| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 96 === Japanese encephalitis virus (JEV) possesses a single, positive-stranded RNA genome. Replication of RNA genome requires trans-acting elements and cis-acting sequences for the formation of replication complex to initiate and complete viral replication cycle. In this study, a host factor, Hdj2 belonging to DnaJ/Hsp40 (heat shock protein) homolog, and two promoters on the antigenome were characterized. NS5 is an RNA- dependent RNA polymerase (RdRp), a central catalytic enzyme in JEV replication. Previous study has been shown that a specific interaction of NS5 with Hdj2 by coimmunoprecipitation and western blot assays. The interaction was further characterized in this study. Both N-terminal (1-121 a.a.) and C-terminal (121-397 a.a.) regions of Hdj2 were found to bind to NS5. Overexpression Hdj2 by transfecting Hdj2 into HEK293T cells increased both plus- and minus-strand RNA synthesis and elevated virus production to 10-fold, suggesting that Hdj2 plays an important role for viral replication. In addition, overexpression of Hdj2 increased apoptotic cells, suggesting that Hdj2 may play a role in the JEV-induced cell death. The second part of this study was to define two RNA regions that may affect viral RNA synthesis: (i) JEV(-)1-160 RNA located at the 3’ end of minus strand is the promoter for genome synthesis, and (ii) the JEV(-)10431-10566 RNA is speculated to be the promoter for small RNA (a 3’-terminal 522-nucleotides genome fragment, representing the highly conserved region of the 3’-untranslated region, in JEV-infected cells) transcription. To address whether these two RNA regions are responsible for genome and small RNA synthesis, JEV(+)1-160 and JEV(+)10431-10566 RNAs were separately transfected into JEV-infected BHK-21 cells and the effects on genome and small RNA accumulation were measured by Northern analysis. Transfecting of JEV(+)1-160 RNA increased the molar ratio of the small RNA to the genome, while transfecting of JEV(+)10431-10566 RNA caused slightly decreased the molar ratio of the small RNA to the genome. These results indicated that both RNA regions may be responsible for genome and small RNA synthesis.
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