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Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers

Paper ID Volume ID Publish Year Pages File Format Full-Text
6381 488 2013 10 PDF Available
Title
Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers
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.

Keywords
Supported lipid bilayers; Membrane fusion; Coronavirus; Fusion kinetics
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Single particle assay of coronavirus membrane fusion with proteinaceous receptor-embedded supported bilayers
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Publisher
Database: Elsevier - ScienceDirect
Journal: Biomaterials - Volume 34, Issue 32, October 2013, Pages 7895–7904
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering
Get Full-Text Now
Don't Miss Today's Special Offer
Price was $35.95
You save - $31
Price after discount Only $4.95
100% Money Back Guarantee
Full-text PDF Download
Online Support
Any Questions? feel free to contact us