Abstract
MicroRNAs are small, noncoding RNAs that posttranscriptionally regulate gene expression. The discovery of this relatively new mode of gene regulation as well as studies showing the prognostic value of viral and cellular miRNAs as biomarkers, such as in cancer progression, has stimulated the development of many methods to characterize miRNAs. EBV encodes 25 viral precursor microRNAs within its genome that are expressed during lytic and latent infection. In addition to viral miRNAs, EBV infection induces the expression of specific cellular oncogenic miRNAs, such as miR-155, miR-146a, miR-21, and others, that can contribute to the persistence of latently infected cells. This chapter describes several current techniques used to identify and detect the expression of viral and cellular miRNAs in EBV-infected cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cameron JE, Fewell C, Yin Q et al (2008) Epstein-Barr virus growth/latency III program alters cellular microRNA expression. Virology 382:257–266
Cameron JE, Yin Q, Fewell C et al (2008) Epstein-Barr virus latent membrane protein 1 induces cellular MicroRNA miR-146a, a modulator of lymphocyte signaling pathways. J Virol 82:1946–1958
Jiang J, Lee EJ, Schmittgen TD (2006) Increased expression of microRNA-155 in Epstein-Barr virus transformed lymphoblastoid cell lines. Genes Chromosomes Cancer 45:103–106
Marquitz AR, Mathur A, Chugh PE et al (2014) Expression profile of microRNAs in Epstein-Barr virus-infected AGS gastric carcinoma cells. J Virol 88:1389–1393
Skalsky RL, Cullen BR (2010) Viruses, microRNAs, and host interactions. Annu Rev Microbiol 64:123–141
Forte E, Luftig MA (2011) The role of microRNAs in Epstein-Barr virus latency and lytic reactivation. Microbes Infect 13:1156–1167
Mrazek J, Kreutmayer SB, Grasser FA et al (2007) Subtractive hybridization identifies novel differentially expressed ncRNA species in EBV-infected human B cells. Nucleic Acids Res 35:e73
Linnstaedt SD, Gottwein E, Skalsky RL et al (2010) Virally induced cellular miR-155 plays a key role in B-cell immortalization by EBV. J Virol 84:11670–11678
Navarro A, Gaya A, Martinez A et al (2008) MicroRNA expression profiling in classic Hodgkin lymphoma. Blood 111:2825–2832
Leucci E, Onnis A, Cocco M et al (2010) B-cell differentiation in EBV-positive Burkitt lymphoma is impaired at posttranscriptional level by miRNA-altered expression. Int J Cancer 126:1316–1326
Calin GA, Ferracin M, Cimmino A et al (2005) A MicroRNA signature associated with prognosis and progression in chronic lymphocytic leukemia. N Engl J Med 353:1793–1801
Zhang G, Zong J, Lin S et al (2015) Circulating Epstein-Barr virus microRNAs miR-BART7 and miR-BART13 as biomarkers for nasopharyngeal carcinoma diagnosis and treatment. Int J Cancer 136:E301–E312
Pfeffer S, Zavolan M, Grasser FA et al (2004) Identification of virus-encoded microRNAs. Science 304:734–736
Grundhoff A, Sullivan CS, Ganem D (2006) A combined computational and microarray-based approach identifies novel microRNAs encoded by human gamma-herpesviruses. RNA 12:733–750
Cai X, Schafer A, Lu S et al (2006) Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed. PLoS Pathog 2:e23
Chen SJ, Chen GH, Chen YH et al (2010) Characterization of Epstein-Barr virus miRNAome in nasopharyngeal carcinoma by deep sequencing. PLoS One 5:e12745
Zhu JY, Pfuhl T, Motsch N et al (2009) Identification of novel Epstein-Barr virus microRNA genes from nasopharyngeal carcinomas. J Virol 83:3333–3341
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136:215–233
Ambros V (2004) The functions of animal microRNAs. Nature 431:350–355
