SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor
Markus Hoffmann, Hannah Kleine-Weber, Simon Schroeder, Nadine Krüger, Tanja Herrler, Sandra Erichsen, Tobias S Schiergens, Georg Herrler, Nai-Huei Wu, Andreas Nitsche, Marcel A Müller, Christian Drosten, Stefan Pöhlmann, Markus Hoffmann, Hannah Kleine-Weber, Simon Schroeder, Nadine Krüger, Tanja Herrler, Sandra Erichsen, Tobias S Schiergens, Georg Herrler, Nai-Huei Wu, Andreas Nitsche, Marcel A Müller, Christian Drosten, Stefan Pöhlmann
Abstract
The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
Keywords: ACE2; COVID-19; SARS-CoV-2; TMPRSS2; coronavirus; entry; neutralization; priming; spike.
Conflict of interest statement
Declaration of Interests The authors declare no competing interests.
Copyright © 2020 Elsevier Inc. All rights reserved.
Figures
References
- Berger Rentsch M., Zimmer G. A vesicular stomatitis virus replicon-based bioassay for the rapid and sensitive determination of multi-species type I interferon. PLoS ONE. 2011;6:e25858.
- Bertram S., Glowacka I., Blazejewska P., Soilleux E., Allen P., Danisch S., Steffen I., Choi S.Y., Park Y., Schneider H. TMPRSS2 and TMPRSS4 facilitate trypsin-independent spread of influenza virus in Caco-2 cells. J. Virol. 2010;84:10016–10025.
- Bertram S., Heurich A., Lavender H., Gierer S., Danisch S., Perin P., Lucas J.M., Nelson P.S., Pöhlmann S., Soilleux E.J. Influenza and SARS-coronavirus activating proteases TMPRSS2 and HAT are expressed at multiple sites in human respiratory and gastrointestinal tracts. PLoS ONE. 2012;7:e35876.
- Bossart K.N., Wang L.F., Flora M.N., Chua K.B., Lam S.K., Eaton B.T., Broder C.C. Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins. J. Virol. 2002;76:11186–11198.
- Brinkmann C., Hoffmann M., Lübke A., Nehlmeier I., Krämer-Kühl A., Winkler M., Pöhlmann S. The glycoprotein of vesicular stomatitis virus promotes release of virus-like particles from tetherin-positive cells. PLoS ONE. 2017;12:e0189073.
- Buchholz U., Müller M.A., Nitsche A., Sanewski A., Wevering N., Bauer-Balci T., Bonin F., Drosten C., Schweiger B., Wolff T. Contact investigation of a case of human novel coronavirus infection treated in a German hospital, October-November 2012. Euro Surveill. 2013;18:20406.
- Chan J.F., Yuan S., Kok K.H., To K.K., Chu H., Yang J., Xing F., Liu J., Yip C.C., Poon R.W. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet. 2020;395:514–523.
- Corman V.M., Lienau J., Witzenrath M. [Coronaviruses as the cause of respiratory infections] Internist (Berl.) 2019;60:1136–1145.
- Corman V.M., Landt O., Kaiser M., Molenkamp R., Meijer A., Chu D.K., Bleicker T., Brünink S., Schneider J., Schmidt M.L. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro Surveill. 2020;25 doi: 10.2807/1560-7917.ES.2020.25.3.2000045.
- de Wit E., van Doremalen N., Falzarano D., Munster V.J. SARS and MERS: recent insights into emerging coronaviruses. Nat. Rev. Microbiol. 2016;14:523–534.
- Ding Y., He L., Zhang Q., Huang Z., Che X., Hou J., Wang H., Shen H., Qiu L., Li Z. Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J. Pathol. 2004;203:622–630.
- Fehr A.R., Channappanavar R., Perlman S. Middle East Respiratory Syndrome: Emergence of a Pathogenic Human Coronavirus. Annu. Rev. Med. 2017;68:387–399.
- Ge X.Y., Li J.L., Yang X.L., Chmura A.A., Zhu G., Epstein J.H., Mazet J.K., Hu B., Zhang W., Peng C. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature. 2013;503:535–538.
