Abstract
Viruses use different strategies to interact with their host and perform a successful viral infection that results in the formation of new infectious viral particles and their propagation to new hosts. Understanding how viruses interact with their hosts requires the use of high-resolution techniques for the direct visualization of these interactions. Here electron microscopy (EM) methods are described that allow the 3D ultrastructural analysis of virus-infected cells. These methods can be implemented with light microscopy (LM) to certainly allocate virus-infected cells or cells displaying a specific/interesting phenotype caused by the interaction of viral proteins with the cellular machinery. Some sample preparation procedures where LM is integrated, known as correlative light electron microscopy (CLEM), are also explained in this chapter. All of these methods are applicable to any kind of cultured cells, including influenza virus-infected cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Romero-Brey I, Bartenschlager R (2015) Viral infection at high magnification: 3D electron microscopy methods to analyze the architecture of infected cells. Viruses 7(12):6316–6345. https://doi.org/10.3390/v7122940
Romero-Brey I, Bartenschlager R (2014) Membranous replication factories induced by plus-strand RNA viruses. Viruses 6(7):2826–2857. https://doi.org/10.3390/v6072826
Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P, Fuller SD, Antony C, Krijnse-Locker J, Bartenschlager R (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5(4):365–375. https://doi.org/10.1016/j.chom.2009.03.007
Romero-Brey I, Merz A, Chiramel A, Lee JY, Chlanda P, Haselman U, Santarella-Mellwig R, Habermann A, Hoppe S, Kallis S, Walther P, Antony C, Krijnse-Locker J, Bartenschlager R (2012) Three-dimensional architecture and biogenesis of membrane structures associated with hepatitis C virus replication. PLoS Pathog 8(12):e1003056. https://doi.org/10.1371/journal.ppat.1003056
Miorin L, Romero-Brey I, Maiuri P, Hoppe S, Krijnse-Locker J, Bartenschlager R, Marcello A (2013) Three-dimensional architecture of tick-borne encephalitis virus replication sites and trafficking of the replicated RNA. J Virol 87(11):6469–6481. https://doi.org/10.1128/JVI.03456-12
Chatel-Chaix L, Cortese M, Romero-Brey I, Bender S, Neufeldt CJ, Fischl W, Scaturro P, Schieber N, Schwab Y, Fischer B, Ruggieri A, Bartenschlager R (2016) Dengue virus perturbs mitochondrial morphodynamics to dampen innate immune responses. Cell Host Microbe 20(3):342–356. https://doi.org/10.1016/j.chom.2016.07.008
Fontana J, Steven AC (2015) Influenza virus-mediated membrane fusion: structural insights from electron microscopy. Arch Biochem Biophys 581:86–97. https://doi.org/10.1016/j.abb.2015.04.011
Chlanda P, Schraidt O, Kummer S, Riches J, Oberwinkler H, Prinz S, Krausslich HG, Briggs JA (2015) Structural analysis of the roles of influenza a virus membrane-associated proteins in assembly and morphology. J Virol 89(17):8957–8966. https://doi.org/10.1128/JVI.00592-15
Gavazzi C, Isel C, Fournier E, Moules V, Cavalier A, Thomas D, Lina B, Marquet R (2013) An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus. Nucleic Acids Res 41(2):1241–1254. https://doi.org/10.1093/nar/gks1181
Berger C, Romero-Brey I, Radujkovic D, Terreux R, Zayas M, Paul D, Harak C, Hoppe S, Gao M, Penin F, Lohmann V, Bartenschlager R (2014) Daclatasvir-like inhibitors of NS5A block early biogenesis of hepatitis C virus-induced membranous replication factories, independent of RNA replication. Gastroenterology 147(5):1094–1105 e1025. https://doi.org/10.1053/j.gastro.2014.07.019
Romero-Brey I, Berger C, Kallis S, Kolovou A, Paul D, Lohmann V, Bartenschlager R (2015) NS5A domain 1 and polyprotein cleavage kinetics are critical for induction of double-membrane vesicles associated with hepatitis C virus replication. MBio 6(4):e00759. https://doi.org/10.1128/mBio.00759-15
