Cellular and Molecular Life Sciences

, Volume 70, Issue 2, pp 277–291 | Cite as

MicroRNA-203 enhances Coxsackievirus B3 replication through targeting zinc finger protein-148

  • Maged Gomaa Hemida
  • Xin Ye
  • Huifang M. Zhang
  • Paul J. Hanson
  • Zhen Liu
  • Bruce M. McManus
  • Decheng Yang
Research article


Coxsackievirus B3 (CVB3) is the primary causal agent of viral myocarditis. During infection, it hijacks host genes to favour its own replication. However, the underlying mechanism is still unclear. Although the viral receptor is an important factor for viral infectivity, other factors such as microRNAs (miRNA) may also play an essential role in its replication after host cell entry. miRNAs are post-transcriptional gene regulators involved in various fundamental biological processes as well as in diseases. To identify miRNAs involved in CVB3 pathogenesis, we performed microarray analysis of miRNAs using CVB3-infected murine hearts and identified miR-203 as one of the most upregulated candidates. We found that miR-203 upregulation is through the activation of protein kinase C/transcription factor AP-1 pathway. We further identified zinc finger protein-148 (ZFP-148), a transcription factor, as a novel target of miR-203. Ectopic expression of miR-203 downregulated ZFP-148 translation, increased cell viability and subsequently enhanced CVB3 replication. Silencing of ZFP-148 by siRNA showed similar effects on CVB3 replication. Finally, analyses of the signalling cascade downstream of ZFP-148 revealed that miR-203-induced suppression of ZFP-148 differentially regulated the expression of prosurvival and proapoptotic genes of the Bcl-2 family proteins as well as the cell cycle regulators. This altered gene expression promoted cell survival and growth, which provided a favourable environment for CVB3 replication, contributing to the further damage of the infected cells. Taken together, this study identified a novel target of miR-203 and revealed, for the first time, the molecular link between miR-203/ZFP-148 and the pathogenesis of CVB3.


MicroRNA-203 ZFP-148 Coxsackievirus B3 Myocarditis 



Coxsackievirus B3


Dilated cardiomyopathy


Dulbecco’s modified Eagle’s medium




Multiplicity of infection


Protein kinase C




Plaque-forming unit


Untranslated region


Zinc finger protein

Supplementary material

18_2012_1104_MOESM1_ESM.doc (562 kb)
Supplementary material 1 (DOC 561 kb)


