The presented research focuses on viral and cellular determinants of the Hepatitis C Virus (HCV) replication. In particular, a novel minus strand replication system was developed to study distinct aspects of the HCV minus strand synthesis initiation. The system enabled a revision of state-of-the-art requirements for the HCV antigenome synthesis when uncoupled from possibly overlapping functions in viral translation and/or plus strand synthesis. So far mainly addressing the genome RNA sequence and structural elements prerequisites, the system displays a universal potential for investigation of replication of other plus strand RNA virus genomes.HCV is a positive-sense single-stranded RNA virus that infects human hepatocytes and causes both acute and chronic hepatitis. The challenge of elimination of HCV as a public health threat is largely complicated by absence of an efficient vaccine and by rapid emergence of virus quasispecies resistant to the existing treatment. Therefore, novel approaches are required for further understanding of molecular mechanisms of the HCV life cycle. Development of the autonomous replicon and full-length HCV cell culture systems has enabled a substantial breakthrough in understanding of the HCV translation and replication. However, these systems allow analysis of cis-elements and trans-acting factors required for genome replication only in presence of both genomic ends. Thus, it remains unclear, whether an element identified in the annotated HCV genome exerts its function when physically present on the plus or the minus strand, or on both. Additionally, given an entangled nature of viral RNA and protein synthesis, a number of overlapping cis-elements cannot be assigned to a specific step of the viral life cycle. To overcome the above limitations, the minus strand replication system was designed to specifically focus on prerequisites for the HCV antigenome production.The system, which represents a set of replication constructs assembled in agreement with the current knowledge on the HCV replication, uncouples to various extents the HCV minus strand synthesis from the plus strand synthesis and translation, thereby empowering diversified applications. The very 5 -end of the annotated HCV genome constituted by the stem-loop (SL) I and II domains was confirmed to be essential for the minus strand synthesis initiation at the genomic 3 -end. The positive regulation of the antigenome production was found to extend up to inclusion of the SL III domain; however, enabling of functional translation initiation from the HCV internal ribosome entry site (IRES) resulted in a profound negative effect on RNA replication. The latter observation has encouraged in-depth analysis of a balance between the HCV genome translation and replication that suggested an importance of the genomic RNA ends communication. Yet lacking an experimental confirmation, a possible circularization of the HCV RNA for efficient replication is supported by the importance of long-range RNA-RNA interactions between cis-elements, which were addressed by mutational analysis. Along with validation of the previously reported regulatory RNA elements, a comprehensive screening for cis-acting replication elements within the protein-coding sequence was undertaken. In addition to addressing the genome sequence and structure determinants, the minus strand replication system was utilized to examine a regulation of the HCV RNA synthesis by selected cellular factors. A positive role of the most of the liver-specific microRNA-122 (miR-122) binding sites on efficiency of the HCV minus strand synthesis initiation was demonstrated.In conclusion, an assay system for the specific analysis of requirements for the HCV minus strand RNA synthesis was developed. Uncoupling of the HCV minus and plus strand replication from each other and from translation enabled a dissection of essential cis-elements and assignment of their functions specifically to antigenome synthesis. Ultimately, the versatility of the system enables further characterization of regulatory trans-factors and investigation of the interplay of molecular processes during the HCV life cycle as well as of other RNA viruses.
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