- Gierer S., Bertram S., Kaup F., Wrensch F., Heurich A., Krämer-Kühl A., Welsch K., Winkler M., Meyer B., Drosten C. The spike protein of the emerging betacoronavirus EMC uses a novel coronavirus receptor for entry, can be activated by TMPRSS2, and is targeted by neutralizing antibodies. J. Virol. 2013;87:5502–5511.
- Glowacka I., Bertram S., Müller M.A., Allen P., Soilleux E., Pfefferle S., Steffen I., Tsegaye T.S., He Y., Gnirss K. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J. Virol. 2011;85:4122–4134.
- Gu J., Gong E., Zhang B., Zheng J., Gao Z., Zhong Y., Zou W., Zhan J., Wang S., Xie Z. Multiple organ infection and the pathogenesis of SARS. J. Exp. Med. 2005;202:415–424.
- Guan Y., Zheng B.J., He Y.Q., Liu X.L., Zhuang Z.X., Cheung C.L., Luo S.W., Li P.H., Zhang L.J., Guan Y.J. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science. 2003;302:276–278.
- Haga S., Yamamoto N., Nakai-Murakami C., Osawa Y., Tokunaga K., Sata T., Yamamoto N., Sasazuki T., Ishizaka Y. Modulation of TNF-alpha-converting enzyme by the spike protein of SARS-CoV and ACE2 induces TNF-alpha production and facilitates viral entry. Proc. Natl. Acad. Sci. USA. 2008;105:7809–7814.
- Hamming I., Timens W., Bulthuis M.L., Lely A.T., Navis G., van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J. Pathol. 2004;203:631–637.
- He Y., Li J., Heck S., Lustigman S., Jiang S. Antigenic and immunogenic characterization of recombinant baculovirus-expressed severe acute respiratory syndrome coronavirus spike protein: implication for vaccine design. J. Virol. 2006;80:5757–5767.
- Hoffmann M., Müller M.A., Drexler J.F., Glende J., Erdt M., Gützkow T., Losemann C., Binger T., Deng H., Schwegmann-Weßels C. Differential sensitivity of bat cells to infection by enveloped RNA viruses: coronaviruses, paramyxoviruses, filoviruses, and influenza viruses. PLoS ONE. 2013;8:e72942.
- Hofmann H., Geier M., Marzi A., Krumbiegel M., Peipp M., Fey G.H., Gramberg T., Pöhlmann S. Susceptibility to SARS coronavirus S protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor. Biochem. Biophys. Res. Commun. 2004;319:1216–1221.
- Hofmann H., Hattermann K., Marzi A., Gramberg T., Geier M., Krumbiegel M., Kuate S., Uberla K., Niedrig M., Pöhlmann S. S protein of severe acute respiratory syndrome-associated coronavirus mediates entry into hepatoma cell lines and is targeted by neutralizing antibodies in infected patients. J. Virol. 2004;78:6134–6142.
- Hofmann H., Pyrc K., van der Hoek L., Geier M., Berkhout B., Pöhlmann S. Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry. Proc. Natl. Acad. Sci. USA. 2005;102:7988–7993.
- Huang C., Wang Y., Li X., Ren L., Zhao J., Hu Y., Zhang L., Fan G., Xu J., Gu X. Lancet; China: 2020. Clinical features of patients infected with 2019 novel coronavirus in Wuhan.
- Imai Y., Kuba K., Rao S., Huan Y., Guo F., Guan B., Yang P., Sarao R., Wada T., Leong-Poi H. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature. 2005;436:112–116.
- Iwata-Yoshikawa N., Okamura T., Shimizu Y., Hasegawa H., Takeda M., Nagata N. TMPRSS2 Contributes to Virus Spread and Immunopathology in the Airways of Murine Models after Coronavirus Infection. J. Virol. 2019;93 doi: 10.1128/JVI.01815-18.
- Kawase M., Shirato K., van der Hoek L., Taguchi F., Matsuyama S. Simultaneous treatment of human bronchial epithelial cells with serine and cysteine protease inhibitors prevents severe acute respiratory syndrome coronavirus entry. J. Virol. 2012;86:6537–6545.
