博士 / 國立臺灣大學 / 動物學研究研究所 / 95 / Here we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during white spot syndrome virus (WSSV) infection. Sixty candidate immediate early (IE) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively. The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using enhanced green fluorescence protein (EGFP) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nucleopolyhedrovirus ie2 promoter. Although the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway is part of the anti-viral response in arthropods such as Drosophila, here we show that WSSV uses shrimp STAT as a transcription factor to enhance viral gene expression in host cells. In a series of deletion and mutation assays using the WSSV immediate early gene ie1 promoter, which is active in shrimp cells and also in insect Sf9 cells, an element containing a STAT binding motif was shown to be important for the overall level of WSSV ie1 promoter activity. In the Sf9 insect cell line, a specific protein-DNA complex was detected by using electrophoresis mobility shift assays (EMSA) with the 32P-labeled STAT binding motif of the WSSV ie1 promoter as the probe. When recombinant Penaeus monodon STAT (rPmSTAT) was overexpressed in Sf9 cells, EMSA with specific antibodies confirmed that the STAT was responsible for the formation of the specific protein-DNA complex. Another EMSA showed that in WSSV-infected P. monodon, levels of activated PmSTAT were higher than in WSSV-free P. monodon. A transactivation assay of the WSSV ie1 promoter demonstrated that increasing the level of rPmSTAT led to dose-dependent increases in ie1 promoter activity. These results show that STAT directly transactivates WSSV ie1 gene expression and contributes to its high promoter activity. We conclude that WSSV has successfully annexed a putative shrimp defense mechanism, which it now uses to enhance the expression of viral immediate early genes.
| Identifer | oai:union.ndltd.org:TW/095NTU05312002 |
| Date | January 2007 |
| Creators | Wang-Jing Liu, 劉宛菁 |
| Contributors | Chu-Fang Lo, Guang-Hsiung Kou, 羅竹芳, 郭光雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 94 |
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