| Summary: | 碩士 === 國立陽明大學 === 生化暨分子生物研究所 === 100 === Hepatitis C virus (HCV) is the major pathogen of transfusion-associated non-A, non-B hepatitis, which might lead to steatosis, cirrhosis and hepatocellular carcinoma (HCC). Although the life cycle of HCV is not fully understood, recent studies have revealed that DDX3 is required for HCV RNA replication. DDX3, the DEAD box RNA helicase that interacts with HCV core, possesses ATP-dependent RNA helicase activity and participates in various aspects of cellular RNA metabolism, cell cycle, innate immunity, tumorigenesis and replication of numerous viruses. In order to elucidate the molecular mechanism by which DDX3 modulated HCV RNA replication, the interactions between DDX3 and HCV UTRs were investigated. Using in vitro RNA pull-down assay, we demonstrated that DDX3 associated with 5’- and 3’-UTR of both HCV genomic/anti-genomic RNA with different affinity, whereas purified His-DDX3 directly interacted with HCV genomic 3’UTR and anti-genomic 5’UTR RNAs. Interestingly, in this study, we present evidence that purified GST-DDX3 could directly interact with poly (U/UC) tract and X-tail region in the HCV genomic 3’UTR, while the N-terminal 226 amino-acid region and C-terminal 125 amino-acid region of DDX3 could directly interact with HCV genomic 3’UTR. Furthermore, the computational prediction of RNA-binding residues (PiRaNhA) and mutagenesis analysis identified three critical residues (Arg585, Lys589 and Arg592) of DDX3 for binding to HCV genomic 3’UTR. In addition, we demonstrated that DDX3 possessed 5’ to 3’ RNA helicase activity to unwind the secondary structure of HCV genomic 3’UTR. Taken together, our results suggested that DDX3 might facilitate HCV RNA replication by binding and rearranging the structure of HCV genomic 3’UTR. These findings should provide the acting mechanism of DDX3-mediated HCV RNA replication and novel insights into the HCV life-cycle.
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