  1. 1.
    Esfandiarei M, McManus BM (2008) Molecular biology and pathogenesis of viral myocarditis. Annu Rev Pathol 3:127–155. doi:10.1146/annurev.pathmechdis.3.121806.151534 PubMedCrossRefGoogle Scholar
  2. 2.
    Kuhl U, Pauschinger M, Seeberg B, Lassner D, Noutsias M, Poller W, Schultheiss HP (2005) Viral persistence in the myocardium is associated with progressive cardiac dysfunction. Circulation 112(13):1965–1970. doi:10.1161/CIRCULATIONAHA.105.548156 PubMedCrossRefGoogle Scholar
  3. 3.
    Cheung PK, Yuan J, Zhang HM, Chau D, Yanagawa B, Suarez A, McManus B, Yang D (2005) Specific interactions of mouse organ proteins with the 5′ untranslated region of Coxsackievirus B3: potential determinants of viral tissue tropism. J Med Virol 77(3):414–424. doi:10.1002/jmv.20470 PubMedCrossRefGoogle Scholar
  4. 4.
    Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RL, Finberg RW (1997) Isolation of a common receptor for coxsackie B viruses and adenoviruses 2 and 5. Science 275(5304):1320–1323PubMedCrossRefGoogle Scholar
  5. 5.
    Bartel DP (2009) MicroRNAs: target recognition and regulatory functions. Cell 136(2):215–233. doi:10.1016/j.cell.2009.01.002 PubMedCrossRefGoogle Scholar
  6. 6.
    Cullen BR (2010) Five questions about viruses and microRNAs. PLoS Pathog 6(2):e1000787. doi:10.1371/journal.ppat.1000787 PubMedCrossRefGoogle Scholar
  7. 7.
    Selbach M, Schwanhausser B, Thierfelder N, Fang Z, Khanin R, Rajewsky N (2008) Widespread changes in protein synthesis induced by microRNAs. Nature 455(7209):58–63. doi:10.1038/nature07228 PubMedCrossRefGoogle Scholar
  8. 8.
    Lewis BP, Burge CB, Bartel DP (2005) Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell 120(1):15–20. doi:10.1016/j.cell.2004.12.035 PubMedCrossRefGoogle Scholar
  9. 9.
    Lim LP, Lau NC, Garrett-Engele P, Grimson A, Schelter JM, Castle J, Bartel DP, Linsley PS, Johnson JM (2005) Microarray analysis shows that some microRNAs downregulate large numbers of target mRNAs. Nature 433(7027):769–773. doi:10.1038/nature03315 PubMedCrossRefGoogle Scholar
  10. 10.
    Furuta M, Kozaki KI, Tanaka S, Arii S, Imoto I, Inazawa J (2010) miR-124 and miR-203 are epigenetically silenced tumor-suppressive microRNAs in hepatocellular carcinoma. Carcinogenesis 31(5):766–776. doi:10.1093/carcin/bgp250 PubMedCrossRefGoogle Scholar
  11. 11.
    Ikeda S, Pu WT (2010) Expression and function of microRNAs in heart disease. Curr Drug Targets 11(8):913–925PubMedCrossRefGoogle Scholar
  12. 12.
    Pedersen IM, Cheng G, Wieland S, Volinia S, Croce CM, Chisari FV, David M (2007) Interferon modulation of cellular microRNAs as an antiviral mechanism. Nature 449(7164):919–922. doi:10.1038/nature06205 PubMedCrossRefGoogle Scholar
  13. 13.
    Ziegelbauer JM, Sullivan CS, Ganem D (2009) Tandem array-based expression screens identify host mRNA targets of virus-encoded microRNAs. Nat Genet 41(1):130–134. doi:10.1038/ng.266 PubMedCrossRefGoogle Scholar
  14. 14.
    Choy EY, Siu KL, Kok KH, Lung RW, Tsang CM, To KF, Kwong DL, Tsao SW, Jin DY (2008) An Epstein–Barr virus-encoded microRNA targets PUMA to promote host cell survival. J Exp Med 205(11):2551–2560. doi:10.1084/jem.20072581 PubMedCrossRefGoogle Scholar
  15. 15.
    Sullivan CS, Grundhoff AT, Tevethia S, Pipas JM, Ganem D (2005) SV40-encoded microRNAs regulate viral gene expression and reduce susceptibility to cytotoxic T cells. Nature 435(7042):682–686. doi:10.1038/nature03576 PubMedCrossRefGoogle Scholar
  16. 16.
    Voellenkle C, van Rooij J, Cappuzzello C, Greco S, Arcelli D, Di Vito L, Melillo G, Rigolini R, Costa E, Crea F, Capogrossi MC, Napolitano M, Martelli F (2010) MicroRNA signatures in peripheral blood mononuclear cells of chronic heart failure patients. Physiol Genomics 42(3):420–426. doi:10.1152/physiolgenomics.00211.2009 PubMedCrossRefGoogle Scholar
  17. 17.
    Cheng PY, Kagawa N, Takahashi Y, Waterman MR (2000) Three zinc finger nuclear proteins, Sp1, Sp3, and a ZBP-89 homologue, bind to the cyclic adenosine monophosphate-responsive sequence of the bovine adrenodoxin gene and regulate transcription. Biochemistry 39(15):4347–4357PubMedCrossRefGoogle Scholar
