白蝦 Yin-Yang 1蛋白質對白點症病毒極早期基因之轉錄調控探討

Transcriptional regulation of white spot syndrome virus immediate-early genes by Litopenaeus vannamei Yin-Yang 1

黃柄翰(Ping-Han Huang)

指導教授 ： 張麗冠

國立臺灣大學/生命科學院/生化科技學系/博士(2018年)

https://doi.org/10.6342/NTU201800035

摘要

白點症病毒(White spot syndrome virus, WSSV)為目前危害對蝦(penaeid shrimp)養殖業最嚴重的病毒，養殖對蝦在受到病毒急性感染後3至10天內即會死亡，最終能導致80%~100%的死亡率，至今仍沒有可實際利用的治療方式。白點症病毒感染宿主後將依序表現極早期基因(immediate early)、早期基因(early)以及晚期基因(late)三種溶裂期(lytic cycle)基因，而最早表現的極早期蛋白質則參與調控下游病毒基因表現，目前已發現二十一種白點症病毒極早期基因，但詳細基因功能都尚待釐清，僅有具轉錄活性的wssv126 (ie1)探討較為透徹。Yin-Yang 1 (YY1)為細胞內的多功能轉錄因子，對於細胞發育及分化極為重要，在YY1蛋白質序列C端具有家族間的保守性鋅指功能區，能結合於專一性DNA序列調控基因轉錄，本研究成功自對蝦選殖出YY1家族同源基因Litopenaeus vannamei Yin Yang 1 (LvYY1)，並探討LvYY1參與白點症病毒極早期基因之轉錄調控機制。啟動子序列分析發現ie1近端啟動子(proximal promoter)具有兩個保守性YY1結合序列，並於冷光報導分析中證實LvYY1活化ie1轉錄，此外也發現突變ie1啟動子(-127/-119)序列上的YY1保守性序列能抑制LvYY1的轉錄活性。藉由EMSA分析證實LvYY1結合於ie1啟動子啟動子(-127/-119)序列，而染色質免疫沉澱分析(Chromatin immunoprecipitation, ChIP)的結果也證實當病毒感染對蝦時LvYY1結合在ie1啟動子上。另外也發現LvYY1能與Litopenaeus vannamei TATA binding protein (LvTBP)結合並活化ie1核心啟動子轉錄。當白蝦受到白點症病毒感染時，利用RNA interference (RNAi)抑制白蝦活體的LvYY1基因表現後，顯著地抑制病毒基因體複製以及ie1基因表現，並降低病毒感染導致的高死亡率。本研究中首次證實對蝦科生物具有YY1家族基因，並發現LvYY1結合於近端啟動子上的YY1保守性結合序列活化ie1轉錄以及參與基礎轉錄因子(general transcription factor, GTF)複合體調控ie1基礎轉錄活性，在白點症病毒溶裂期時促進病毒基因表現以及複製，並期待上述研究成果對於未來探討白點症病毒與宿主交互作用能有更多的貢獻。

關鍵字

白點症病毒 ； 極早期基因ie1 ； Yin Yang 1蛋白質 ； TATA結合蛋白質 ； 轉錄調控

並列摘要

White spot syndrome virus (WSSV), which causes the shrimp white spot syndrome (WSD), is the most threatening virus to the shrimp aquaculture. WSD leads to the high mortality rate up to 100% in 3 to 10 days after infection. Even now, there is still no effective treatment for WSD. After infection, WSSV undergoes a productive replication state called lytic cycle and expresses the immediate early (IE) genes, early genes, and late genes in order. The IE genes express firstly and regulate downstream viral genes expression. So far, 21 WSSV IE genes were identified, but the exact function of these genes are still unknown. Only the gene function and expression regulation of wssv126, which encodes the possible transcription factor IE1, were most studied. Yin-Yang 1 (YY1) is a multifunctional transcription factor that regulates many key cellular processes, including cell proliferation and differentiation. The YY1 family contains a conserved zincfinger domain, which recognizes a specific DNA sequence. In this study, YY1 from Litopenaeus vannamei (LvYY1) was cloned and analyzed. Furthermore the WSSV IE gene transcription regulation mechanisms, which LvYY1 involves, were studied. According to promoter sequence analysis, this study found two possible conserved YY1 binding sites, which are located in the proximal promoter of ie1. Transient transfection revealed that LvYY1 activates ie1transcription apparently. Moreover, point mutations introduced into one of the conserved YY1-binding sites between positions -119 and -126 in the ie1 promoter represses this capability. Electrophoretic mobility shift assay (EMSA) results further indicated that LvYY1 binds to the conserved YY1-binding site in the region between -119 to -126 in the ie1 promoter. Chromatin immunoprecipitation (ChIP) analysis also confirmed that LvYY1 binds to the ie1 promoter in WSSV-infected shrimp. Additionally, reporter assay results suggested that LvYY1 is involved in basal transcriptional regulation via an interaction with L. vannamei TATA-binding protein, (LvTBP). Knockdown of LvYY1 expression by RNA interference (RNAi) in WSSV-infected shrimp reduced viral replication and viral gene expression significantly. The cumulative mortality of infected shrimp also declined with LvYY1 dsRNA injection. In the present study, the penaeid shrimp YY1 homolog, LvYY1, was reported and analyzed for the first time. The results indicate that WSSV uses host LvYY1 to enhance ie1 expression via an YY1-binding site and the TATA box, thereby facilitating lytic activation and viral replication. These findings are expected to contribute to future studies involving WSSV-host interactions.

並列關鍵字

WSSV ； immediate-early gene 1 (ie1) ； Yin Yang 1 ； TATA-binding protein ； transcriptional regulation

參考文獻

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