- Kim T.S., Heinlein C., Hackman R.C., Nelson P.S. Phenotypic analysis of mice lacking the Tmprss2-encoded protease. Mol. Cell. Biol. 2006;26:965–975.
- Kleine-Weber H., Elzayat M.T., Hoffmann M., Pöhlmann S. Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein. Sci. Rep. 2018;8:16597.
- Kleine-Weber H., Elzayat M.T., Wang L., Graham B.S., Müller M.A., Drosten C., Pöhlmann S., Hoffmann M. Mutations in the Spike Protein of Middle East Respiratory Syndrome Coronavirus Transmitted in Korea Increase Resistance to Antibody-Mediated Neutralization. J. Virol. 2019;93 doi: 10.1128/JVI.01381-18.
- Kuba K., Imai Y., Rao S., Gao H., Guo F., Guan B., Huan Y., Yang P., Zhang Y., Deng W. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat. Med. 2005;11:875–879.
- Kumar S., Stecher G., Li M., Knyaz C., Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;35:1547–1549.
- Lau S.K., Woo P.C., Li K.S., Huang Y., Tsoi H.W., Wong B.H., Wong S.S., Leung S.Y., Chan K.H., Yuen K.Y. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. USA. 2005;102:14040–14045.
- Li W., Moore M.J., Vasilieva N., Sui J., Wong S.K., Berne M.A., Somasundaran M., Sullivan J.L., Luzuriaga K., Greenough T.C. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature. 2003;426:450–454.
- Li F., Li W., Farzan M., Harrison S.C. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science. 2005;309:1864–1868.
- Li W., Zhang C., Sui J., Kuhn J.H., Moore M.J., Luo S., Wong S.K., Huang I.C., Xu K., Vasilieva N. Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J. 2005;24:1634–1643.
- Li W., Hulswit R.J.G., Widjaja I., Raj V.S., McBride R., Peng W., Widagdo W., Tortorici M.A., van Dieren B., Lang Y. Identification of sialic acid-binding function for the Middle East respiratory syndrome coronavirus spike glycoprotein. Proc. Natl. Acad. Sci. USA. 2017;114:E8508–E8517.
- Lin J.T., Zhang J.S., Su N., Xu J.G., Wang N., Chen J.T., Chen X., Liu Y.X., Gao H., Jia Y.P. Safety and immunogenicity from a phase I trial of inactivated severe acute respiratory syndrome coronavirus vaccine. Antivir. Ther. (Lond.) 2007;12:1107–1113.
- Liu W., Fontanet A., Zhang P.H., Zhan L., Xin Z.T., Baril L., Tang F., Lv H., Cao W.C. Two-year prospective study of the humoral immune response of patients with severe acute respiratory syndrome. J. Infect. Dis. 2006;193:792–795.
- Matsuyama S., Nagata N., Shirato K., Kawase M., Takeda M., Taguchi F. Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2. J. Virol. 2010;84:12658–12664.
- Menachery V.D., Dinnon K.H., III, Yount B.L., Jr., McAnarney E.T., Gralinski L.E., Hale A., Graham R.L., Scobey T., Anthony S.J., Wang L. Trypsin treatment unlocks barrier for zoonotic bat coronaviruses infection. J. Virol. 2020;94 doi: 10.1128/JVI.01774-19.
- Munster V.J., Koopmans M., van Doremalen N., van Riel D., de Wit E. A Novel Coronavirus Emerging in China - Key Questions for Impact Assessment. N. Engl. J. Med. 2020;382:692–694.
- Park J.E., Li K., Barlan A., Fehr A.R., Perlman S., McCray P.B., Jr., Gallagher T. Proteolytic processing of Middle East respiratory syndrome coronavirus spikes expands virus tropism. Proc. Natl. Acad. Sci. USA. 2016;113:12262–12267.