Skalsky RL, Corcoran DL, Gottwein E et al (2012) The viral and cellular microRNA targetome in lymphoblastoid cell lines. PLoS Pathog 8:e1002484
Skalsky RL, Kang D, Linnstaedt SD et al (2014) Evolutionary conservation of primate lymphocryptovirus microRNA targets. J Virol 88:1617–1635
Gottwein E, Corcoran DL, Mukherjee N et al (2011) Viral microRNA targetome of KSHV-infected primary effusion lymphoma cell lines. Cell Host Microbe 10:515–526
Gottwein E, Cullen BR (2007) Protocols for expression and functional analysis of viral microRNAs. Methods Enzymol 427:229–243
Dolken L, Malterer G, Erhard F et al (2010) Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Cell Host Microbe 7:324–334
Riley KJ, Rabinowitz GS, Yario TA et al (2012) EBV and human microRNAs co-target oncogenic and apoptotic viral and human genes during latency. EMBO J 31:2207–2221
Kang D, Skalsky RL, Cullen BR (2015) EBV BART MicroRNAs target multiple pro-apoptotic cellular genes to promote epithelial cell survival. PLoS Pathog 11:e1004979
Hafner M, Landthaler M, Burger L, et al. (2010) PAR-CliP-a method to identify transcriptome-wide the binding sites of RNA binding proteins. J Vis Exp 41: pii: 2034
Flores O, Kennedy EM, Skalsky RL et al (2014) Differential RISC association of endogenous human microRNAs predicts their inhibitory potential. Nucleic Acids Res 42:4629–4639
Hook LM, Landais I, Hancock MH et al (2014) Techniques for characterizing cytomegalovirus-encoded miRNAs. Methods Mol Biol 1119:239–265
Keene JD, Komisarow JM, Friedersdorf MB (2006) RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts. Nat Protoc 1:302–307
Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25
Friedlander MR, Mackowiak SD, Li N et al (2012) miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades. Nucleic Acids Res 40:37–52
Robinson MD, McCarthy DJ, Smyth GK (2010) edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26:139–140
Anders S, McCarthy DJ, Chen Y et al (2013) Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat Protoc 8:1765–1786
Manzano M, Forte E, Raja AN et al (2015) Divergent target recognition by coexpressed 5′-isomiRs of miR-142-3p and selective viral mimicry. RNA 21:1606–1620
Manzano M, Shamulailatpam P, Raja AN et al (2013) Kaposi’s sarcoma-associated herpesvirus encodes a mimic of cellular miR-23. J Virol 87:11821–11830
Motsch N, Alles J, Imig J et al (2012) MicroRNA profiling of Epstein-Barr virus-associated NK/T-cell lymphomas by deep sequencing. PLoS One 7:e42193
Feederle R, Haar J, Bernhardt K et al (2011) The members of an Epstein-Barr virus microRNA cluster cooperate to transform B lymphocytes. J Virol 85:9801–9810
Feederle R, Linnstaedt SD, Bannert H et al (2011) A viral microRNA cluster strongly potentiates the transforming properties of a human herpesvirus. PLoS Pathog 7:e1001294
Acknowledgments
R.L.S. is supported by NIH grant K99-CA175181. The author thanks the current and former members of Dr. Bryan Cullen’s laboratory at Duke University, the members of Dr. Jack Keene’s lab at Duke University, and the members of Dr. Jay Nelson’s laboratory at Oregon Health and Science University for the discussions, troubleshooting, and optimizing protocols over the years.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media New York
About this protocol
Cite this protocol
Skalsky, R.L. (2017). Analysis of Viral and Cellular MicroRNAs in EBV-Infected Cells. In: Minarovits, J., Niller, H. (eds) Epstein Barr Virus. Methods in Molecular Biology, vol 1532. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6655-4_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6655-4_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6653-0
Online ISBN: 978-1-4939-6655-4
eBook Packages: Springer Protocols