白點症病毒WSSV108蛋白質對極早期基因wssv304之轉錄調控

Abstract

白點症病毒 (White spot syndrome virus, WSSV) 為一DNA病毒，對於海洋甲殼類具有廣泛之感染性，特別對於對蝦類 (Penaeid shrimp) 有極高之致死率。WSSV首次於1990年代初期在中國漳浦爆發後，疫情便迅速地擴散到世界各地，造成全世界蝦類養殖業嚴重之經濟損失，至今仍無有效治療方式。大型DNA病毒如人類皰疹病毒，在感染宿主後，會潛伏於宿主細胞中，在接收到適當的環境刺激後，病毒會由潛伏期 (latency) 進入溶裂期 (lytic cycle)，溶裂期基因會以階段性的方式表現，依序表現極早期 (immediate-early)、早期 (early) 以及晚期 (late) 基因。目前白點症病毒已被報導具有21個極早期基因，其中WSSV108被報導具有轉錄因子活性，而本研究進一步探討WSSV108對於白點症病毒極早期基因的調控。冷光報導分析 (Luciferase reporter assay) 顯示WSSV108在Sf21昆蟲細胞株中能促進wssv304基因啟動子轉錄活性至6.5倍。接著利用wssv304啟動子序列刪除株進行冷光報導分析，結果發現WSSV108蛋白質會透過wssv304啟動子上-93到-36的位置活化啟動子活性。並利用電泳游動性轉移分析 (Electrophoretic mo-bility shift assay) 發現WSSV108蛋白質能結合到wssv304啟動子上-65到-46的位置。同時，將wssv304啟動子-59到-50之序列突變後，WSSV108即無法調控轉錄活性，進一步確認WSSV108利用此段DNA序列調控wssv304啟動子。除此之外，本研究利用GST pull-down assay發現WSSV108會形成多聚體，同時也會與宿主轉錄因子LvYY1直接結合，推測WSSV108可能以二聚體形式結合上DNA，並與LvYY1有協同活化的功能，其相互作用可能與致病機轉相關。本研究首次發現WSSV108蛋白質具有調控白點症病毒極早期基因wssv304轉錄活性的能力，對於病毒極早期基因的相互調控有了進一步的了解。

White spot syndrome virus (WSSV) is a large DNA virus with highly infectious and high mortalities to Penaeid shrimp. Since the first outbreaks of WSSV in southeast China in early 1990s, it spreads all over the world and causes serious economic losses. However, there’s no treatment for WSSV. After infection of DNA virus, it remains la-tency and switches into lytic cycle with appropriate environmental stimulation, subse-quently expressing immediate-early genes, early genes and late genes. So far, twenty one WSSV immediate-early genes (IE genes) have been found. Among these IE genes, wssv108 contains transcriptional activity. Transient transfection experiments showed that WSSV108 activated the promoter of wssv304 about 6.5-fold in insect cells. Dele-tion analysis of the promoter suggested that nucleotides -93 to -36 in wssv304 promoter is important to the activation by WSSV108. Moreover, electrophoretic mobility shift assay verified that wssv108 binds to the nucleotide -65 to -46 in wssv304 promoter. Meanwhile, site-directed mutagenesis proved that -59 to -50 in wssv304 promoter is important to the activation by WSSV108. In addition, GST pull-down assay showed that wssv108 forms dimer and interacts with LvYY1 directly. Therefore, this study demonstrated that wssv108 enhances the transcription of wssv304, suggesting that WSSV108 acts as a transcription factor in the regulation of other viral genes.