- Park Y.J., Walls A.C., Wang Z., Sauer M.M., Li W., Tortorici M.A., Bosch B.J., DiMaio F., Veesler D. Structures of MERS-CoV spike glycoprotein in complex with sialoside attachment receptors. Nat. Struct. Mol. Biol. 2019;26:1151–1157.
- Raj V.S., Mou H., Smits S.L., Dekkers D.H., Müller M.A., Dijkman R., Muth D., Demmers J.A., Zaki A., Fouchier R.A. Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC. Nature. 2013;495:251–254.
- Shieh W.J., Hsiao C.H., Paddock C.D., Guarner J., Goldsmith C.S., Tatti K., Packard M., Mueller L., Wu M.Z., Rollin P. Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan. Hum. Pathol. 2005;36:303–309.
- Shirato K., Kanou K., Kawase M., Matsuyama S. Clinical Isolates of Human Coronavirus 229E Bypass the Endosome for Cell Entry. J. Virol. 2016;91 doi: 10.1128/JVI.01387-16.
- Shirato K., Kawase M., Matsuyama S. Wild-type human coronaviruses prefer cell-surface TMPRSS2 to endosomal cathepsins for cell entry. Virology. 2018;517:9–15.
- Shulla A., Heald-Sargent T., Subramanya G., Zhao J., Perlman S., Gallagher T. A transmembrane serine protease is linked to the severe acute respiratory syndrome coronavirus receptor and activates virus entry. J. Virol. 2011;85:873–882.
- Simmons G., Gosalia D.N., Rennekamp A.J., Reeves J.D., Diamond S.L., Bates P. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc. Natl. Acad. Sci. USA. 2005;102:11876–11881.
- Wang C., Horby P.W., Hayden F.G., Gao G.F. A novel coronavirus outbreak of global health concern. Lancet. 2020;395:470–473.
- WHO . 2004. Summary of probable SARS cases with onset of illness from 1 November 2002 to 31 July 2003.
- WHO . 2020. Novel Coronavirus(2019-nCoV) Situation Report 23.
- Wu N.H., Yang W., Beineke A., Dijkman R., Matrosovich M., Baumgärtner W., Thiel V., Valentin-Weigand P., Meng F., Herrler G. The differentiated airway epithelium infected by influenza viruses maintains the barrier function despite a dramatic loss of ciliated cells. Sci. Rep. 2016;6:39668.
- Yamamoto M., Matsuyama S., Li X., Takeda M., Kawaguchi Y., Inoue J.I., Matsuda Z. Identification of Nafamostat as a Potent Inhibitor of Middle East Respiratory Syndrome Coronavirus S Protein-Mediated Membrane Fusion Using the Split-Protein-Based Cell-Cell Fusion Assay. Antimicrob. Agents Chemother. 2016;60:6532–6539.
- Yang Y., Du L., Liu C., Wang L., Ma C., Tang J., Baric R.S., Jiang S., Li F. Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. USA. 2014;111:12516–12521.
- Yang Y., Liu C., Du L., Jiang S., Shi Z., Baric R.S., Li F. Two Mutations Were Critical for Bat-to-Human Transmission of Middle East Respiratory Syndrome Coronavirus. J. Virol. 2015;89:9119–9123.
- Yeager C.L., Ashmun R.A., Williams R.K., Cardellichio C.B., Shapiro L.H., Look A.T., Holmes K.V. Human aminopeptidase N is a receptor for human coronavirus 229E. Nature. 1992;357:420–422.
- Zhou Y., Vedantham P., Lu K., Agudelo J., Carrion R., Jr., Nunneley J.W., Barnard D., Pöhlmann S., McKerrow J.H., Renslo A.R., Simmons G. Protease inhibitors targeting coronavirus and filovirus entry. Antiviral Res. 2015;116:76–84.
- Zhou P., Yang X.L., Wang X.G., Hu B., Zhang L., Zhang W., Si H.R., Zhu Y., Li B., Huang C.L. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature. 2020 doi: 10.1038/s41586-020-2012-7.
- Zhu N., Zhang D., Wang W., Li X., Yang B., Song J., Zhao X., Huang B., Shi W., Lu R. A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med. 2020;382:727–733.
Source: PubMed