蝦白點症病毒極早期基因ie1之研究

Studies on shrimp white spot syndrome virus (WSSV) immediate-early gene ie1

劉宛菁(Wang-Jing Liu)

指導教授 ： 羅竹芳 ； 郭光雄

國立臺灣大學/生命科學院/動物學研究研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.01508

摘要

本研究首度成功的應用放線菌酮(cycloheximide)抑制了白點症病毒(white spot syndrome virus)感染細胞後病毒新生蛋白質的合成，再結合白點症病毒開放譯讀區(open reading frame)微陣列進行病毒基因表現整體分析，篩選出六十個候選的極早期(immediate early, IE)基因，進一步藉由反轉錄-聚合酶鏈反應(reverse transcriptase-polymerase chain reaction)，確認出此六十個基因中在白點症病毒台灣分離株基因體上編號分別為126、242和418號的開放譯讀區，其基因表現確實不受到放線菌酮處理影響，將此三個基因分別命名為ie1, ie2和ie3。這三個基因的序列同樣也出現在其他的白點症病毒分離株，在中國分離株基因體上三個基因均可分別對應到其所預測的開放譯讀區，但在泰國分離株則僅有相對於ie1的序列被預測為是一個開放譯讀區。另外在秋夜盜蛾(Spodoptera frugiperda)細胞株Sf9中，以增強型綠螢光蛋白(enhanced green fluorescence protein)基因為報導基因進行啟動子(promoter)活性分析顯示，白點症病毒ie1的啟動子展現為一個極強勢的啟動子，其在此昆蟲細胞內所啟動增強型綠螢光蛋白表現量，甚而還高過昆蟲病毒–花旗松毒蛾核多角體病毒(Orgyia pseudotsugata multicapsid nucleopolyhedrovirus)極早期基因ie2啟動子所啟動的基因表現。在節肢動物例如果蠅中，Janus kinase-signal transducer and activator of transcription (JAK-STAT)訊息傳導路徑是其對抗病毒感染的機制上重要的角色，但在本研究中發現白點症病毒卻是可以利用草蝦的STAT作為一個轉錄因子來促進病毒基因ie1在感染細胞中的表現。由於白點症病毒ie1的啟動子不僅可在蝦細胞也可在昆蟲細胞中被活化，我們將ie1啟動子區域核苷酸序列進行其可能的功能預測，並在Sf9昆蟲細胞中對此啟動子區域進行一系列的核苷酸序列刪除及突變分析，發現此啟動子的區域中包含了一個STAT結合的位置，而同時也證實此結合位對於此一啟動子的整體活性是非常重要的。另外，利用Sf9昆蟲細胞以32P標定的白點症病毒ie1的啟動子中與STAT結合區域的DNA片段當作探針進行電泳流動性轉移分析(electrophoresis mobility shift assay)，可偵測到一個特殊的蛋白質-DNA複合體的存在；將重組草蝦STAT在Sf9昆蟲細胞中進行大量表現，經電泳流動性轉移分析後再利用專一性抗體反應，證實了此一複合物中的蛋白質為STAT；另外的電泳流動性轉移分析也顯示，白點症病毒感染後的草蝦細胞內被活化的STAT的量多於未被病毒感染的草蝦。活化作用(transactivation)的分析顯示，STAT的劑量與ie1啟動子活性具有正相關性，當昆蟲細胞內重組草蝦STAT蛋白的表現量增加時，白點症病毒ie1的啟動子的活性也跟著增加。這些結果顯示STAT能夠直接活化白點症病毒ie1的表現，同時也顯示STAT在ie1啟動子呈現為一個強勢啟動子上扮演重要的角色，由此推論白點症病毒可以成功的利用一個可能是宿主的防禦機制，來加強病毒極早期基因的表現。

關鍵字

放線菌酮 ； 白點症病毒 ； 極早期基因

並列摘要

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.

並列關鍵字

cycloheximide ； white spot syndrome virus ； immediate early gene

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