| Summary: | 碩士 === 國立金門大學 === 食品科學系 === 107 === Nervous necrosis virus ( NNV) genome consists of two positive sense single-strand RNA molecules with 5’cap structures and without poly(A) tails.RNA1 ( 3.1 k) encodes RNA dependent RNA polymerase, whereas, RNA2 ( 1.4 k)encodes capsid protein. After virus infection, RNA1 transcribes a 0.4 k subgenomic RNA3 molecule, which translates B2 protein for blocking host RNAi. After high moi infection, we found that positive and negative RNA genomes and subgenomes were detected as early as 4 hpi and the signal of RNA3 always higher than that of RNA1 in the early stage of infection. It was found that the nucleotide sequence starting from the 2,286 position formed an 8 nucleotides paired stem structure with sequence starting from 2,720 nucleotide at the 2 nucleotides upstream of the transcription start codon of the positive-strand RNA3. We speculate that this stem structure is important for the transcription of RNA3. To address this issue, we established a recombinant virus model to investigate the transcription of RNA3. Capped RNA 1 and RNA2 were co-transfected into GB cells to produce virus particle with comparable infectious efficacy. Furthermore, a recombinant chimeric virus was produced, and a sequence of Myc-tag was added before the stop codon of RdRp. The recombinant chimeric virus infected GB cells expressed the RdRp-myc which recognized by the anti Myc antibody. Nevertheless, we found that transfected RNAs were degraded after transfection into GB cells. To overcome this RNA degradation problem, we preformed the rescue experiment by pre-expression of B2 protein before RNA transfection into GB cells. The result shows that the signal of negative-strand RNA3 increased with time procession. Taken together, III these results showed that the host RNAi defense mechanism of GB cells has a high response to RNA1 transfection, furthermore, the recombinant chimeric virus may useful to the studies of replication and transcription of RNA1 and RNA3.
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