Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein
Identifieur interne : 002011 ( Ncbi/Merge ); précédent : 002010; suivant : 002012Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein
Auteurs : Hannah Kleine-Weber [Allemagne] ; Mahmoud Tarek Elzayat [Allemagne] ; Markus Hoffmann [Allemagne] ; Stefan Pöhlmann [Allemagne]Source :
- Scientific Reports [ 2045-2322 ] ; 2018.
Descripteurs français
- KwdFr :
- Animaux, Cathepsine L (génétique), Cathepsine L (métabolisme), Cellules Vero, Coronavirus du syndrome respiratoire du Moyen-Orient (physiologie), Furine (génétique), Furine (métabolisme), Glycoprotéine de spicule des coronavirus (génétique), Glycoprotéine de spicule des coronavirus (métabolisme), Humains, Infections à coronavirus (virologie), Mutation, Protéolyse, Pénétration virale, Serine endopeptidases (génétique), Serine endopeptidases (métabolisme).
- MESH :
- génétique : Cathepsine L, Furine, Glycoprotéine de spicule des coronavirus, Serine endopeptidases.
- métabolisme : Cathepsine L, Furine, Glycoprotéine de spicule des coronavirus, Serine endopeptidases.
- physiologie : Coronavirus du syndrome respiratoire du Moyen-Orient.
- virologie : Infections à coronavirus.
- Animaux, Cellules Vero, Humains, Mutation, Protéolyse, Pénétration virale.
English descriptors
- KwdEn :
- Animals, Cathepsin L (genetics), Cathepsin L (metabolism), Chlorocebus aethiops, Coronavirus Infections (virology), Furin (genetics), Furin (metabolism), Humans, Middle East Respiratory Syndrome Coronavirus (physiology), Mutation, Proteolysis, Serine Endopeptidases (genetics), Serine Endopeptidases (metabolism), Spike Glycoprotein, Coronavirus (genetics), Spike Glycoprotein, Coronavirus (metabolism), Vero Cells, Virus Internalization.
- MESH :
- chemical , genetics : Cathepsin L, Furin, Serine Endopeptidases, Spike Glycoprotein, Coronavirus.
- chemical , metabolism : Cathepsin L, Furin, Serine Endopeptidases, Spike Glycoprotein, Coronavirus.
- physiology : Middle East Respiratory Syndrome Coronavirus.
- virology : Coronavirus Infections.
- Animals, Chlorocebus aethiops, Humans, Mutation, Proteolysis, Vero Cells, Virus Internalization.
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.
Url:
DOI: 10.1038/s41598-018-34859-w
PubMed: 30413791
PubMed Central: 6226446
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PMC:6226446Le document en format XML
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<term>Cathepsin L (metabolism)</term>
<term>Chlorocebus aethiops</term>
<term>Coronavirus Infections (virology)</term>
<term>Furin (genetics)</term>
<term>Furin (metabolism)</term>
<term>Humans</term>
<term>Middle East Respiratory Syndrome Coronavirus (physiology)</term>
<term>Mutation</term>
<term>Proteolysis</term>
<term>Serine Endopeptidases (genetics)</term>
<term>Serine Endopeptidases (metabolism)</term>
<term>Spike Glycoprotein, Coronavirus (genetics)</term>
<term>Spike Glycoprotein, Coronavirus (metabolism)</term>
<term>Vero Cells</term>
<term>Virus Internalization</term>
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<keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term>
<term>Cathepsine L (génétique)</term>
<term>Cathepsine L (métabolisme)</term>
<term>Cellules Vero</term>
<term>Coronavirus du syndrome respiratoire du Moyen-Orient (physiologie)</term>
<term>Furine (génétique)</term>
<term>Furine (métabolisme)</term>
<term>Glycoprotéine de spicule des coronavirus (génétique)</term>
<term>Glycoprotéine de spicule des coronavirus (métabolisme)</term>
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<term>Infections à coronavirus (virologie)</term>
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<term>Serine Endopeptidases</term>
<term>Spike Glycoprotein, Coronavirus</term>
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<term>Furin</term>
<term>Serine Endopeptidases</term>
<term>Spike Glycoprotein, Coronavirus</term>
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<term>Furine</term>
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<term>Serine endopeptidases</term>
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<term>Furine</term>
<term>Glycoprotéine de spicule des coronavirus</term>
<term>Serine endopeptidases</term>
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<front><div type="abstract" xml:lang="en"><p id="Par1">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.</p>
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<author><name sortKey="Elzayat, Mahmoud Tarek" sort="Elzayat, Mahmoud Tarek" uniqKey="Elzayat M" first="Mahmoud Tarek" last="Elzayat">Mahmoud Tarek Elzayat</name>
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<author><name sortKey="Kleine Weber, Hannah" sort="Kleine Weber, Hannah" uniqKey="Kleine Weber H" first="Hannah" last="Kleine-Weber">Hannah Kleine-Weber</name>
