range, and it is also a main target for neutralizing antibodies and vaccine design. Although the S proteins of SARS-CoV-2 and SARS-CoV are highly homologous, polyclonal rabbit anti-SARS S1 antibodies T62 did not bind to SARS-CoV-2 S protein well, and poorly neutralized SARS-CoV-2 S protein-mediated virus entry. Further analysis reveals that major immune-epitopes for T62 antibodies likely lie in the region of RBD (Fig. 4a). We further evaluated cross-neutralization of SARS-CoV and SARS-CoV-2 using convalescent sera from SARS and COVID-19 patients. We only saw moderate cross-neutralization activities between SARS-CoV-2 and SARS-CoV convalescent sera, suggesting that those previously recovered from SARS-CoV infection may not be fully protected against SARS-CoV-2 infection, and vice versa.
While this manuscript was under review,human ACE2 is the entry receptor of SARS-CoV-2, and that serine protease TMPRSS2 is important for SARS-CoV-2 S activation, consistent with our findings. In agreement with our work, sera from convalescent SARS patients neutralized SARS-CoV-2 S pseudovirion entry with lower efficiency than SARS-CoV S pseudovirion entry, showing moderate cross-neutralization.
In conclusion, we demonstrated that SARS-CoV-2 S protein entry on 293/hACE2 cells is mainly mediated through endocytosis, and that PIKfyve, TPC2, and cathepsin L are critical for virus entry. We further found that SARS-CoV-2 S protein could trigger syncytia in 293/hACE2 cells independent of exogenous protease. Finally, there was limited cross-neutralization activity between convalescent sera from SARS and COVID-19 patients. Our findings provide potential targets for development of drugs and vaccines against this newly emerging lineage B beta-CoV.
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