  18. 18.
    Yuan J, Liu Z, Lim T, Zhang H, He J, Walker E, Shier C, Wang Y, Su Y, Sall A, McManus B, Yang D (2009) CXCL10 inhibits viral replication through recruitment of natural killer cells in Coxsackievirus B3-induced myocarditis. Circ Res 104(5):628–638. doi:10.1161/CIRCRESAHA.108.192179 PubMedCrossRefGoogle Scholar
  19. 19.
    Luo L, Ye L, Liu G, Shao G, Zheng R, Ren Z, Zuo B, Xu D, Lei M, Jiang S, Deng C, Xiong Y, Li F (2010) Microarray-based approach identifies differentially expressed microRNAs in porcine sexually immature and mature testes. PLoS One 5(8):e11744. doi:10.1371/journal.pone.0011744 PubMedCrossRefGoogle Scholar
  20. 20.
    Yuan J, Stein DA, Lim T, Qiu D, Coughlin S, Liu Z, Wang Y, Blouch R, Moulton HM, Iversen PL, Yang D (2006) Inhibition of Coxsackievirus B3 in cell cultures and in mice by peptide-conjugated morpholino oligomers targeting the internal ribosome entry site. J Virol 80(23):11510–11519. doi:10.1128/JVI.00900-06 PubMedCrossRefGoogle Scholar
  21. 21.
    Mestdagh P, Feys T, Bernard N, Guenther S, Chen C, Speleman F, Vandesompele J (2008) High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA. Nucleic Acids Res 36(21):e143. doi:10.1093/nar/gkn725 PubMedCrossRefGoogle Scholar
  22. 22.
    Paloheimo O, Ihalainen TO, Tauriainen S, Valilehto O, Kirjavainen S, Niskanen EA, Laakkonen JP, Hyoty H, Vihinen-Ranta M (2011) Coxsackievirus B3-induced cellular protrusions: structural characteristics and functional competence. J Virol 85(13):6714–6724. doi:10.1128/JVI.00247-10 PubMedCrossRefGoogle Scholar
  23. 23.
    Ye X, Liu Z, Hemida MG, Yang D (2011) Targeted delivery of mutant tolerant anti-coxsackievirus artificial microRNAs using folate conjugated bacteriophage Phi29 pRNA. PLoS One 6(6):e21215. doi:10.1371/journal.pone.0021215 PubMedCrossRefGoogle Scholar
  24. 24.
    Sonkoly E, Wei T, Pavez Lorie E, Suzuki H, Kato M, Torma H, Stahle M, Pivarcsi A (2010) Protein kinase C-dependent upregulation of miR-203 induces the differentiation of human keratinocytes. J Invest Dermatol 130(1):124–134. doi:10.1038/jid.2009.294 PubMedCrossRefGoogle Scholar
  25. 25.
    Moffatt CE, Lamont RJ (2011) Porphyromonas gingivalis induction of microRNA-203 expression controls suppressor of cytokine signaling 3 in gingival epithelial cells. Infect Immun 79(7):2632–2637. doi:10.1128/IAI.00082-11 PubMedCrossRefGoogle Scholar
  26. 26.
    Si X, Gao G, Wong J, Wang Y, Zhang J, Luo H (2008) Ubiquitination is required for effective replication of Coxsackievirus B3. PLoS One 3(7):e2585. doi:10.1371/journal.pone.0002585 PubMedCrossRefGoogle Scholar
  27. 27.
    Lowe SW, Sherr CJ (2003) Tumor suppression by Ink4a-Arf: progress and puzzles. Curr Opin Genet Dev 13(1):77–83PubMedCrossRefGoogle Scholar
  28. 28.
    Zhang CZ, Chen GG, Lai PB (2010) Transcription factor ZBP-89 in cancer growth and apoptosis. Biochim Biophys Acta 1806(1):36–41. doi:10.1016/j.bbcan.2010.03.002 PubMedGoogle Scholar
  29. 29.
    Shi Y, Chen C, Lisewski U, Wrackmeyer U, Radke M, Westermann D, Sauter M, Tschope C, Poller W, Klingel K, Gotthardt M (2009) Cardiac deletion of the Coxsackievirus-adenovirus receptor abolishes Coxsackievirus B3 infection and prevents myocarditis in vivo. J Am Coll Cardiol 53(14):1219–1226. doi:10.1016/j.jacc.2008.10.064 PubMedCrossRefGoogle Scholar
  30. 30.
    Hess J, Angel P, Schorpp-Kistner M (2004) AP-1 subunits: quarrel and harmony among siblings. J Cell Sci 117(Pt 25):5965–5973. doi:10.1242/jcs.01589 PubMedCrossRefGoogle Scholar
  31. 31.
    Shaulian E, Karin M (2001) AP-1 in cell proliferation and survival. Oncogene 20(19):2390–2400. doi:10.1038/sj.onc.1204383 PubMedCrossRefGoogle Scholar
  32. 32.
    Yi R, Poy MN, Stoffel M, Fuchs E (2008) A skin microRNA promotes differentiation by repressing ‘stemness’. Nature 452(7184):225–229. doi:10.1038/nature06642 PubMedCrossRefGoogle Scholar
  33. 33.
    Stanczyk J, Ospelt C, Karouzakis E, Filer A, Raza K, Kolling C, Gay R, Buckley CD, Tak PP, Gay S, Kyburz D (2011) Altered expression of microRNA-203 in rheumatoid arthritis synovial fibroblasts and its role in fibroblast activation. Arthr Rheum 63(2):373–381. doi:10.1002/art.30115 CrossRefGoogle Scholar
