Structural and functional mechanism of SARS-CoV-2 cell entry

The novel coronavirus SARS-CoV-2 emerged as a human pathogen in China at the end of 2019 and has since spread across the globe. Understanding the structure and function of this virus is essential to target vaccines and therapies to tackle the COVID-19 disease. 


Overview

  

SARS-CoV-2 virus

Rapid analysis of the viral pathogen at the beginning of this pandemic revealed that it belongs to the B beta-CoV lineage1,2. Coronaviruses are enveloped, single-strand RNA viruses characterized by club-like spikes projecting from their surface and an unusually large RNA genome3. The SARS-CoV-2 genome encodes four major structural proteins: the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein and the envelope (E) protein, each of which is essential to compose the viral particle 3.  



Binding of SARS-CoV-2 to the host cell receptor

​Like all coronaviruses, SARS-CoV-2 utilizes the S glycoprotein to promote entry into the host cell. This protein contains two functional domains: an S1 receptor-binding domain (RBD), and a second S2 domain that mediates the fusion of the viral and host cell membranes4.​

SARS-CoV-2 S protein binds to the ACE2 receptor on the host cell, initially through the S1 receptor binding domain. The S1 domain is then shed from the viral surface, allowing the S2 domain to fuse to the host cell membrane. This process is dependent upon activation of the S protein, by cleavage at two sites (S1/S2 and S2’) via the proteases Furin and TMPRSS2. Furin cleavage at the S1/S2 site may lead to conformational changes in the viral S protein that exposes the RBD and/or the S2 domain. TMPRSS2 cleavage of the SARS-CoV-2 S protein is believed to enable the fusion of the viral capsid with the host cell to permit viral entry5,6. 

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Exposure of the RBD in the S1 protein subunit creates an unstable subunit conformation. Consequently, during binding, this subunit undergoes conformational rearrangement between two states, known as the up and down conformations. The down state transiently hides the RBD, while the up state exposes the RBD, but temporarily destabilizes the protein subunit7,8,9. Within the trimeric S protein, only one of the three RBD is present in the accessible conformation to bind the human Angiotensin 2 (hACE2) host cell receptor10.

The critical functions of the SARS-CoV-2 S protein, ACE2, and the Furin and TMPRSS2 enzymes in binding and mediating viral entry to the host cell make these proteins key targets for drug development and viral inhibition in the efforts against COVID-19.

Alternative SARS-CoV-2 cell entry mechanisms

In addition to binding ACE2, research evidence is building concerning SARS-CoV-2 that suggests that the virus can also bind other cell surface molecules to gain cell entry. The increased transmissibility of SARS-CoV-2 compared with SARS-CoV could potentially be explained by an increased number of cellular receptors allowing the virus to penetrate host cells.

Both Neuropilin-1 and Neuropilin-2 have been shown to bind the cleaved form of the SARS-CoV-2 S protein to mediate host cell entry 11, 12.  Neuropilin proteins are expressed in neurons, providing an entry system for the virus into the nervous system and this has been observed in the neuropathological analysis of human COVID-19 autopsies 12. Antibody blockade of the NP receptors, receptor mutagenesis and structural studies all support the role of NP as a cell receptor for SARS-CoV-2 S protein 11, 12. Binding to NP receptors on the cell surface, in addition to the known ACE2 receptor, was shown to potentiate SARS-CoV-2 infection and may explain the increased tissue tropism seen in SARS-CoV-2 infection, compared to SARS-CoV 11, 12.

An initial study suggested that the SARS-CoV-2 S protein was able to bind to CD147 on the cell surface and subsequently enter the cell 13. As part of this study, in vitro antiviral tests indicated that meplazumab, an anti-CD147 humanized antibody that blocks the interaction of the S protein with the CD147 cell surface receptor, significantly inhibited viral cell entry. Clinical trials into the potential of this antibody to treat COVID-19 are ongoing 14. As higher blood sugar levels are known to upregulate CD147 expression, this could help to explain why diabetes is a factor for poor prognosis in cases of COVID-19 15.

