Characterization of microRNA-protein complexes involved in Hepatitis C Virus translation regulation by the liver-specific microRNA-122
Final Report Abstract
Translation of Hepatitis C Virus (HCV) RNA is directed by an internal ribosome entry site (IRES) in the 5´-untranslated region (5´-UTR). HCV overall genome amplification is stimulated by the liver-specific microRNA-122 (miR-122) that binds to two binding sites between the stem-loops I and II near the 5´-end of the 5´-UTR. This binding was reported to enhance HCV RNA replication, translation and stability. We have analyzed binding of miR-122/Argonaute 2 (Ago2) complexes to these sites using anti-Ago-co-immunoprecipitation of radioactively labeled HCV RNAs along with ectopic miR- 122 in HeLa cells (D. Conrad). We showed that Argonaute (Ago) 2 protein binds to the HCV 5´-UTR in a miR-122-dependent manner. miR-122 also recruits Ago1 to the HCV 5’-UTR. Only miRNA duplex precursors of the correct length stimulate HCV translation, indicating that the duplex miR-122 precursors are unwound by a complex that measures their length. Partially masking the miR-122 binding sites in a stem-loop structure impairs Ago2 binding and translation stimulation by miR-122. These results suggest that Ago protein is directly involved in loading miR-122 to the HCV RNA and mediating translation stimulation. Using constructs with two independently mutated miR-122 target sites in the HCV 5´UTR, we aimed to analyze possible cooperative effects of binding of the miR-122/Ago2 complexes, as well as the possible influence of each of the complex on downstream effector functions (i.e., translation stimulation and RNA stability) (A. Nieder-Röhrmann). Our results show that the miR-122 target sites can be addressed separately, but in these cases binding of each single complex is weak. In the binding assays as well as in the translation assays, binding site 1 has a stronger effect than binding site 2. However, when both target sites were addressed simultaneously, we observed a synergistic binding of both miR/Ago2 complexes. Consistently, simultaneous binding of both miR-122/Ago2 complexes results in cooperative translation stimulation. In contrast to the working hypothesis featured in the grant application, we found that any mutation in any of the miR-122 target sites always affects translation stimulation, indicating that the cooperative binding of both miR-122/Ago2 complexes does not allow to discriminate between different effects of either of the two miR-122/Ago2 complexes on such effector functions. We also analyzed the overall RNA stability as well as the 5´end integrity of these HCV RNAs in the presence of miR-122. Surprisingly, using short HCV reporter RNAs we did not find effects of miR-122 binding on overall RNA stability or 5´end integrity over up to 36 hours. In contrast, using full-length HCV genomes that are incapable of replication, we found a slightly positive influence of miR-122 on RNA stability, indicating that features of the full-length HCV genome that do not reside in the 5´- and 3´UTRs may render HCV RNA genome stability miR-122-dependent. Additional work closely associated with this project (by G. Gerresheim and N. Dünnes) showed that the putative miR-122 target sites in the NS5B region and in the 3´UTR bind miR- 122/Ago2 complexes. The miR-122 target sites in the NS5B coding region are accessible to varying extents, depending on the availability of the flanking sequences.
Publications
- Stimulation of Hepatitis C Virus RNA translation bymicroRNA-122 occurs under different conditions in vivo and in vitro. Virus Res. 2012 Aug;167(2):343-52
Goergen D, Niepmann M
(See online at https://doi.org/10.1016/j.virusres.2012.05.022) - Hepatitis C virus RNA translation. Curr. Top. Microbiol. Immunol. 2013;369:143-66
Niepmann M
(See online at https://dx.doi.org/10.1007/978-3-642-27340-7_6) - MicroRNA-122 dependent binding of Ago2 protein to hepatitis C virus RNA is associated with enhanced RNA stability and translation stimulation. PLoS One. 2013;8(2):e56272
Conrad KD, Giering F, Erfurth C, Neumann A, Fehr C, Meister G, Niepmann M
(See online at https://doi.org/10.1371/journal.pone.0056272) - The role of microRNAs in hepatitis C virus RNA replication. Arch. Virol. 2014 May;159(5):849-62
Conrad KD, Niepmann M
(See online at https://doi.org/10.1007/s00705-013-1883-4) - Conserved RNA secondary structures and long-range interactions in hepatitis C viruses. RNA. 2015 Jul;21(7):1219-32
Fricke M, Dünnes N, Zayas M, Bartenschlager R, Niepmann M, Marz M
(See online at https://doi.org/10.1261/rna.049338.114) - Cooperative enhancement of translation by two adjacent microRNA- 122/Argonaute 2 complexes binding to the 5' untranslated region of hepatitis C virus RNA. J. Gen. Virol. 2017 Feb;98(2):212-224
Nieder-Röhrmann A, Dünnes N, Gerresheim GK, Shalamova LA, Herchenröther A, Niepmann M
(See online at https://doi.org/10.1099/jgv.0.000697) - 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. Cell. Mol. Life Sci. 2017 Feb;74(4):747-760
Gerresheim GK, Dünnes N, Nieder-Röhrmann A, Shalamova LA, Fricke M, Hofacker I, Höner Zu Siederdissen C, Marz M, Niepmann M
(See online at https://doi.org/10.1007/s00018-016-2377-9) - Signals involved in regulation of Hepatitis C Virus RNA genome replication. Review. Frontiers in Microbiology
Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O
(See online at https://doi.org/10.3389/fmicb.2018.00395)
