| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 92 === Japanese encephalitis virus (JEV) belongs to genus Flavivirus, family Flaviviridae. JEV genome possesses a positive-stranded RNA of 10,976 nts in length and encodes only one open reading frame. For RNA viruses, the synthesis of viral genome requires as a first step of interaction of the terminal sequences (usually the untranslated regions) with cellular and viral proteins. To analyze the initial interaction between the terminal sequences and cellular or viral proteins, electrophoretic mobility shift assays (EMSA) were performed. The purified recombinant viral NS5 protein that functions in RNA-dependent RNA polymerase was first demonstrated to interact specifically with 3’ ends of both plus- and minus-strand RNAs. The specificity of RNA-protein interaction was confirmed by EMSA and competition assays. Three ribonucleoprotein complexes were formed when S10 cell extracts from BHK-21 cells were used for the interaction with the 3’ terminal stem-loop (3’-SL), CS1 and CS2 motifs of plus strand. Proteins with molecular weight of 35, 50, 105 and 160 kDa were detected by UV-induced cross-linking to the 3’ end of plus-strand RNA. These proteins were subjected to gel purified and digested with trypsin and followed by MALDI-TOF analysis. Profiles of Mass spectrum in conjunction with databank search analyses suggested a general transcription factor II might be one of cellular protein candidates that interacts with the 3’-SL of plus-strand RNA. These results demonstrated that 3’-terminal sequences of plus-strand and minus-strand viral RNAs do associate with NS5 protein and some uncharacterized cellular factors may play roles during viral life cycle.
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