AbstractsBiology & Animal Science

Investigation of microRNA-protein complex recruitment to the hepatitis C virus untranslated regions

by Karl Dominik Conrad

Institution: Universität Giessen
Department: FB 08 - Biologie und Chemie
Degree: PhD
Year: 2013
Record ID: 1105918
Full text PDF: http://geb.uni-giessen.de/geb/volltexte/2013/10144


Hepatitis C virus belongs to the family of Flaviviridae and contains a single stranded RNA genome in positive orientation. The genome carries a single polyprotein open reading frame (ORF), which is flanked by two highly structured RNA stretches, the 5’- and 3’-untranslated regions (UTRs). In these UTRs several secondary structures were identified to be necessary for HCV translation and replication. The internal ribosome entry site (IRES), which is required for translation, comprises three of the four stem loops present in the 5’-UTR. In addition two microRNA-122 (miR-122) binding sites are located upstream of the IRES. The 3’-UTR comprises a variable region (VR), a poly U/C tract and three conserved stem loops. In the VR an additional miR-122 binding site was identified. miR-122 was shown to stimulate HCV translation and replication possibly by protecting the HCV RNA from degradation. This work focused on the question whether miR-122 recruits a microRNA-protein complex to its binding sites on the HCV RNA and which components this complex contains. Towards this end an anti-Argonaute (Ago) protein-specific – HCV RNA co-immunoprecipitation was developed. Using this method it could be shown that two of the four members of the Ago protein family, Ago1 and Ago2, are recruited to the HCV RNA in a miR-122 dependent manner. For the miR-122 binding site in the VR of the 3’-UTR it has also been shown that Ago2 can be recruited by miR-122. This however is only the case when four nucleotides located in a stem loop in the NS5B coding region are present. These findings give rise to the assumption that for this miR-122 binding site not only the seed sequence but also four additional nucleotides located close to the 3’-end of the miR-122 bind to the HCV RNA. Finally, employing a time course assay it has been shown that miR-122 prolongs the half-life of a wild type HCV reporter RNA compared to a mutant that is not able to bind miR-122 or compared to the wild type in presence of a control miR-122. This experiment also shows that, besides stabilizing the HCV RNA, miR-122 seems to directly stimulate HCV translation. Thus, it seems that miR-122 recruits a microRNA-protein complex and positively influences the HCV life cycle via two different mechanisms. Firstly, miR-122 enhances HCV RNA stability, most likely by protecting it from degradation. Secondly, miR-122 improves HCV translation efficiency probably by changing the IRES structure to a conformation favorable for translation. Das Hepatitis C Virus (HCV) gehört zu der Familie der Flaviviridae und besitzt ein einzelsträngiges RNA-Genom in (+)-Strang-Orientierung. Dieses Genom enthält einen einzelnen offen Leserahmen für das Polyprotein. Dieser ist von 5‘- und 3‘-nicht translatierten Regionen (UTRs) flankiert, in welchen sich viele Sekundärstrukturen befinden, die unerlässlich für die Translation und Replikation des Virus sind. Die sogenannte „internal ribosome entry site“ (IRES) in der 5‘-UTR besteht aus drei Haarnadelstrukturen und ist unerlässlich für die Translationsinitiation des…