Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein
TL;DR: It is found that an intact S1/S2 site was only required for efficient entry into cells expressing endogenous TMPRSS2, and findings suggest that cleavage at S2′ is carried out by proteases recognizing a single arginine, most likely TMPR SS2 and cathepsin L.
Abstract: The Middle East respiratory syndrome-related coronavirus (MERS-CoV) can cause severe disease and has pandemic potential. Therefore, development of antiviral strategies is an important task. The activation of the viral spike protein (S) by host cell proteases is essential for viral infectivity and the responsible enzymes are potential therapeutic targets. The cellular proteases furin, cathepsin L and TMPRSS2 can activate MERS-S and may cleave the S protein at two distinct sites, termed S1/S2 and S2′. Moreover, a potential cathepsin L cleavage site in MERS-S has been reported. However, the relative importance of these sites for MERS-S activation is incompletely understood. Here, we used mutagenic analysis and MERS-S-bearing vectors to study the contribution of specific cleavage sites to S protein-driven entry. We found that an intact S1/S2 site was only required for efficient entry into cells expressing endogenous TMPRSS2. In keeping with a previous study, pre-cleavage at the S1/S2 motif (RSVR) was important although not essential for subsequent MERS-S activation by TMPRSS2, and indirect evidence was obtained that this motif is processed by a protease depending on an intact RXXR motif, most likely furin. In contrast, the S2′ site (RSAR) was required for robust viral entry into all cell lines tested and the integrity of one of the two arginines was sufficient for efficient entry. These findings suggest that cleavage at S2′ is carried out by proteases recognizing a single arginine, most likely TMPRSS2 and cathepsin L. Finally, mutation of the proposed cathepsin L site did not impact viral entry and double mutation of S1/S2 and S2′ site was compatible with cathepsin L- but not TMPRSS2-dependent host cell entry, indicating that cathepsin L can process the S protein at auxiliary sites. Collectively, our results indicate a rigid sequence requirement for S protein activation by TMPRSS2 but not cathepsin L.
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TL;DR: It is demonstrated that SARS-CoV-2 uses the SARS -CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming, and it is shown that the sera from convalescent SARS patients cross-neutralized Sars-2-S-driven entry.
15,362 citations
TL;DR: A SARS-CoV-2 variant carrying the Spike protein amino acid change D614G has become the most prevalent form in the global pandemic, and it is found that the G614 variant grows to higher titer as pseudotyped virions.
3,302 citations
Cites background from "Functional analysis of potential cl..."
...TMPRSS2, a type-II transmembrane serine protease, cleaves the viral Spike after receptor binding to enhance entry of MERSCoV, SARS-CoV, and SARS-CoV-2 (Hoffmann et al., 2020b; Kleine-Weber et al., 2018; Matsuyama et al., 2020; Millet and Whittaker, 2014; Park et al., 2016; Shulla et al., 2011; Zang et al., 2020)....
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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 "Functional analysis of potential cl..."
...Thus, the Middle East respiratory syndrome coronavirus spike protein (MERS-S) is activated by a two-step process: MERS-S is first cleaved by furin at the S1/S2 site in infected cells, which is required for subsequent TMPRSS2-mediated cleavage at the S2 0 site ( Figure 1A ) during viral entry into lung cells (Kleine-Weber et al., 2018; Park et al., 2016; Millet and Whittaker, 2014) ....
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Technische Universität München1, German Center for Neurodegenerative Diseases2, University of Helsinki3, University of Göttingen4, Ludwig Maximilian University of Munich5, Life Sciences Institute6, University of Tartu7, National Institute for Health and Welfare8, University of Queensland9, ETH Zurich10, University of California, Santa Barbara11, Discovery Institute12, University of Zurich13
TL;DR: It is found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1.
Abstract: The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.
1,304 citations
TL;DR: In this article, the authors show that SARS-CoV-2/COVID-19 variants B.1.7 (UK), B.351 (South Africa), and P.1 (Brazil) harbor mutations in the viral spike (S) protein that may alter virus-host cell interactions and confer resistance to inhibitors and antibodies.
754 citations
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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
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
TL;DR: The Middle East respiratory syndrome coronavirus (MERS-CoV) is a lethal zoonotic pathogen that was first identified in humans in Saudi Arabia and Jordan in 2012.
1,030 citations
TL;DR: Biochemical studies demonstrate that CatB and CatL mediate entry by carrying out proteolysis of the EboV GP subunit GP1 and support a multistep mechanism that explains the relative contributions of these enzymes to infection.
Abstract: Ebola virus (EboV) causes rapidly fatal hemorrhagic fever in humans and there is currently no effective treatment. We found that the infection of African green monkey kidney (Vero) cells by vesicular stomatitis viruses bearing the EboV glycoprotein (GP) requires the activity of endosomal cysteine proteases. Using selective protease inhibitors and protease-deficient cell lines, we identified an essential role for cathepsin B (CatB) and an accessory role for cathepsin L (CatL) in EboV GP-dependent entry. Biochemical studies demonstrate that CatB and CatL mediate entry by carrying out proteolysis of the EboV GP subunit GP1 and support a multistep mechanism that explains the relative contributions of these enzymes to infection. CatB and CatB/CatL inhibitors diminish the multiplication of infectious EboV-Zaire in cultured cells and may merit investigation as anti-EboV drugs.
806 citations
"Functional analysis of potential cl..." refers background in this paper
...In this context, it is noteworthy that Caco-2 cell entry driven by the S protein of SARS-CoV also relied on TMPRSS2 while entry driven by the glycoprotein of Ebola virus, which is known to depend on cathepsin B and cathepsin L activity 30 , was blocked by the cathepsin B/L inhibitor MDL28170 (data not shown)....
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TL;DR: This review focuses on the diversity of strategies coronaviruses have evolved to proteolytically activate their fusion protein during spike protein biosynthesis and the critical entry step of their life cycle, and highlights important findings on how proteolytic activation of coronavirus spike influences tissue and cell tropism, host range and pathogenicity.
753 citations
"Functional analysis of potential cl..." refers background in this paper
...The S protein is synthesized as an inactive precursor and is converted into its active form upon cleavage by host cell proteases 8, 9 ....
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...SCientifiC REPORtS | (2018) 8 :16597 | DOI: 10 .1038/s41598-018-34859-w sufficient for S protein-driven entry....
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