Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2
01 Dec 2013-Journal of Virology (American Society for Microbiology)-Vol. 87, Iss: 23, pp 12552-12561
TL;DR: It is suggested that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line and cathepsin L in the endosome.
Abstract: The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-L-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.
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Xiuyuan Ou1, Yan Liu1, Xiaobo Lei1, Pei Li1, Dan Mi1, Lili Ren1, Li Guo1, Ruixuan Guo1, Ting Chen1, Jiaxin Hu1, Zichun Xiang1, Zhixia Mu1, Xing Chen1, Jieyong Chen, Keping Hu1, Qi Jin1, Jianwei Wang1, Zhaohui Qian1 •
TL;DR: It is shown that the SARS-CoV-2 spike protein is less stable than that of SARS -CoV, and limited cross-neutralization activities between SARS and COVID-19 patients’ sera showlimited cross- neutralization activities, suggesting that recovery from one infection might not protect against the other.
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.
2,622 citations
TL;DR: This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions.
Abstract: The coronavirus spike protein is a multifunctional molecular machine that mediates coronavirus entry into host cells. It first binds to a receptor on the host cell surface through its S1 subunit and then fuses viral and host membranes through its S2 subunit. Two domains in S1 from different coronaviruses recognize a variety of host receptors, leading to viral attachment. The spike protein exists in two structurally distinct conformations, prefusion and postfusion. The transition from prefusion to postfusion conformation of the spike protein must be triggered, leading to membrane fusion. This article reviews current knowledge about the structures and functions of coronavirus spike proteins, illustrating how the two S1 domains recognize different receptors and how the spike proteins are regulated to undergo conformational transitions. I further discuss the evolution of these two critical functions of coronavirus spike proteins, receptor recognition and membrane fusion, in the context of the corresponding fu...
2,075 citations
TL;DR: It is reported that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells and identifies furin as a potential target for therapeutic intervention.
1,443 citations
Cites background from "Middle East Respiratory Syndrome Co..."
...Our previous work revealed that the activity of the cellular serine protease TMPRSS2, which activates several coronaviruses Gierer et al., 2013; Glowacka et al., 2011; Matsuyama et al., 2010; Shirato et al., 2013 Shirato et al., , 2016 Shulla et al., 2011) , is also required for robust SARS-CoV-2 infection of human lung cells (Hoffmann et al., 2020) ....
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TL;DR: The epidemiology, virology, clinical features and current treatment strategies of SARS and MERS are summarized, and the discovery and development of new virus-based and host-based therapeutic options for CoV infections are discussed.
Abstract: Severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), which are caused by coronaviruses, have attracted substantial attention owing to their high mortality rates and potential to cause epidemics. Yuen and colleagues discuss progress with treatment options for these syndromes, including virus- and host-targeted drugs, and the challenges that need to be overcome in their further development. In humans, infections with the human coronavirus (HCoV) strains HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1 usually result in mild, self-limiting upper respiratory tract infections, such as the common cold. By contrast, the CoVs responsible for severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), which were discovered in Hong Kong, China, in 2003, and in Saudi Arabia in 2012, respectively, have received global attention over the past 12 years owing to their ability to cause community and health-care-associated outbreaks of severe infections in human populations. These two viruses pose major challenges to clinical management because there are no specific antiviral drugs available. In this Review, we summarize the epidemiology, virology, clinical features and current treatment strategies of SARS and MERS, and discuss the discovery and development of new virus-based and host-based therapeutic options for CoV infections.
1,388 citations
Shutoku Matsuyama1, Naganori Nao1, Kazuya Shirato1, Miyuki Kawase1, Shinji Saito1, Ikuyo Takayama1, Noriyo Nagata1, Tsuyoshi Sekizuka1, Hiroshi Katoh1, Fumihiro Kato1, Masafumi Sakata1, Maino Tahara1, Satoshi Kutsuna, Norio Ohmagari, Makoto Kuroda1, Tadaki Suzuki1, Tsutomu Kageyama1, Makoto Takeda1 •
TL;DR: It is shown that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to Sars-CoV-2 infection, making it useful for isolating and propagating SARS-Cov-2.
