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Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers
Total internal reflection microscopy combined with microfluidics and supported bilayers is a powerful, single particle tracking (SPT) platform for host-pathogen membrane fusion studies. But one major inadequacy of this platform has been capturing the complexity of the cell membrane, including membra...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Published by Elsevier Ltd.
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111216/ https://www.ncbi.nlm.nih.gov/pubmed/23886734 http://dx.doi.org/10.1016/j.biomaterials.2013.06.034 |
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author | Costello, Deirdre A. Millet, Jean K. Hsia, Chih-Yun Whittaker, Gary R. Daniel, Susan |
author_facet | Costello, Deirdre A. Millet, Jean K. Hsia, Chih-Yun Whittaker, Gary R. Daniel, Susan |
author_sort | Costello, Deirdre A. |
collection | PubMed |
description | Total internal reflection microscopy combined with microfluidics and supported bilayers is a powerful, single particle tracking (SPT) platform for host-pathogen membrane fusion studies. But one major inadequacy of this platform has been capturing the complexity of the cell membrane, including membrane proteins. Because of this, viruses requiring proteinaceous receptors, or other unknown cellular co-factors, have been precluded from study. Here we describe a general method to integrate proteinaceous receptors and cellular components into supported bilayers for SPT fusion studies. This method is general to any enveloped virus-host cell pair, but demonstrated here for feline coronavirus (FCoV). Supported bilayers are formed from mammalian cell membrane vesicles that express feline aminopeptidase N (the viral receptor) using a cell blebbing technique. SPT is then used to identify fusion intermediates and measure membrane fusion kinetics for FCoV. Overall, the fusion results recapitulate what is observed in vivo, that coronavirus entry requires binding to specific receptors, a low-pH environment, and that membrane fusion is receptor- and protease-dependent. But this method also provides quantitative kinetic rate parameters for intermediate steps in the coronavirus fusion pathway, which to our knowledge have not been obtained before. Moreover, the platform offers versatile, precise control over the sequence of triggers for fusion; these triggers may define the fusion pathway, tissue tropism, and pathogenicity of coronaviruses. Systematically varying these triggers in this platform provides a new route to study how viruses rapidly adapt to other hosts, and to identify factors that led to the emergence of zoonotic viruses, such as human SARS-CoV and the newly emerging human MERS-CoV. |
format | Online Article Text |
id | pubmed-7111216 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Published by Elsevier Ltd. |
record_format | MEDLINE/PubMed |
spelling | pubmed-71112162020-04-02 Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers Costello, Deirdre A. Millet, Jean K. Hsia, Chih-Yun Whittaker, Gary R. Daniel, Susan Biomaterials Article Total internal reflection microscopy combined with microfluidics and supported bilayers is a powerful, single particle tracking (SPT) platform for host-pathogen membrane fusion studies. But one major inadequacy of this platform has been capturing the complexity of the cell membrane, including membrane proteins. Because of this, viruses requiring proteinaceous receptors, or other unknown cellular co-factors, have been precluded from study. Here we describe a general method to integrate proteinaceous receptors and cellular components into supported bilayers for SPT fusion studies. This method is general to any enveloped virus-host cell pair, but demonstrated here for feline coronavirus (FCoV). Supported bilayers are formed from mammalian cell membrane vesicles that express feline aminopeptidase N (the viral receptor) using a cell blebbing technique. SPT is then used to identify fusion intermediates and measure membrane fusion kinetics for FCoV. Overall, the fusion results recapitulate what is observed in vivo, that coronavirus entry requires binding to specific receptors, a low-pH environment, and that membrane fusion is receptor- and protease-dependent. But this method also provides quantitative kinetic rate parameters for intermediate steps in the coronavirus fusion pathway, which to our knowledge have not been obtained before. Moreover, the platform offers versatile, precise control over the sequence of triggers for fusion; these triggers may define the fusion pathway, tissue tropism, and pathogenicity of coronaviruses. Systematically varying these triggers in this platform provides a new route to study how viruses rapidly adapt to other hosts, and to identify factors that led to the emergence of zoonotic viruses, such as human SARS-CoV and the newly emerging human MERS-CoV. Published by Elsevier Ltd. 2013-10 2013-07-22 /pmc/articles/PMC7111216/ /pubmed/23886734 http://dx.doi.org/10.1016/j.biomaterials.2013.06.034 Text en Copyright © 2013 Published by Elsevier Ltd. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active. |
spellingShingle | Article Costello, Deirdre A. Millet, Jean K. Hsia, Chih-Yun Whittaker, Gary R. Daniel, Susan Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
title | Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
title_full | Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
title_fullStr | Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
title_full_unstemmed | Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
title_short | Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
title_sort | single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7111216/ https://www.ncbi.nlm.nih.gov/pubmed/23886734 http://dx.doi.org/10.1016/j.biomaterials.2013.06.034 |
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