白蝦 YY1 調控白點症病毒極早期基因 wssv108 的機轉

Regulation of the immediate-early gene wssv108 of white spot syndrome virus by Litopenaeus vannamei Yin-yang 1

蔡沛賜(Pei-Si Cai)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/生化科技學系/碩士(2015年)

https://doi.org/10.6342/NTU.2015.01860

摘要

白點症病毒 (White spot syndrome virus) 為大型雙股DNA病毒，對於對蝦類 宿主 (Penaeidea) 具有高致病性。目前白點症病毒已被報導具有21個極早期基因，其中極早期基因wssv108的胺基酸序列與Bad/Rrf2家族轉錄因子具有25%之相似性。此外，前人研究顯示白點症病毒會挾持蝦宿主轉錄因子活化其極早期基因進而走向病毒溶裂期。而本研究室先前自白蝦mRNA中選殖出一Yin-yang 1 (YY1)相似基因LvYY1，YY1為廣泛表現於各種組織之轉錄調控因子，在各種物種中已被報導能調控數種病毒極早期基因，因此本研究假設白蝦基因LvYY1會參與白點症病毒極早期基因的調控。結果發現LvYY1靜默化能夠抑制白點症病毒複製數以及延遲病毒感染白蝦的死亡率，而luciferase reporter assay分析顯示LvYY1在Drosophila Schneider 2昆蟲細胞株中能促進白點症病毒極早基因wssv108啟動子的轉錄活性至3.9倍，且啟動子截切分析 (promoter deletion analysis) 顯示wssv108啟動子上的30-mer DNA序列 (-102 nt/-73 nt) 為LvYY1的可能調控區段。但是經由線上資料庫的序列比對，此DNA調控區並不具有YY1家族轉錄因子之保守性DNA結合序列，因此製備分段競爭者序列以及位點突變競爭者序列，再以electrophoretic mobility shift assay證實wssv108轉錄起始點上游序列第91到83個核苷酸之間的序列為LvYY1的結合位，並以LvYY1結合位突變株證明LvYY1是藉由此段序列活化wssv108的啟動子。最後發現當白蝦體內LvYY1的表現量受到抑制時，也會導致病毒的wssv108表現量隨之下降。總結以上，本研究證實白蝦的LvYY1經由非典型的YY1的DNA結合序列促進白點症病毒極早期基因wssv108的轉錄活性，其機轉可能與白點症病毒的致病相關。

關鍵字

白點症病毒 ； 白蝦 ； Yin-yang 1 蛋白質 ； 極早期基因 ； wssv108基因

並列摘要

White spot syndrome virus (WSSV) is a deadly pathogen that causes shrimp death with a mortality rate of 90-100%. So far, twenty-one WSSV immediate-early genes (IE genes) had been found. The protein sequence of the wssv108 IE gene displays 25% identify with Bad/Rrf2 family transcription factor. Previous studies showed that WSSV could annex shrimp transcription factors to activate its own immediate-early genes to get into lytic phase. Recently, LvYY1 (Yin-yang 1) was cloned from Litopenaeus vannamei. A ubiquitously distributed transcription factor YY1 was known to regulate viral immediate-early genes. This study assumed that LvYY1 participates in the pathogenesis of WSSV. Firstly, knockdown of LvYY1 expression in WSSV-infected shrimp caused lower copy numbers of WSSV and delayed cumulative mortality. Moreover, the reporter assay revealed that the promoter activity of wssv108 was activated 3.9 fold by LvYY1 in S2 cell. Deletion analysis of the promoter suggested that 30-mer DNA region (-102 nt/-73 nt) is important to the activation. According to the sequence alignment, this 30-mer DNA region in the wssv108 promoter has no consensus sequence of YY1 binding sites. The electrophoretic mobility shift assay using mutant sequences as competitive oligonucleotides confirmed that recombinant LvYY1 from Sf9 insect cell binds on the sequence from -91 nt to -83 nt of the wssv108 promoter. Furthermore, site-directed mutagenesis of the promoter verified the YY1 binding site is crucial for transcriptional regulation of the wssv108 promoter by LvYY1. Finally, silencing of LvYY1 expression in WSSV-infected shrimp reduced the levels of wssv108 mRNA. Therefore, this study demonstrated that LvYY1 enhances the transactivation activity of wssv108 via an atypical YY1-binding site to affect WSSV infection.

並列關鍵字

white spot syndrome virus ； Litopenaeus vannamei ； Yin-yang 1 ； immediate-early gene ； wssv108

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