Studies on the Replication of Bamboo Mosaic Virus: an Optimized in vitro Viral Transcription System Applying on the Studies of BaMV Cis-acting Elements and Host Factors
| Autor: | Jen-Wen Lin, 林振文 |
|---|---|
| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus. Positive-sense RNA viruses usually accumulate more plus-strand RNA and less minus-strand RNA, possibly due to a stronger promoter at the 3’-end of minus-strand RNA. In vitro BaMV RNA-dependent RNA polymerase (RdRp) assay system has been used to study the activity and the structure of viral RNA promoters. Results derived from time-course experiments indicated that the 5 days post inoculation (dpi) leaves with 0.5 μg of BaMV inoculum accumulated the highest level of RdRp activity. Results of structural probing and in vitro RdRp assays suggested that the sequences within the lower stem of the major stem-loop in the 3’-terminus of minus-strand RNA is important for BaMV plus-strand RNA synthesis. In addition, the 3’-end UUUUC deletion of the minus-strand RNA dramatically reduced the plus-strand RNA synthesis, while further deletions to expose the middle UUUUC repeats at the 3’-end partially restored the RNA synthesis, strengthening the importance of the exposure of 3’-end UUUUC in the regulation of plus-strand RNA synthesis. In combination with the results of in vivo protoplast assay, it was suggested that three cis-acting elements of the minus-strand RNA, including the 3’-terminal UUUUC, the sequences in the major stem-loop, and the distance between these two regions, are involved in the plus-strand RNA synthesis. We also used BaMV 3’ untranslated region (3’ UTR) to probe the associated host factors. A 51-kDa protein (p51) binding to the pseudoknot region and a 43-kDa protein (p43) binding to the poly(A) tail were found. Results of the RdRp assay indicated that the minus-strand RNA synthesis was specifically suppressed by a p51-enriched extract, probably due to the occupancy of the pseudoknot by p51. Results of mass spectrometry analysis indicated that p51 was elongation factor 1a (EF1a) and that p43 was chloroplast phosphoglycerate kinase (PGK). Finally, a virus-induced gene silencing (VIGS) system was utilized to test the involvement of chloroplast PGK in BaMV replication. Results showed that the plants with efficient chloroplast PGK gene-knockdown accumulated lower level of BaMV coat protein than control plants. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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