Abstract
Influenza virus enters host cells by fusion of viral and endosomal membranes mediated by the influenza hemagglutinin (HA). The pathway of HA-catalyzed fusion has been widely investigated in influenza virus membrane fusion with liposomes. In this chapter we describe methodology for studying the virus-liposome fusion system using a combination of fluorescence dequenching assays and cryo-electron tomography (cryo-ET). In particular, the fluorescence dequenching is used to monitor the efficiency of membrane fusion between whole influenza viruses labeled with a lipophilic dye (DiD) in the membrane and liposomes labeled with a water-soluble dye (sulforhodamine B). By simultaneously monitoring the two fluorescent signals, we can determine the relative time scales of liposomal content leakage or transfer vs. lipid merging. In addition, cryo-ET offers a means of imaging three-dimensional snapshots of different stages of virus-liposome fusion such as prefusion, fusion intermediates, and postfusion.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
White JM, Delos SE, Brecher M, Schornberg K (2008) Structures and mechanisms of viral membrane fusion proteins: multiple variations on a common theme. Crit Rev Biochem Mol Biol 43:189–219
Garcia NK, Guttman M, Ebner JL, Lee KK (2015) Dynamic changes during acid-induced activation of influenza hemagglutinin. Structure 23:665–676
Skehel JJ, Wiley DC (2000) Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin. Annu Rev Biochem 69:531–569
Harrison SC (2015) Viral membrane fusion. Virology 479-480:498–507. https://doi.org/10.1016/j.virol.2015.03.043
Lakadamyali M, Rust MJ (2003) Visualizing infection of individual influenza viruses. Proc Natl Acad Sci U S A 100(16):9280–9285
Floyd DL, Ragains JR, Skehel JJ (2008) Single-particle kinetics of influenza virus membrane fusion. Proc Natl Acad Sci U S A 105(40):15382–15387
Ivanovic T, Choi JL, Whelan SP et al (2013) Influenza-virus membrane fusion by cooperative fold-back of stochastically induced hemagglutinin intermediates. eLife 2:e00333
Subramaniam S, Bartesaghi A, Liu J et al (2007) Electron tomography of viruses. Curr Opin Struct Biol 17:596–602
Lee KK, Gui L (2016) Dissecting virus infectious cycles by cryo-electron microscopy. PLoS Pathog 12:e1005625–e1005628
Baumeister W, Grimm R, Walz J (1999) Electron tomography of molecules and cells. Trends Cell Biol 9:81–85
Gui L, Ebner JL, Mileant A et al (2016) Visualization and sequencing of membrane remodeling leading to influenza virus fusion. J Virol 90(15):6948–6962
Chen PS, Toribara TY, Warner H (1956) Microdetermination of phosphorus. Anal Chem 28:1756–1758
Suloway C, Shi J, Cheng A et al (2009) Fully automated, sequential tilt-series acquisition with Leginon. J Struct Biol 167(1):11–18
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116:71–76
Karmakar S, Sarangi BR, Raghunathan VA (2006) Phase behavior of lipid–cholesterol membranes. Solid State Commun 139:630–634
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Gui, L., Lee, K.K. (2018). Influenza Virus-Liposome Fusion Studies Using Fluorescence Dequenching and Cryo-electron Tomography. In: Yamauchi, Y. (eds) Influenza Virus. Methods in Molecular Biology, vol 1836. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8678-1_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8678-1_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-8677-4
Online ISBN: 978-1-4939-8678-1
eBook Packages: Springer Protocols