Abstract
Influenza viruses exhibit a complex life cycle that is still poorly understood. It involves independent replication of each of the eight segments that make up its genome and subsequent coordinated assembly as they egress from the host cell. Fast, time-resolved volumetric live cell imaging offers a powerful tool for understanding the various host mechanisms hijacked by the virus. Here, we describe the methods necessary for generating influenza viruses that carry a fluorescently tagged polymerase complex, infection of biologically relevant cells with these viruses, and finally protocols for live cell imaging and analysis.
Key words
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Eisfeld AJ, Neumann G, Kawaoka Y (2015) At the centre: influenza A virus ribonucleoproteins. Nat Rev Microbiol 13(1):28–41. https://doi.org/10.1038/nrmicro3367
Yamauchi Y, Helenius A (2013) Virus entry at a glance. J Cell Sci 126(Pt 6):1289–1295. https://doi.org/10.1242/jcs.119685
Brauer R, Chen P (2015) Influenza virus propagation in embryonated chicken eggs. J Vis Exp (97). https://doi.org/10.3791/52421
Dos Santos Afonso E, Escriou N, Leclercq I, van der Werf S, Naffakh N (2005) The generation of recombinant influenza A viruses expressing a PB2 fusion protein requires the conservation of a packaging signal overlapping the coding and noncoding regions at the 5′ end of the PB2 segment. Virology 341(1):34–46. https://doi.org/10.1016/j.virol.2005.06.040
Avilov SV, Moisy D, Munier S, Schraidt O, Naffakh N, Cusack S (2012) Replication-competent influenza A virus that encodes a split-green fluorescent protein-tagged PB2 polymerase subunit allows live-cell imaging of the virus life cycle. J Virol 86(3):1433–1448. https://doi.org/10.1128/Jvi.05820-11
Lakdawala SS, Wu Y, Wawrzusin P, Kabat J, Broadbent AJ, Lamirande EW, Fodor E, Altan-Bonnet N, Shroff H, Subbarao K (2014) Influenza A virus assembly intermediates fuse in the cytoplasm. PLoS Pathog 10(3):e1003971. https://doi.org/10.1371/journal.ppat.1003971
Murray ZBJI (2011) Live imaging of Caenorhabditis elegans embryogenesis. In: Sharpe J, Wong RO (eds) Imaging in developmental biology: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Cole RW, Jinadasa T, Brown CM (2011) Measuring and interpreting point spread functions to determine confocal microscope resolution and ensure quality control. Nat Protoc 6(12):1929–1941. https://doi.org/10.1038/nprot.2011.407
Wu Y, Wawrzusin P, Senseney J, Fischer RS, Christensen R, Santella A, York AG, Winter PW, Waterman CM, Bao Z, Colon-Ramos DA, McAuliffe M, Shroff H (2013) Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy. Nat Biotechnol 31(11):1032–1038. https://doi.org/10.1038/nbt.2713
Hoffmann E, Neumann G, Kawaoka Y, Hobom G, Webster RG (2000) A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97(11):6108–6113. https://doi.org/10.1073/pnas.100133697
Fodor E, Smith M (2004) The PA subunit is required for efficient nuclear accumulation of the PB1 subunit of the influenza A virus RNA polymerase complex. J Virol 78(17):9144–9153. https://doi.org/10.1128/JVI.78.17.9144-9153.2004
Kumar A, Wu Y, Christensen R, Chandris P, Gandler W, McCreedy E, Bokinsky A, Colon-Ramos DA, Bao Z, McAuliffe M, Rondeau G, Shroff H (2014) Dual-view plane illumination microscopy for rapid and spatially isotropic imaging. Nat Protoc 9(11):2555–2573. https://doi.org/10.1038/nprot.2014.172
ASI diSPIM Assembly (2015) YouTube. https://youtu.be/TAgbr6IrTqw
diSPIM Alignment Video (2015) YouTube. https://youtu.be/qnOrg30NNuE
Zerial M. MotionTracking. http://motiontracking.mpi-cbg.de/get/
Jaqaman K, Loerke D, Mettlen M, Kuwata H, Grinstein S, Schmid SL, Danuser G (2008) Robust single-particle tracking in live-cell time-lapse sequences. Nat Methods 5(8):695–702
Directed and Undirected Graphs. https://www.mathworks.com/help/matlab/math/directed-and-undirected-graphs.html?requestedDomain=www.mathworks.com
Bertrand E, Chartrand P, Schaefer M, Shenoy SM, Singer RH, Long RM (1998) Localization of ASH1 mRNA particles in living yeast. Mol Cell 2(4):437–445
York A, Hengrung N, Vreede FT, Huiskonen JT, Fodor E (2013) Isolation and characterization of the positive-sense replicative intermediate of a negative-strand RNA virus. Proc Natl Acad Sci U S A 110(45):E4238–E4245. https://doi.org/10.1073/pnas.1315068110
Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333(6042):642–646. https://doi.org/10.1126/science.1207339
Santangelo PJ, Lifland AW, Curt P, Sasaki Y, Bassell GJ, Lindquist ME, Crowe JE Jr (2009) Single molecule-sensitive probes for imaging RNA in live cells. Nat Methods 6(5):347–349. https://doi.org/10.1038/nmeth.1316
WHO Global Influenza Surveillance Network (2011) Manual for the laboratory diagnosis and virological surveillance of influenza. WHO Press, Geneva
Eisfeld AJ, Neumann G, Kawaoka Y (2014) Influenza A virus isolation, culture and identification. Nat Protoc 9(11):2663–2681. https://doi.org/10.1038/nprot.2014.180
Kamiyama D, Sekine S, Barsi-Rhyne B, Hu J, Chen B, Gilbert LA, Ishikawa H, Leonetti MD, Marshall WF, Weissman JS, Huang B (2016) Versatile protein tagging in cells with split fluorescent protein. Nat Commun 7:11046. https://doi.org/10.1038/ncomms11046
Long F, Zeng S, Huang ZL (2012) Localization-based super-resolution microscopy with an sCMOS camera part II: experimental methodology for comparing sCMOS with EMCCD cameras. Opt Express 20(16):17741–17759. https://doi.org/10.1364/OE.20.017741
Aitken CE, Marshall RA, Puglisi JD (2008) An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. Biophys J 94(5):1826–1835. https://doi.org/10.1529/biophysj.107.117689
Schmied C, Steinbach P, Pietzsch T, Preibisch S, Tomancak P (2016) An automated workflow for parallel processing of large multiview SPIM recordings. Bioinformatics 32(7):1112–1114. https://doi.org/10.1093/bioinformatics/btv706
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
Bhagwat, A.R., Le Sage, V., Lakdawala, S.S. (2018). Live Imaging of Influenza Viral Ribonucleoproteins Using Light-Sheet Microscopy. 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_15
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8678-1_15
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