Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers.
Identifieur interne : 001A62 ( PubMed/Checkpoint ); précédent : 001A61; suivant : 001A63Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers.
Auteurs : Deirdre A. Costello [États-Unis] ; Jean K. Millet ; Chih-Yun Hsia ; Gary R. Whittaker ; Susan DanielSource :
- Biomaterials [ 1878-5905 ] ; 2013.
Descripteurs français
- KwdFr :
- Animaux, Chats, Coronavirus félin (métabolisme), Cricetinae, Fusion membranaire (physiologie), Humains, Lignée cellulaire, Membrane cellulaire (métabolisme), Membrane cellulaire (virologie), Microfluidique, Protéines de l'enveloppe virale (métabolisme), Protéines membranaires (métabolisme), Récepteurs viraux (métabolisme), Techniques d'analyse microfluidique, Traitement d'image par ordinateur.
- MESH :
- métabolisme : Coronavirus félin, Membrane cellulaire, Protéines de l'enveloppe virale, Protéines membranaires, Récepteurs viraux.
- physiologie : Fusion membranaire.
- virologie : Membrane cellulaire.
- Animaux, Chats, Cricetinae, Humains, Lignée cellulaire, Microfluidique, Techniques d'analyse microfluidique, Traitement d'image par ordinateur.
English descriptors
- KwdEn :
- Animals, CD13 Antigens (metabolism), Cats, Cell Line, Cell Membrane (metabolism), Cell Membrane (virology), Coronavirus, Feline (metabolism), Cricetinae, Humans, Image Processing, Computer-Assisted, Membrane Fusion (physiology), Membrane Proteins (metabolism), Microfluidic Analytical Techniques, Microfluidics, Receptors, Virus (metabolism), Viral Envelope Proteins (metabolism).
- MESH :
- chemical , metabolism : CD13 Antigens, Membrane Proteins, Receptors, Virus, Viral Envelope Proteins.
- metabolism : Cell Membrane, Coronavirus, Feline.
- physiology : Membrane Fusion.
- virology : Cell Membrane.
- Animals, Cats, Cell Line, Cricetinae, Humans, Image Processing, Computer-Assisted, Microfluidic Analytical Techniques, Microfluidics.
Abstract
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.
DOI: 10.1016/j.biomaterials.2013.06.034
PubMed: 23886734
Affiliations:
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pubmed:23886734Le document en format XML
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<front><div type="abstract" xml:lang="en">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.</div>
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<Abstract><AbstractText>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.</AbstractText>
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