Frequent cellular targets of the SARS-CoV-2 virus within the human host are neuronal cells, the endothelial cells of blood vessels, and epithelial cells within the respiratory system and gastrointestinal tract 16. However, ACE2 is known to be expressed only at low levels within the brain 17. A structural modeling study indicated that SARS-CoV-2 can bind to sialic acid glycoproteins and gangliosides on the cell surface 18, and sialic acid is known to be expressed at high levels on the surface of all of the cell types targeted by SARS-CoV-2 19, including neuronal cells. This is a common cell entry mechanism for other viruses, including influenza 20, MERS, SARS-CoV and HCoV-OC43 21, and could offer potential new therapeutic strategies using drugs to lower sialic acid levels in COVID-19 patients to help prevent SARS-CoV-2 cell entry 22. 

SARS-CoV-2 recommended products


NameAbIDApplication
Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody [CR3022]ab273073Neutralising, ELISA
Recombinant Anti-SARS-CoV-2 Spike Glycoprotein S1 antibody [CR3022] - Chimericab273074Neutralising, ELISA
Anti-SARS spike glycoprotein antibody [1A9]ab273433ELISA, WB, ICC/IF, Flow Cyt, IP
Anti-SARS nucleocapsid protein antibody [6H3]ab273434ELISA, WB, ICC/IF, Flow Cyt, IP
Recombinant Human coronavirus SARS-CoV-2 nucleocapsid protein (His tag)ab273530ELISA, WB
Human ACE2 ELISA Kitab235649ELISA
Recombinant Anti-ACE2 antibody [EPR4435(2)]ab108252IHC-P, IP, WB
Anti-ACE2 antibody [EPR4436]ab108209WB, IP, IHC-P
Recombinant Human ACE2 protein (Fc chimera)ab273687WB, ELISA, HPLC

Recombinant Human ACE2 (mutated H374 N + H378 N) protein (Fc Chimera)

ab273885WB, ELISA, HPLC
Recombinant Anti-TMPRSS2 antibody [EPR3862]ab109131ICC/IF, WB, IHC-P
Recombinant anti-TMPRSS2 antibody [EPR3861]ab92323WB, IHC-P
DX600 ACE2 inhibitorab273525Inhibitor
Camostat mesylate, TMPRSS2 inhibitorab145709Inhibitor
Recombinant Anti-Furin antibody [EPR14674]ab183495ICC/IF, IHC-P, WB
Naphthofluorescein, Furin inhibitorab145383Inhibitor
Recombinant Anti-Neuropilin 1 antibody [EPR3113]ab81321ICC/IF, Flow Cyt, IP, IHC-P, WB
Recombinant anti-CD147 antibody [EPR4053]ab108308ICC/IF, IHC-P, WB
Recombinant Anti-SARS nucleocapsid protein antibody [CR-3018 (03-018]ab275983ELISA
Recombinant Anti-SARS nucleocapsid protein antibody [CR-3009 (03-009)]ab275984ELISA
​

Abcam’s products are intended for research use only, not for use in diagnostic, therapeutic or any other purposes.


