Abstract
Influenza A virus is an enveloped virus with a segmented genome consisting of eight negative-sense, single-stranded RNAs. Accumulating evidence has revealed that influenza viruses selectively package their genomes. However, less is known about how different viral RNA segments are selected for incorporation into progeny virions. Understanding the trafficking routes and assembly process of various viral RNA segments during infection will shed light on the mechanisms of selective genome packaging for influenza A viruses. This chapter describes the single-molecule sensitivity RNA fluorescence in situ hybridization assay (smFISH) for influenza viral RNAs, a method used to analyze the distributions and trafficking of viral RNAs in infected cells with segment specificity. Hybridization using 20 or more short fluorescently labeled DNA probes allows the detection of viral RNAs with single-molecule sensitivity. The following imaging analyses provide information regarding quantitative measurements of vRNA abundance and the relative positions of the different viral RNA segments in cells. This chapter also includes a protocol for combining immunofluorescence techniques with smFISH, which is useful to analyze the positions of viral RNAs relative to viral/cellular proteins in infected cells.
Key words
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Shaw ML, Palese P (2007) Orthomyxoviridae: the viruses and their replication. In: Knipe DM, Howley PM (eds) Fields Virology, 5th edn. Lippincott, Williams and Wilkins, Philadelphia, pp 1647–1689
Lakdawala SS, Fodor E, Subbarao K (2016) Moving on out: transport and packaging of influenza viral RNA into virions. Annu Rev Virol 3(1):411–427. https://doi.org/10.1146/annurev-virology-110615-042345
Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, Sessions WM, Xu X, Skepner E, Deyde V, Okomo-Adhiambo M, Gubareva L, Barnes J, Smith CB, Emery SL, Hillman MJ, Rivailler P, Smagala J, de Graaf M, Burke DF, Fouchier RA, Pappas C, Alpuche-Aranda CM, Lopez-Gatell H, Olivera H, Lopez I, Myers CA, Faix D, Blair PJ, Yu C, Keene KM, Dotson PD Jr, Boxrud D, Sambol AR, Abid SH, St George K, Bannerman T, Moore AL, Stringer DJ, Blevins P, Demmler-Harrison GJ, Ginsberg M, Kriner P, Waterman S, Smole S, Guevara HF, Belongia EA, Clark PA, Beatrice ST, Donis R, Katz J, Finelli L, Bridges CB, Shaw M, Jernigan DB, Uyeki TM, Smith DJ, Klimov AI, Cox NJ (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325(5937):197–201. https://doi.org/10.1126/science.1176225
Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, Pybus OG, Ma SK, Cheung CL, Raghwani J, Bhatt S, Peiris JS, Guan Y, Rambaut A (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459(7250):1122–1125. https://doi.org/10.1038/nature08182
Fujii K, Fujii Y, Noda T, Muramoto Y, Watanabe T, Takada A, Goto H, Horimoto T, Kawaoka Y (2005) Importance of both the coding and the segment-specific noncoding regions of the influenza A virus NS segment for its efficient incorporation into virions. J Virol 79(6):3766–3774. https://doi.org/10.1128/JVI.79.6.3766-3774.2005
Marsh GA, Hatami R, Palese P (2007) Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions. J Virol 81(18):9727–9736. https://doi.org/10.1128/JVI.01144-07
Hutchinson EC, Curran MD, Read EK, Gog JR, Digard P (2008) Mutational analysis of cis-acting RNA signals in segment 7 of influenza A virus. J Virol 82(23):11869–11879. https://doi.org/10.1128/JVI.01634-08
Marsh GA, Rabadan R, Levine AJ, Palese P (2008) Highly conserved regions of influenza a virus polymerase gene segments are critical for efficient viral RNA packaging. J Virol 82(5):2295–2304. https://doi.org/10.1128/JVI.02267-07
Gao Q, Chou YY, Doganay S, Vafabakhsh R, Ha T, Palese P (2012) The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging. J Virol 86(13):7043–7051. https://doi.org/10.1128/JVI.00662-12
Sherry L, Punovuori K, Wallace LE, Prangley E, DeFries S, Jackson D (2016) Identification of cis-acting packaging signals in the coding regions of the influenza B virus HA gene segment. J Gen Virol 97(2):306–315. https://doi.org/10.1099/jgv.0.000358
Noda T, Sugita Y, Aoyama K, Hirase A, Kawakami E, Miyazawa A, Sagara H, Kawaoka Y (2012) Three-dimensional analysis of ribonucleoprotein complexes in influenza A virus. Nat Commun 3:639. https://doi.org/10.1038/ncomms1647
Nakatsu S, Sagara H, Sakai-Tagawa Y, Sugaya N, Noda T, Kawaoka Y (2016) Complete and incomplete genome packaging of influenza A and B viruses. MBio 7(5):e01248. https://doi.org/10.1128/mBio.01248-16
Hutchinson EC, von Kirchbach JC, Gog JR, Digard P (2010) Genome packaging in influenza A virus. J Gen Virol 91(Pt 2):313–328. https://doi.org/10.1099/vir.0.017608-0
Isel C, Munier S, Naffakh N (2016) Experimental approaches to study genome packaging of influenza a viruses. Viruses 8(8). https://doi.org/10.3390/v8080218
Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. https://doi.org/10.1038/nmeth.1253
Chou YY, Heaton NS, Gao Q, Palese P, Singer RH, Lionnet T (2013) Colocalization of different influenza viral RNA segments in the cytoplasm before viral budding as shown by single-molecule sensitivity FISH analysis. PLoS Pathog 9(5):e1003358. https://doi.org/10.1371/journal.ppat.1003358
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
Lionnet T, Czaplinski K, Darzacq X, Shav-Tal Y, Wells AL, Chao JA, Park HY, de Turris V, Lopez-Jones M, Singer RH (2011) A transgenic mouse for in vivo detection of endogenous labeled mRNA. Nat Methods 8(2):165–170. https://doi.org/10.1038/nmeth.1551
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
Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini DM (2006) CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biol 7(10):R100. https://doi.org/10.1186/gb-2006-7-10-r100
Author information
Authors and Affiliations
Corresponding authors
Editor information
Editors and Affiliations
1 Electronic Supplementary Material
Supplementary Table 1
An example probe set designed for the PB1 segment of influenza A virus A/Puerto Rico/8/34, PR8 strain (DOCX 114 kb)
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Chou, Yy., Lionnet, T. (2018). Single-Molecule Sensitivity RNA FISH Analysis of Influenza Virus Genome Trafficking. 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_10
Download citation
DOI: https://doi.org/10.1007/978-1-4939-8678-1_10
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