Identification, functional assay and application of ICP11, the most highly expressed novel protein of shrimp white spot syndrome virus (WSSV)

蝦類水產養殖是亞洲地區之重要產業之一，而此產業持續地受到幾種蝦類病毒性疾病的危害，尤為白點症病毒 (white spot syndrome virus, WSSV)所造成的白點症(white spot syndrome)。本研究係探討白點症病毒在兩種蝦類—草蝦 (Penaeus monodon) 及白蝦 (Litopenaeus vannamei) —寄主細胞內病毒基因表現的層次。藉由微陣列分析及草蝦蝦苗EST資料庫，分析WSSV感染之草蝦體內之病毒基因表現圖譜，從中鑑定出WSSV icp11 (infective cytosolic protein 11) 係為具有最高表現量的病毒基因 (其基因表現量為WSSV主要結構性蛋白質基因vp28的三倍)。而WSSV感染之白蝦之蛋白質表現上，依感染後之白蝦鰓組織之蛋白質二維電泳及液相層析串聯式質譜儀進行蛋白質鑑定所得結果，顯示ICP11同樣在轉譯層次上具有高度表現性。然而屬於白點症病毒非結構性蛋白質的ICP11之胺基酸序列與現在任何已知蛋白質序列不具有同源性，而認為ICP11為一個新穎蛋白質。我們利用蛋白質結構和功能分析，成功地鑑定出ICP11多重生物性功能。ICP11具有形成多聚體特性，且可同時存在於感染病毒之草蝦血細胞及轉染ICP11之昆蟲細胞 (Sf9) 的細胞質與細胞核中。而經由Far-Western blot反應顯示ICP11可與histone 相結合。除此之外，ICP11亦具有鎂離子依存之DNA水解酵素的特性，而只要存在有Mg2+及足夠量的ICP11下，這兩種功能皆可以同時存在的。因此，我們假設ICP11與寄主DNA競爭與histone結合，使表現或複製中的寄主DNA持續裸露外，亦因此結合而使寄主無法形成完整的核小體構造，而其餘的ICP11則可利用DNase活性以切割寄主DNA，此種作用機制或許就是導致寄主細胞死亡的主要原因。This study investigates white spot syndrome virus (WSSV) gene expression levels in the cells of two hosts (Penaeus monodon and Litopenaeus vannamei). Microarray and EST analysis of the mRNA profiles in WSSV-infected P. monodon cells were used to identify WSSV genes that were very highly expressed. Results showed that the mRNA of the WSSV icp11 gene consistently had the highest copy number of all other viral transcripts (eg. 3x higher than the transcripts of the major envelope protein, VP28). At the protein level in WSSV-infected L. vannamei, 2-DE gel analysis and MS/MS protein identification also showed that this WSSV non-structural protein has the highest expression levels so far reported in WSSV-infected cells. However, this novel protein has no sequence homology to any other known protein, and its function remains unknown. Therefore, by using structural and functional analysis, we successfully characterized its multiple biological roles. ICP11 is capable of homo-oligomerization and localizing in both the cytoplasm and nucleus of the host cell and transfected insect cell. A Far-Western blot confirmed ICP11 can interact with the histone protein. We also found that ICP11 exhibits Mg2+ dependent nuclease activity. Additional competitive binding assays further suggested that in the presence of Mg2+ and sufficient ICP11, both of these functions are fulfilled. We therefore hypothesize that ICP11 competes with host DNA to bind to histone protein, and when all of the eligible DNA binding sites are filled, any excess ICP11 will remain unbound and act as a DNase to digest host DNA. These functions could easily result in nucleosome disorder and lead to cell death.中文摘要………………………………………………………………………………1 英文摘要………………………………………………………………………………3 第一章 蝦白點症及其病原體-蝦白點症病毒相關文獻回顧……….……………...4 蝦白點症及其病原體 -蝦白點症病毒..………………………………………...5 參考文獻.…………………...………………………………….………………16 第二章 蝦白點症病毒極高表現新穎蛋白質ICP11 之鑑定、功能分析與應用..26 前言 …………………………………………………………………………….27 材料與方法 …………………………………………………………………….33 結果 …………………………………………………………………………….44 討論 …………………………………………………………………………….57 總結 …………………………………………………………………………….68 參考文獻.……………………………………………………………………….69 Table 1.………………………………………………………………………....78 Table 2………………………………………………………………………….79 Figures …………………………………………………………………………81 附錄 …………………………………………………………………………….94 參與發表文章 ……………………………………………………………………….96 期刊論文 ……………………………………………………………………….97 研討會論文 ...…………………………………………………………………..99 專利及專書論文....……………………………………………………………10