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Kellnerweg 4, 37077 Göttingen, Germany</nlm:aff>
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<placeName><region type="land" nuts="2">Basse-Saxe</region>
<settlement type="city">Göttingen</settlement>
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<affiliation wicri:level="3"><nlm:aff id="Aff2">Faculty of Biology and Psychology, Wilhelm-Weber-Str. 2, University Göttingen, 37073 Göttingen, Germany</nlm:aff>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Faculty of Biology and Psychology, Wilhelm-Weber-Str. 2, University Göttingen, 37073 Göttingen</wicri:regionArea>
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<author><name sortKey="Elzayat, Mahmoud Tarek" sort="Elzayat, Mahmoud Tarek" uniqKey="Elzayat M" first="Mahmoud Tarek" last="Elzayat">Mahmoud Tarek Elzayat</name>
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<settlement type="city">Göttingen</settlement>
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<author><name sortKey="Hoffmann, Markus" sort="Hoffmann, Markus" uniqKey="Hoffmann M" first="Markus" last="Hoffmann">Markus Hoffmann</name>
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<settlement type="city">Göttingen</settlement>
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<author><name sortKey="Pohlmann, Stefan" sort="Pohlmann, Stefan" uniqKey="Pohlmann S" first="Stefan" last="Pöhlmann">Stefan Pöhlmann</name>
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Kellnerweg 4, 37077 Göttingen, Germany</nlm:aff>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Kellnerweg 4, 37077 Göttingen</wicri:regionArea>
<placeName><region type="land" nuts="2">Basse-Saxe</region>
<settlement type="city">Göttingen</settlement>
</placeName>
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<affiliation wicri:level="3"><nlm:aff id="Aff2">Faculty of Biology and Psychology, Wilhelm-Weber-Str. 2, University Göttingen, 37073 Göttingen, Germany</nlm:aff>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Faculty of Biology and Psychology, Wilhelm-Weber-Str. 2, University Göttingen, 37073 Göttingen</wicri:regionArea>
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<series><title level="j">Scientific Reports</title>
<idno type="eISSN">2045-2322</idno>
<imprint><date when="2018">2018</date>
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<front><div type="abstract" xml:lang="en"><p id="Par1">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.</p>
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<pubmed><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein.</title>
<author><name sortKey="Kleine Weber, Hannah" sort="Kleine Weber, Hannah" uniqKey="Kleine Weber H" first="Hannah" last="Kleine-Weber">Hannah Kleine-Weber</name>
<affiliation wicri:level="3"><nlm:affiliation>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany.</nlm:affiliation>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
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<author><name sortKey="Elzayat, Mahmoud Tarek" sort="Elzayat, Mahmoud Tarek" uniqKey="Elzayat M" first="Mahmoud Tarek" last="Elzayat">Mahmoud Tarek Elzayat</name>
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<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
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<author><name sortKey="Hoffmann, Markus" sort="Hoffmann, Markus" uniqKey="Hoffmann M" first="Markus" last="Hoffmann">Markus Hoffmann</name>
<affiliation wicri:level="3"><nlm:affiliation>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany. mhoffmann@dpz.eu.</nlm:affiliation>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
<placeName><region type="land" nuts="2">Basse-Saxe</region>
<settlement type="city">Göttingen</settlement>
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</author>
<author><name sortKey="Pohlmann, Stefan" sort="Pohlmann, Stefan" uniqKey="Pohlmann S" first="Stefan" last="Pöhlmann">Stefan Pöhlmann</name>
<affiliation wicri:level="3"><nlm:affiliation>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany. spoehlmann@dpz.eu.</nlm:affiliation>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
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<settlement type="city">Göttingen</settlement>
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<sourceDesc><biblStruct><analytic><title xml:lang="en">Functional analysis of potential cleavage sites in the MERS-coronavirus spike protein.</title>
<author><name sortKey="Kleine Weber, Hannah" sort="Kleine Weber, Hannah" uniqKey="Kleine Weber H" first="Hannah" last="Kleine-Weber">Hannah Kleine-Weber</name>
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<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
<placeName><region type="land" nuts="2">Basse-Saxe</region>
<settlement type="city">Göttingen</settlement>
