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.
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.
Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany.
Gesche K
GK
Gerresheim
Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany.
Nadia
N
Dünnes
Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany.
Anika
A
Nieder-Röhrmann
Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany.
Lyudmila A
LA
Shalamova
Faculty of Mathematics and Computer Science, Friedrich-Schiller-University, 07743, Jena, Germany.
Markus
M
Fricke
Institute for Theoretical Chemistry, University of Vienna, 1090, Vienna, Austria.
Ivo
I
Hofacker
Institute for Theoretical Chemistry, University of Vienna, 1090, Vienna, Austria.|Bioinformatics Group, Department of Computer Science, and Interdisciplinary Center for Bioinformatics, Universität Leipzig, 04107, Leipzig, Germany.
Christian
C
Höner Zu Siederdissen
Faculty of Mathematics and Computer Science, Friedrich-Schiller-University, 07743, Jena, Germany.|FLI Leibniz Institute for Age Research, 07743, Jena, Germany.
Manja
M
Marz
Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University, Friedrichstrasse 24, 35392, Giessen, Germany. michael.niepmann@biochemie.med.uni-giessen.de.
Michael
0000-0002-0846-8658
M
Niepmann
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D060565:Argonaute Proteins / Q000378:metabolism; D001483:Base Sequence; D015967:Gene Expression Regulation, Viral; D016679:Genome, Viral; D006367:HeLa Cells; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006526:Hepatitis C / Q000235:genetics / Q000378:metabolism* / Q000821:virology; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000235:genetics*; D014779:Virus Replication; D012324:RNA-Dependent RNA Polymerase
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D060565:Argonaute Proteins; D035683:MicroRNAs; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C466422:AGO2 protein, human; C531907:MIRN122 microRNA, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase
Accessibility; Ago2; Regulation; Translation; microRNA
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