Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fus...

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Autores principales:	Christine Burkard, Monique H Verheije, Oliver Wicht, Sander I van Kasteren, Frank J van Kuppeveld, Bart L Haagmans, Lucas Pelkmans, Peter J M Rottier, Berend Jan Bosch, Cornelis A M de Haan
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2014
Materias:	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
Acceso en línea:	https://doaj.org/article/1d8f17335c7942ac9824e89e4be9d62e
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spelling	oai:doaj.org-article:1d8f17335c7942ac9824e89e4be9d62e2021-11-25T05:45:52ZCoronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.1553-73661553-737410.1371/journal.ppat.1004502https://doaj.org/article/1d8f17335c7942ac9824e89e4be9d62e2014-11-01T00:00:00Zhttps://doi.org/10.1371/journal.ppat.1004502https://doaj.org/toc/1553-7366https://doaj.org/toc/1553-7374Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.Christine BurkardMonique H VerheijeOliver WichtSander I van KasterenFrank J van KuppeveldBart L HaagmansLucas PelkmansPeter J M RottierBerend Jan BoschCornelis A M de HaanPublic Library of Science (PLoS)articleImmunologic diseases. AllergyRC581-607Biology (General)QH301-705.5ENPLoS Pathogens, Vol 10, Iss 11, p e1004502 (2014)
institution	DOAJ
collection	DOAJ
language	EN
topic	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5
spellingShingle	Immunologic diseases. Allergy RC581-607 Biology (General) QH301-705.5 Christine Burkard Monique H Verheije Oliver Wicht Sander I van Kasteren Frank J van Kuppeveld Bart L Haagmans Lucas Pelkmans Peter J M Rottier Berend Jan Bosch Cornelis A M de Haan Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
description	Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.
format	article
author	Christine Burkard Monique H Verheije Oliver Wicht Sander I van Kasteren Frank J van Kuppeveld Bart L Haagmans Lucas Pelkmans Peter J M Rottier Berend Jan Bosch Cornelis A M de Haan
author_facet	Christine Burkard Monique H Verheije Oliver Wicht Sander I van Kasteren Frank J van Kuppeveld Bart L Haagmans Lucas Pelkmans Peter J M Rottier Berend Jan Bosch Cornelis A M de Haan
author_sort	Christine Burkard
title	Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
title_short	Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
title_full	Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
title_fullStr	Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
title_full_unstemmed	Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
title_sort	coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.
publisher	Public Library of Science (PLoS)
publishDate	2014
url	https://doaj.org/article/1d8f17335c7942ac9824e89e4be9d62e
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Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

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