References

  1. ​Zhou P, Yang XL, Wang XG, et al.  A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). 
  2. Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020). 
  3. Fehr AR and Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol. 1282:1-23 (2015).
  4. Walls AC, Park YJ, Tortorici MA, et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181(2), 281-292.e6 (2020).
  5. Bestle D, Heindl MR, Limbu H et al. TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets. bioRxiv doi: https://doi.org/10.1101/2020.04.15.042085 (2020). 
  6. Yuan M, Wu MC, Zhu X, et al. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science 10.1126/science.abb7269 (2020).
  7. Gui M, Song W, Zhou H, et al. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell Res. 27, 119–129 (2017).
  8. Walls AC, Xiong X, Park YJ, et al. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion. Cell 176, 1026–1039.e15 (2019).
  9. Walls AC, Tortorici MA, Snijder J, et al. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc. Natl. Acad. Sci. U.S.A. 114, 11157–11162 (2017).
  10. Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 36(6483), 1260-1263 (2020).
  11. Daly et al. Neuropilin-1 is a host factor for SARS-CoV-2 infection. bioRxiv doi:10.1101/2020.06.05.134114
  12. Cantuti-Castelvetri et al. Neuropilin-1 facilitates SARS-CoV-2 cell entry and provides a possible pathway into the central nervous system. bioRxiv. doi:10.1101/2020.06.07.137802
  13. Wang et al. SARS-CoV-2 invades host cells via a novel route: CD147-spike protein. bioRxiv. [doi:10.1101/2020.03.14988345]
  14. Clinicaltrials.gov Clinical Study of Anti-CD147 Humanized Meplazumab for Injection to Treat With 2019-nCoV Pneumonia [Accessed: 21/07/2020]
  15. Bao et al. Monocyte CD147 is induced by advanced glycation end products and high glucose concentration: possible role in diabetic complications. Am J Physiol Cell Physiol 299(5):C1212-9. (2010).
  16. Zhang et al. New understanding of the damage of SARS-CoV-2 infection outside the respiratory system. Biomed Pharmacother. 127: 110195 (2020).
  17. Alenina M and Bader M. ACE2 in Brain Physiology and Pathophysiology: Evidence from Transgenic Animal Models. Neurochem Res 44(6):1323-1329 (2019).
  18. Fantini et al. Structural and molecular modelling studies reveal a new mechanism of action of chloroquine and hydroxychloroquine against SARS-CoV-2 infection. Int J Antimicorb Agents 55(5):105960 (2020).
  19. Roe K. High COVID‐19 virus replication rates, the creation of antigen–antibody immune complexes and indirect haemagglutination resulting in thrombosis. Transbound Emerg Dis.  67(4):1418-1421 (2020).
  20. Yang et al. Mutations during the Adaptation of H9N2 Avian Influenza Virus to the Respiratory Epithelium of Pigs Enhance Sialic Acid Binding Activity and Virulence in Mice. J Virol. 91(8): e02125-16 (2017).
  21. Devaux et al. New insights on the antiviral effects of chloroquine against coronavirus: what to expect for COVID-19? Int J Antimicorb Agents 55(5):105938 (2020).
  22. Li et al. Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein. PNAS. 114(40) E8508-E8517 (2017).
Zhou P, Yang XL, Wang XG, et al.  A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020). 
Wu F, Zhao S, Yu B, et al. A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020). 
Fehr AR and Perlman S. Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol. 1282:1-23 (2015).
Walls AC, Park YJ, Tortorici MA, et al. Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein. Cell 181(2), 281-292.e6 (2020).
Bestle D, Heindl MR, Limbu H et al. TMPRSS2 and furin are both essential for proteolytic activation and spread of SARS-CoV-2 in human airway epithelial cells and provide promising drug targets. bioRxiv doi: https://doi.org/10.1101/2020.04.15.042085 (2020). 
Yuan M, Wu MC, Zhu X, et al. A highly conserved cryptic epitope in the receptor-binding domains of SARS-CoV-2 and SARS-CoV. Science 10.1126/science.abb7269 (2020).
Gui M, Song W, Zhou H, et al. Cryo-electron microscopy structures of the SARS-CoV spike glycoprotein reveal a prerequisite conformational state for receptor binding. Cell Res. 27, 119–129 (2017).
Walls AC, Xiong X, Park YJ, et al. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion. Cell 176, 1026–1039.e15 (2019).
Walls AC, Tortorici MA, Snijder J, et al. Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion. Proc. Natl. Acad. Sci. U.S.A. 114, 11157–11162 (2017).
Wrapp D, Wang N, Corbett KS, et al. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 36(6483), 1260-1263 (2020).