Preview

PDF, English Download (88MB) | Terms of use

Abstract

Hepatitis C Virus (HCV) is a positive stranded RNA virus, grouped into the family of Flaviviridae. The HCV genome encodes a single polyprotein, which is co- and posttranslationally cleaved into ten structural and non-structural (NS) proteins by cellular and viral proteases. The coding sequence is flanked by 5’ and 3’ untranslated regions (UTRs), which contain essential cis-acting elements, regulating translation and RNA synthesis, e.g. an internal ribosome entry site (IRES) for cap-independent translation. HCV RNA replication involves the synthesis of a negative strand replication intermediate, serving as a template for the generation of multiple strands of genomic RNA. This process requires a concerted action of several viral nonstructural proteins, cis-acting replication elements and host factors, and is poorly understood at the molecular level. The first part of this study aimed to characterize the viral non-structural proteins comprising the replicase complex in vitro and their mode of action during (-)-strand RNA synthesis. Since the natural 3’(+)-end is a poor template for the viral polymerase NS5B, supporting roles of the viral protease/helicase NS3 and the phosphoprotein NS5A were hypothesized. Optimal conditions for NS3 activity were established by an in vitro helicase assay. By combining the individual proteins with different RNA templates, it was observed that initiation and processivity of NS5B were stimulated by active NS3, but not by inactive mutants. Inhibition of NS3 helicase activity did not impair the stimulatory effect on NS5B, but led to an altered mode of initiation. Addition of purified NS5A further augmented the effect of NS3. In conclusion, this work demonstrates that NS3 and NS5A can improve RNA dependent RNA polymerase activity on a natural template, thereby providing an experimental model to study the molecular mechanisms governing initiation of RNA synthesis. Liver -specific microRNA (miR)-122 is an important host factor of HCV replication, and recognizes two conserved target sites within the first 45nt of the HCV 5’ UTR, close to the IRES. Previous studies suggested a role of miR-122 in RNA stability, translation, and RNA synthesis. The mechanisms, by which miR-122 exerts these functions, remain enigmatic. Insertion of a heterologous IRES element, allowing for miRindependent translation of the non-structural proteins, was sufficient to enable replication in miR122deficient Hep3B cells, suggesting a substantial role of miR-122 in IRES-dependent translation. The miR122 binding region is engaged in a strong secondary in the complementary negative strand. Additionally, we found that a similar structure was predicted inthe positive strand, which would interfere with IRES formation. We therefore hypothesized that miR-122 binding in this region might prevent such alternative structures, thereby facilitating IRES-mediated translation. Indeed, mutations in the miR-122 binding region, but not the IRES sequence, which were designed to stabilize or destabilize the IRES, enhanced or decreased initial translation, respectively, independent of miR-122. Translation enhancement was independent from RNA stability, but short-lived, suggesting additional roles of miR-122, e.g. recruitment of host proteins facilitating steady state translation. Moreover, structural analysis suggested that the HCV IRES folds into a number of conformers in solution, which can be modified by miR-122 under certain conditions. Apart from the 5’ UTR, HCV also contains several seed-matches for miR-122 in the coding sequence of NS5B, and the 3’ UTR, with unknown functional significance. Two novel sites were identified to be conserved over a number of genotypes. The functional characterization of these miR-122 binding sites was evaluated by insertion of point mutations, abrogating miR-122 binding to single and multiple sites, revealing a previously unappreciated role in virion assembly or release. However, assembly of the mutants could not be rescued by a corresponding mutant miR, suggesting a specific need for wild type miR-122. Conclusively, this study provides evidence for miR-122 involvement in almost every intracellular stage of HCV infection, and defines translation enhancement by suppression of RNA structures interfering with IRES activity as a key function of miR-122.