  34. 34.
    Schmittgen TD, Jiang J, Liu Q, Yang L (2004) A high-throughput method to monitor the expression of microRNA precursors. Nucleic Acids Res 32(4):e43. doi:10.1093/nar/gnh040 PubMedCrossRefGoogle Scholar
  35. 35.
    Sera T (2005) Inhibition of virus DNA replication by artificial zinc finger proteins. J Virol 79(4):2614–2619. doi:10.1128/JVI.79.4.2614-2619.2005 PubMedCrossRefGoogle Scholar
  36. 36.
    Zimmerman KA, Fischer KP, Joyce MA, Tyrrell DL (2008) Zinc finger proteins designed to specifically target duck hepatitis B virus covalently closed circular DNA inhibit viral transcription in tissue culture. J Virol 82(16):8013–8021. doi:10.1128/JVI.00366-08 PubMedCrossRefGoogle Scholar
  37. 37.
    Law DJ, Labut EM, Merchant JL (2006) Intestinal overexpression of ZNF148 suppresses ApcMin/+ neoplasia. Mamm Genome 17(10):999–1004. doi:10.1007/s00335-006-0052-4 PubMedCrossRefGoogle Scholar
  38. 38.
    Yuan J, Cheung PK, Zhang H, Chau D, Yanagawa B, Cheung C, Luo H, Wang Y, Suarez A, McManus BM, Yang D (2004) A phosphorothioate antisense oligodeoxynucleotide specifically inhibits Coxsackievirus B3 replication in cardiomyocytes and mouse hearts. Lab Invest 84(6):703–714. doi:10.1038/labinvest.3700083 PubMedCrossRefGoogle Scholar
  39. 39.
    Rajewsky N (2006) microRNA target predictions in animals. Nat Genet 38(Suppl):S8–S13. doi:10.1038/ng1798 PubMedCrossRefGoogle Scholar
  40. 40.
    He L, Hannon GJ (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7):522–531. doi:10.1038/nrg1379 PubMedCrossRefGoogle Scholar
  41. 41.
    Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP (2007) MicroRNA targeting specificity in mammals: determinants beyond seed pairing. Mol Cell 27(1):91–105. doi:10.1016/j.molcel.2007.06.017 PubMedCrossRefGoogle Scholar
  42. 42.
    Feng Y, Wang X, Xu L, Pan H, Zhu S, Liang Q, Huang B, Lu J (2009) The transcription factor ZBP-89 suppresses p16 expression through a histone modification mechanism to affect cell senescence. FEBS J 276(15):4197–4206. doi:10.1111/j.1742-4658.2009.07128.x PubMedCrossRefGoogle Scholar
  43. 43.
    Salmon M, Owens GK, Zehner ZE (2009) Over-expression of the transcription factor, ZBP-89, leads to enhancement of the C2C12 myogenic program. Biochim Biophys Acta 1793(7):1144–1155. doi:10.1016/j.bbamcr.2009.01.019 PubMedCrossRefGoogle Scholar
  44. 44.
    Bai L, Yoon SO, King PD, Merchant JL (2004) ZBP-89-induced apoptosis is p53-independent and requires JNK. Cell Death Differ 11(6):663–673. doi:10.1038/sj.cdd.4401393 PubMedGoogle Scholar
  45. 45.
    Merchant JL, Bai L, Okada M (2003) ZBP-89 mediates butyrate regulation of gene expression. J Nutr 133(7 Suppl):2456S–2460SPubMedGoogle Scholar
  46. 46.
    Klopfleisch R, Gruber AD (2009) Differential expression of cell cycle regulators p21, p27 and p53 in metastasizing canine mammary adenocarcinomas versus normal mammary glands. Res Vet Sci 87(1):91–96. doi:10.1016/j.rvsc.2008.12.010 PubMedCrossRefGoogle Scholar
  47. 47.
    Hauck L, Harms C, Grothe D, An J, Gertz K, Kronenberg G, Dietz R, Endres M, von Harsdorf R (2007) Critical role for FoxO3a-dependent regulation of p21CIP1/WAF1 in response to statin signaling in cardiac myocytes. Circ Res 100(1):50–60. doi:10.1161/01.RES.0000254704.92532.b9 PubMedCrossRefGoogle Scholar
  48. 48.
    Chiarle R, Pagano M, Inghirami G (2001) The cyclin dependent kinase inhibitor p27 and its prognostic role in breast cancer. Breast Cancer Res 3(2):91–94PubMedCrossRefGoogle Scholar

Copyright information

© Springer Basel AG 2012

Authors and Affiliations

  • Maged Gomaa Hemida
    • 1
  • Xin Ye
    • 1
  • Huifang M. Zhang
    • 1
  • Paul J. Hanson
    • 1
  • Zhen Liu
    • 1
  • Bruce M. McManus
    • 1
  • Decheng Yang
    • 1
  1. 1.Department of Pathology and Laboratory Medicine, The James Hogg Research Center, The Institute for Heart and Lung Health, St. Paul’s HospitalUniversity of British ColumbiaVancouverCanada

Personalised recommendations