Polo-Like Kinase 1 (PLK1) Is Involved in Hepatitis C Virus Replication by Hyperphosphorylating NS5A
| Autor: | Yung-Chia Chen, 陳永佳 |
|---|---|
| Rok vydání: | 2010 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 98 The thesis contains two sections. In Section 1, we focused on the role of polo-like kinase 1 (Plk1) in hepatitis C virus (HCV) replication. About 3% of people in the world are infected with HCV. HCV infection is associated with liver cirrhosis and hepatocellular carcinoma. However, current treatment is ineffective in some patients. Therefore, identification of new targets for HCV therapy is an important issue. By using RNAi library screening, we previously identified Plk1 as a potential host factor regulating HCV replication. Here we showed that the silencing of Plk1 reduced HCV RNA replication and protein expression, as well as infectious virus production. Interestingly, we found that silencing of Plk1 reduced the hyperphosphorylation of HCV non-structural protein NS5A. Since hyperphosphorylation of NS5A has been pointed out to regulate HCV RNA replication, our results suggested that Plk1 may control HCV replication through hyperphosphorylating NS5A. Indeed, we found that Plk1 interacted and partially co-localized with NS5A. Furthermore, Plk1 could hyperphosphorylate NS5A in vitro. Finally, mutations on the hyperphosphorylation sites on NS5A abolished the regulation by Plk1. Taken together, this study showed that Plk1 is a NS5A- phosphokinase and thereby regulates HCV replication. This study increased our understanding on HCV replication and suggested a potential drug target, Plk1, for anti-HCV therapy. In Section 2, we focused on the characterization and functional assay of the coronavirus non-structural protein 15 (Nsp15). Coronaviruses utilizes a discontinuous transcription mechanism for subgenomic mRNA synthesis. The transcripts share a common 5’-leader sequence, which is derived from the 5’-end of the RNA genome. The detailed mechanism of transcription is unclear, but Nsp15 has been suggested to be involved in this process. Here we expressed and purified the Nsp15 of mouse hepatitis virus (MHV), which is a model organism of coronavirus, and tested if Nsp15 could cleave the 5’-end of viral genome. The results showed that Nsp15 cleaved the 1-331 nt RNA at the 3’-side of 216th uridylate, which is located on a stem-loop structure and beside a bulge. Alteration of the bulge structure severely blocked the cleavage, indicating that the bulge structure is required for the specificity of Nsp15 recognition. Finally, we introduced the mutations which blocked the 216th uridylate to the defective-interfering (DI) RNA for functional assay. These mutations down-regulated the transcription of DI RNA, suggesting that Nsp15 cleavage site on 216th uridylate of the 5’-end of the genome sequence may play a role in the MHV transcription mechanism. This study identified an RNA secondary structure preferred by Nsp15 and suggested a possible role of Nsp15 in coronavirus replication. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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