Abstract: A novel betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which caused a large respiratory outbreak in Wuhan, China in December 2019, is currently spreading across many countries globally. Here, we show that a TMPRSS2-expressing VeroE6 cell line is highly susceptible to SARS-CoV-2 infection, making it useful for isolating and propagating SARS-CoV-2. Our results reveal that, in common with SARS- and Middle East respiratory syndrome-CoV, SARS-CoV-2 infection is enhanced by TMPRSS2.
1,139 citations
Cites background from "Middle East Respiratory Syndrome Co..."
...SARS-CoV and Middle East respiratory syndrome (MERS)-CoV can enter cells via endocytosis and use cathepsin in endosomes for activation (12) (13) (14) ....
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...However, TMPRSS2 expression greatly promotes replication and syncytium formation in these viruses in vitro and in vivo (5, 6, 11, 12, 15) ....
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References
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TL;DR: The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
Abstract: A previously unknown coronavirus was isolated from the sputum of a 60-year-old man who presented with acute pneumonia and subsequent renal failure with a fatal outcome in Saudi Arabia. The virus (called HCoV-EMC) replicated readily in cell culture, producing cytopathic effects of rounding, detachment, and syncytium formation. The virus represents a novel betacoronavirus species. The closest known relatives are bat coronaviruses HKU4 and HKU5. Here, the clinical data, virus isolation, and molecular identification are presented. The clinical picture was remarkably similar to that of the severe acute respiratory syndrome (SARS) outbreak in 2003 and reminds us that animal coronaviruses can cause severe disease in humans.
4,809 citations
"Middle East Respiratory Syndrome Co..." refers methods in this paper
...The virus was propagated by using African green monkey and rhesus macaque kidney epithelial cells (Vero and LLC-MK2 cell lines, respectively) and was termed the human betacoronavirus EMC/ 2012 (HCoV-EMC) (1)....
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V. Stalin Raj1, Huihui Mou2, Saskia L. Smits1, Dick H. W. Dekkers1, Marcel A. Müller3, Ronald Dijkman4, Doreen Muth3, Jeroen Demmers1, Ali Moh Zaki, Ron A. M. Fouchier1, Volker Thiel5, Volker Thiel4, Christian Drosten3, Peter J. M. Rottier2, Albert D. M. E. Osterhaus1, Berend Jan Bosch2, Bart L. Haagmans1 •
TL;DR: Dipeptidyl peptidase 4 (DPP4; also known as CD26) is identified as a functional receptor for hCoV-EMC and will contribute critically to the understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.
Abstract: Most human coronaviruses cause mild upper respiratory tract disease but may be associated with more severe pulmonary disease in immunocompromised individuals. However, SARS coronavirus caused severe lower respiratory disease with nearly 10% mortality and evidence of systemic spread. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was identified in patients with severe and sometimes lethal lower respiratory tract infection. Viral genome analysis revealed close relatedness to coronaviruses found in bats. Here we identify dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional receptor for hCoV-EMC. DPP4 specifically co-purified with the receptor-binding S1 domain of the hCoV-EMC spike protein from lysates of susceptible Huh-7 cells. Antibodies directed against DPP4 inhibited hCoV-EMC infection of primary human bronchial epithelial cells and Huh-7 cells. Expression of human and bat (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells enabled infection by hCoV-EMC. The use of the evolutionarily conserved DPP4 protein from different species as a functional receptor provides clues about the host range potential of hCoV-EMC. In addition, it will contribute critically to our understanding of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention strategies.
1,743 citations
"Middle East Respiratory Syndrome Co..." refers background in this paper
...Next, the factors critical for MERS-CoV entry into cells were identified, including dipeptidyl peptidase 4 (DPP4) as a functional virus receptor (5) and certain cellular proteases (e....
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...Furthermore, DPP4 was identified as the functional receptor for MERS-CoV (5)....