Spronken MI, Short KR, Herfst S, Bestebroer TM, Vaes VP, van der Hoeven B, Koster AJ, Kremers GJ, Scott DP, Gultyaev AP, Sorell EM, de Graaf M, Barcena M, Rimmelzwaan GF, Fouchier RA (2015) Optimisations and challenges involved in the creation of various bioluminescent and fluorescent influenza a virus strains for in vitro and in vivo applications. PLoS One 10(8):e0133888. https://doi.org/10.1371/journal.pone.0133888
Vale-Costa S, Alenquer M, Sousa AL, Kellen B, Ramalho J, Tranfield EM, Amorim MJ (2016) Influenza A virus ribonucleoproteins modulate host recycling by competing with Rab11 effectors. J Cell Sci 129(8):1697–1710. https://doi.org/10.1242/jcs.188409
Kremer JR, Mastronarde DN, McIntosh JR (1996) Computer visualization of three-dimensional image data using IMOD. J Struct Biol 116(1):71–76. https://doi.org/10.1006/jsbi.1996.0013
Paul-Gilloteaux P, Heiligenstein X, Belle M, Domart MC, Larijani B, Collinson L, Raposo G, Salamero J (2017) eC-CLEM: flexible multidimensional registration software for correlative microscopies. Nat Methods 14(2):102–103. https://doi.org/10.1038/nmeth.4170
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez JY, White DJ, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7):676–682. https://doi.org/10.1038/nmeth.2019
Cardona A, Saalfeld S, Schindelin J, Arganda-Carreras I, Preibisch S, Longair M, Tomancak P, Hartenstein V, Douglas RJ (2012) TrakEM2 software for neural circuit reconstruction. PLoS One 7(6):e38011. https://doi.org/10.1371/journal.pone.0038011
Kreshuk A, Straehle CN, Sommer C, Koethe U, Cantoni M, Knott G, Hamprecht FA (2011) Automated detection and segmentation of synaptic contacts in nearly isotropic serial electron microscopy images. PLoS One 6(10):e24899. https://doi.org/10.1371/journal.pone.0024899
Kreshuk A, Walecki R, Koethe U, Gierthmuehlen M, Plachta D, Genoud C, Haastert-Talini K, Hamprecht FA (2015) Automated tracing of myelinated axons and detection of the nodes of Ranvier in serial images of peripheral nerves. J Microsc 259(2):143–154. https://doi.org/10.1111/jmi.12266
Belevich I, Joensuu M, Kumar D, Vihinen H, Jokitalo E (2016) Microscopy image browser: a platform for segmentation and analysis of multidimensional datasets. PLoS Biol 14(1):e1002340. https://doi.org/10.1371/journal.pbio.1002340
Walther P, Ziegler A (2002) Freeze substitution of high-pressure frozen samples: the visibility of biological membranes is improved when the substitution medium contains water. J Microsc 208(Pt 1):3–10
Webb RI, Schieber NL (2017) Volume scanning Electron microscopy. Serial block-face scanning Electron microscopy and Focussed ion beam scanning Electron microscopy. In: Hanssen E (ed) Three dimensional electron microscopy for cellular biology. Springer, New York
Villinger C, Neusser G, Kranz C, Walther P, Mertens T (2015) 3D analysis of HCMV induced-nuclear membrane structures by FIB/SEM tomography: insight into an unprecedented membrane morphology. Viruses 7(11):5686–5704. https://doi.org/10.3390/v7112900
Robinson DG, Ehlers U, Herken R, Herrmann B, Mayer F, Schuermann F-W (1987) Methods of preparation for Electron microscopy. An introduction for the biomedical sciences. Springer-Verlag, Germany
Acknowledgments
Thank you to the excellent technical support provided by Uta Haselmann and the staff members of the Electron Microscopy Core Facility at the European Molecular Biology Laboratory (EMBL, Heidelberg) and at the University of Heidelberg. Special thanks to Nicole Schieber and Rachel Mellwig (EMBL, Heidelberg) for their critical reading of this manuscript and their helpful suggestions. I also would like to express my gratitude to Prof. Dr. Ralf Bartenschlager, chief of the Molecular Virology Department at the University of Heidelberg for his great support, as well as to all the members of the department. Also to Dr. Yannick Schwab and his team at EMBL, specially to Dr. Anna Steyer.
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
Romero-Brey, I. (2018). 3D Electron Microscopy (EM) and Correlative Light Electron Microscopy (CLEM) Methods to Study Virus-Host Interactions. 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_11
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8678-1_11
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