Abstract
Double-stranded RNA (dsRNA) is the genomic material or replicate intermediate of RNA viruses, and is also a crucial signal molecule to trigger innate immune response and RNA silencing in eukaryotic organisms. Studying the subcellular localization and dynamic of dsRNA provides significant information for understanding RNA virus replication, host responses to virus infection, and viral diagnosis. Several antibody-dependent or -independent methods have been developed to in vivo or in vitro visualize dsRNA in cells. Here, we provide a step-by-step protocol for efficiently visualizing the distribution and dynamics of dsRNA in living plant cells.
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References
Jacobs BL, Langland JO (1996) When two strands are better than one: the mediators and modulators of the cellular responses to double-stranded RNA. Virology 219:339–349
Dewitte-Orr SJ, Mossman KL (2011) The antiviral effects of extracellular dsRNA. In: Mossman KL (ed) Viruses and interferon: current research. Academic Press, Caister, pp 1–18
Maida Y, Masutomi K (2011) RNA-dependent RNA polymerases in RNA silencing. Biol Chem 392:299–304
Qi X, Bao FS, Xie Z (2009) Small RNA deep sequencing reveals role for Arabidopsis thaliana RNA-dependent RNA polymerases in viral siRNA biogenesis. PLoS One 4:e4971
Sijen T, Fleenor J, Simmer F et al (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465–476
Rosok O, Sioud M (2004) Systematic identification of sense-antisense transcripts in mammalian cells. Nat Biotechnol 22:104–108
Vance V, Vaucheret H (2001) RNA silencing in plants—defense and counterdefense. Science 292:2277–2280
Peisley A, Hur S (2013) Multi-level regulation of cellular recognition of viral dsRNA. Cell Mol Life Sci 70:1949–1963
Niehl A, Wyrsch I, Boller T et al (2016) Double-stranded RNAs induce a pattern-triggered immune signaling pathway in plants. New Phytol 211:1008–1019
Niehl A, Heinlein M (2009) Perception of double-stranded RNA in plant antiviral immunity. Mol Plant Pathol 20:1203–1210
Ishibashi K, Saruta M, Shimizu T et al (2019) Soybean antiviral immunity conferred by dsRNase targets the viral replication complex. Nat Commun 10:4033
Cuellar WJ, Kreuze JF, Rajamäki M-L et al (2009) Elimination of antiviral defense by viral RNase III. Proc Natl Acad Sci U S A 106:10354–10358
Triantafilou K, Vakakis E, Kar S et al (2012) Visualisation of direct interaction of MDA5 and the dsRNA replicative intermediate form of positive strand RNA viruses. J Cell Sci 125:4761–4769
Son K-N, Liang Z, Lipton HL (2015) Double-strand RNA is detected by immunofluorescence analysis in RNA and DNA virus infections including those by negative-strand RNA viruses. J Virol 89:9383–8392
Weber F, Wagner V, Rasmussen SB et al (2006) Double-stranded RNA is produced by positive-strand RNA viruses and DNA viruses but not in detectable amounts by negative-strand RNA viruses. J Virol 80:5059–5064
Stollar BD, Stollar V (1970) Immunofluorescent demonstration of double-stranded RNA in the cytoplasm of sindbis virus-infected cells. Virology 42:276–280
Schonborn J, Oberstraβ J, Breyel E et al (1991) Monoclonal antibodies to double-stranded RNA as probes of RNA structure in crude nucleic acid extracts. Nucleic Acids Res 19:2993–3000
Bolten R, Egger D, Gosert R et al (1998) Intracellular localization of poliovirus plus- and minus-strand RNA visualized by strand-specific fluorescent in situ hybridization. J Virol 72:8578–8585
Monsion B, Incarbone M, Hleibieh K, Poignavent V, Ghannam A, Dunoyer P, Daeffler L, Tilsner J, Ritzenthaler C (2018) Efficient detection of long dsRNA in vitro and in vivo using the dsRNA binding domain from FHV B2 protein. Front Plant Sci 9:70
Cheng X, Deng P, Cui H et al (2015) Visualizing double-stranded RNA distribution and dynamics in living cells by dsRNA binding-dependent fluorescence complementation. Virology 485:439–451
Cotton S, Grangeon R, Thivierge K et al (2009) Turnip mosaic virus RNA replication complex vesicles are mobile, align with microfilaments, and are each derived from a single viral genome. J Virol 83:10460–10471
Lu Q, Tang X, Tian G et al (2010) Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin. Plant J 61:259–270
Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (31671998) and the Natural Science Foundation of Heilongjiang Province (ZD2018002) to XC.
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Cheng, X., Luan, Y., Wang, A. (2022). In Vivo Detection of Double-Stranded RNA by dRBFC Assay. In: Wang, A., Li, Y. (eds) Plant Virology . Methods in Molecular Biology, vol 2400. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1835-6_1
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DOI: https://doi.org/10.1007/978-1-0716-1835-6_1
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-1835-6
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