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Raoul J. de Groot1, Susan C. Baker2, Ralph S. Baric3, Caroline Brown, Christian Drosten4, Luis Enjuanes5, Ron A. M. Fouchier6, Monica Galiano7, Alexander E. Gorbalenya8, Ziad A. Memish, Stanley Perlman9, Leo L.M. Poon10, Eric J. Snijder8, Gwen Stephens, Patrick C. Y. Woo10, Ali Moh Zaki11, Maria Zambon7, John Ziebuhr12 •
Utrecht University1, Loyola University Medical Center2, University of North Carolina at Chapel Hill3, University of Bonn4, Autonomous University of Madrid5, Erasmus University Rotterdam6, Health Protection Agency7, Leiden University Medical Center8, University of Iowa9, University of Hong Kong10, Ain Shams University11, University of Giessen12
TL;DR: During the summer of 2012, in Jeddah, Saudi Arabia, a hitherto unknown coronavirus was isolated from the sputum of a patient with acute pneumonia and renal failure and was provisionally called human coronav virus Erasmus Medical Center (EMC).
Abstract: During the summer of 2012, in Jeddah, Saudi Arabia, a hitherto unknown coronavirus (CoV) was isolated from the sputum of a patient with acute pneumonia and renal failure ([1][1], [2][2]). The isolate was provisionally called human coronavirus Erasmus Medical Center (EMC) ([3][3]). Shortly thereafter
1,054 citations
"Middle East Respiratory Syndrome Co..." refers background in this paper
...Recently, this virus was renamed the Middle East respiratory syndrome coronavirus (MERS-CoV) (2)....
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TL;DR: This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells.
Abstract: The distribution of the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor, an angiotensin-converting enzyme 2 (ACE2), does not strictly correlate with SARS-CoV cell tropism in lungs; therefore, other cellular factors have been predicted to be required for activation of virus infection. In the present study, we identified transmembrane protease serine 2 (TMPRSS2), whose expression does correlate with SARS-CoV infection in the upper lobe of the lung. In Vero cells expressing TMPRSS2, large syncytia were induced by SARS-CoV infection. Further, the lysosome-tropic reagents failed to inhibit, whereas the heptad repeat peptide efficiently inhibited viral entry into cells, suggesting that TMPRSS2 affects the S protein at the cell surface and induces virus-plasma membrane fusion. On the other hand, production of virus in TMPRSS2-expressing cells did not result in S-protein cleavage or increased infectivity of the resulting virus. Thus, TMPRSS2 affects the entry of virus but not other phases of virus replication. We hypothesized that the spatial orientation of TMPRSS2 vis-a-vis S protein is a key mechanism underling this phenomenon. To test this, the TMPRSS2 and S proteins were expressed in cells labeled with fluorescent probes of different colors, and the cell-cell fusion between these cells was tested. Results indicate that TMPRSS2 needs to be expressed in the opposing (target) cell membrane to activate S protein rather than in the producer cell, as found for influenza A virus and metapneumoviruses. This is the first report of TMPRSS2 being required in the target cell for activation of a viral fusion protein but not for the S protein synthesized in and transported to the surface of cells. Our findings suggest that the TMPRSS2 expressed in lung tissues may be a determinant of viral tropism and pathogenicity at the initial site of SARS-CoV infection.
684 citations
"Middle East Respiratory Syndrome Co..." refers background or result in this paper
...cells (19, 22) or virus release from the cell surface (21)....
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...This is in contrast to the findings for SARS-CoV-induced syncytium formation, which took 36 h to occur in our previous study (19)....
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TL;DR: Findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.
Abstract: Spike (S) proteins, the defining projections of the enveloped coronaviruses (CoVs), mediate cell entry by connecting viruses to plasma membrane receptors and by catalyzing subsequent virus-cell membrane fusions. The latter membrane fusion requires an S protein conformational flexibility that is facilitated by proteolytic cleavages. We hypothesized that the most relevant cellular proteases in this process are those closely linked to host cell receptors. The primary receptor for the human severe acute respiratory syndrome CoV (SARS) CoV is angiotensin-converting enzyme 2 (ACE2). ACE2 immunoprecipitation captured transmembrane protease/serine subfamily member 2 (TMPRSS2), a known human airway and alveolar protease. ACE2 and TMPRSS2 colocalized on cell surfaces and enhanced the cell entry of both SARS S-pseudotyped HIV and authentic SARS-CoV. Enhanced entry correlated with TMPRSS2-mediated proteolysis of both S and ACE2. These findings indicate that a cell surface complex comprising a primary receptor and a separate endoprotease operates as a portal for activation of SARS-CoV cell entry.
563 citations