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<author><name sortKey="Elzayat, Mahmoud Tarek" sort="Elzayat, Mahmoud Tarek" uniqKey="Elzayat M" first="Mahmoud Tarek" last="Elzayat">Mahmoud Tarek Elzayat</name>
<affiliation wicri:level="3"><nlm:affiliation>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany.</nlm:affiliation>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
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<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
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<settlement type="city">Göttingen</settlement>
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<author><name sortKey="Pohlmann, Stefan" sort="Pohlmann, Stefan" uniqKey="Pohlmann S" first="Stefan" last="Pöhlmann">Stefan Pöhlmann</name>
<affiliation wicri:level="3"><nlm:affiliation>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen, Germany. spoehlmann@dpz.eu.</nlm:affiliation>
<country xml:lang="fr">Allemagne</country>
<wicri:regionArea>Infection Biology Unit, German Primate Center - Leibniz Institute for Primate Research, Kellnerweg 4, 37077, Göttingen</wicri:regionArea>
<placeName><region type="land" nuts="2">Basse-Saxe</region>
<settlement type="city">Göttingen</settlement>
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<series><title level="j">Scientific reports</title>
<idno type="eISSN">2045-2322</idno>
<imprint><date when="2018" type="published">2018</date>
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<profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Animals</term>
<term>Cathepsin L (genetics)</term>
<term>Cathepsin L (metabolism)</term>
<term>Chlorocebus aethiops</term>
<term>Coronavirus Infections (virology)</term>
<term>Furin (genetics)</term>
<term>Furin (metabolism)</term>
<term>Humans</term>
<term>Middle East Respiratory Syndrome Coronavirus (physiology)</term>
<term>Mutation</term>
<term>Proteolysis</term>
<term>Serine Endopeptidases (genetics)</term>
<term>Serine Endopeptidases (metabolism)</term>
<term>Spike Glycoprotein, Coronavirus (genetics)</term>
<term>Spike Glycoprotein, Coronavirus (metabolism)</term>
<term>Vero Cells</term>
<term>Virus Internalization</term>
</keywords>
<keywords scheme="KwdFr" xml:lang="fr"><term>Animaux</term>
<term>Cathepsine L (génétique)</term>
<term>Cathepsine L (métabolisme)</term>
<term>Cellules Vero</term>
<term>Coronavirus du syndrome respiratoire du Moyen-Orient (physiologie)</term>
<term>Furine (génétique)</term>
<term>Furine (métabolisme)</term>
<term>Glycoprotéine de spicule des coronavirus (génétique)</term>
<term>Glycoprotéine de spicule des coronavirus (métabolisme)</term>
<term>Humains</term>
<term>Infections à coronavirus (virologie)</term>
<term>Mutation</term>
<term>Protéolyse</term>
<term>Pénétration virale</term>
<term>Serine endopeptidases (génétique)</term>
<term>Serine endopeptidases (métabolisme)</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>Cathepsin L</term>
<term>Furin</term>
<term>Serine Endopeptidases</term>
<term>Spike Glycoprotein, Coronavirus</term>
</keywords>
<keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>Cathepsin L</term>
<term>Furin</term>
<term>Serine Endopeptidases</term>
<term>Spike Glycoprotein, Coronavirus</term>
</keywords>
<keywords scheme="MESH" qualifier="génétique" xml:lang="fr"><term>Cathepsine L</term>
<term>Furine</term>
<term>Glycoprotéine de spicule des coronavirus</term>
<term>Serine endopeptidases</term>
</keywords>
<keywords scheme="MESH" qualifier="métabolisme" xml:lang="fr"><term>Cathepsine L</term>
<term>Furine</term>
<term>Glycoprotéine de spicule des coronavirus</term>
<term>Serine endopeptidases</term>
</keywords>
<keywords scheme="MESH" qualifier="physiologie" xml:lang="fr"><term>Coronavirus du syndrome respiratoire du Moyen-Orient</term>
</keywords>
<keywords scheme="MESH" qualifier="physiology" xml:lang="en"><term>Middle East Respiratory Syndrome Coronavirus</term>
</keywords>
<keywords scheme="MESH" qualifier="virologie" xml:lang="fr"><term>Infections à coronavirus</term>
</keywords>
<keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Coronavirus Infections</term>
</keywords>
<keywords scheme="MESH" xml:lang="en"><term>Animals</term>
<term>Chlorocebus aethiops</term>
<term>Humans</term>
<term>Mutation</term>
<term>Proteolysis</term>
<term>Vero Cells</term>
<term>Virus Internalization</term>
</keywords>
<keywords scheme="MESH" xml:lang="fr"><term>Animaux</term>
<term>Cellules Vero</term>
<term>Humains</term>
<term>Mutation</term>
<term>Protéolyse</term>
<term>Pénétration virale</term>
</keywords>
</textClass>
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<front><div type="abstract" xml:lang="en">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.</div>
</front>
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