Cellular and Molecular Life Sciences

, Volume 74, Issue 4, pp 747–760 | Cite as

microRNA-122 target sites in the hepatitis C virus RNA NS5B coding region and 3′ untranslated region: function in replication and influence of RNA secondary structure

  • Gesche K. Gerresheim
  • Nadia Dünnes
  • Anika Nieder-Röhrmann
  • Lyudmila A. Shalamova
  • Markus Fricke
  • Ivo Hofacker
  • Christian Höner zu Siederdissen
  • Manja Marz
  • Michael Niepmann
Original Article

Abstract

We have analyzed the binding of the liver-specific microRNA-122 (miR-122) to three conserved target sites of hepatitis C virus (HCV) RNA, two in the non-structural protein 5B (NS5B) coding region and one in the 3′ untranslated region (3′UTR). miR-122 binding efficiency strongly depends on target site accessibility under conditions when the range of flanking sequences available for the formation of local RNA secondary structures changes. Our results indicate that the particular sequence feature that contributes most to the correlation between target site accessibility and binding strength varies between different target sites. This suggests that the dynamics of miRNA/Ago2 binding not only depends on the target site itself but also on flanking sequence context to a considerable extent, in particular in a small viral genome in which strong selection constraints act on coding sequence and overlapping cis-signals and model the accessibility of cis-signals. In full-length genomes, single and combination mutations in the miR-122 target sites reveal that site 5B.2 is positively involved in regulating overall genome replication efficiency, whereas mutation of site 5B.3 showed a weaker effect. Mutation of the 3′UTR site and double or triple mutants showed no significant overall effect on genome replication, whereas in a translation reporter RNA, the 3′UTR target site inhibits translation directed by the HCV 5′UTR. Thus, the miR-122 target sites in the 3′-region of the HCV genome are involved in a complex interplay in regulating different steps of the HCV replication cycle.

Keywords

microRNA Accessibility Ago2 Translation Regulation 

Notes

Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft, Germany (DFG; IRTG 1384, MA-5082/1, SFB 1021, NI-604/2-2) and the Carl-Zeiss-Stiftung. We thank Volker Lohmann for providing plasmid pFK\I389/NS3-39/Hygubi/5.1, Charles M. Rice for plasmid pFL-J6-JFH-1, Thomas Pietschmann and Ralf Bartenschlager for the Jc1 clone, and Yann Ponty for programming the VARNAv3-92 update including Vienna style handling.

Supplementary material

18_2016_2377_MOESM1_ESM.pdf (11.8 mb)
Supplementary material 1 (PDF 12059 kb)

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Copyright information

© Springer International Publishing 2016

Authors and Affiliations

  • Gesche K. Gerresheim
    • 1
  • Nadia Dünnes
    • 1
  • Anika Nieder-Röhrmann
    • 1
  • Lyudmila A. Shalamova
    • 1
  • Markus Fricke
    • 2
  • Ivo Hofacker
    • 4
  • Christian Höner zu Siederdissen
    • 4
    • 5
  • Manja Marz
    • 2
    • 3
  • Michael Niepmann
    • 1
  1. 1.Institute of Biochemistry, Faculty of MedicineJustus-Liebig-UniversityGiessenGermany
  2. 2.Faculty of Mathematics and Computer ScienceFriedrich-Schiller-UniversityJenaGermany
  3. 3.FLI Leibniz Institute for Age ResearchJenaGermany
  4. 4.Institute for Theoretical ChemistryUniversity of ViennaViennaAustria
  5. 5.Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for BioinformaticsUniversität LeipzigLeipzigGermany

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