蝦白點症病毒基因轉錄及轉譯機制之研究

Study of the transcriptional and translational mechanisms of shrimp white spot syndrome virus (WSSV) genes

康詩婷(Shih-Ting Kang)

指導教授 ： 郭光雄 ； 羅竹芳

國立臺灣大學/生命科學院/動物學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.00427

摘要

根據白點症病毒台灣株基因體之分析顯示，許多結構性蛋白基因及非結構性蛋白基因之位置排列極為相近，且具有同一轉錄方向，推測這些基因可能形成同向轉錄基因群 (gene cluster)。本論文利用北方轉印法 (Northern blot analysis) 進行分析，結果證實至少有四個基因群會轉錄出多順反子mRNA，因此本研究針對其中一個基因群vp31/vp39b/vp11之轉錄及轉譯機制進行深入探討。vp31/vp39b/vp11基因群會產生兩個轉錄本，分別為包含三個基因的3.4-kb多順反子mRNA，以及僅帶有vp11的單順反子mRNA (1.6 kb)；然而，沒有vp39b之單順反子mRNA被偵測到。根據試管內轉錄及轉譯反應 (TNT) 以及試管內轉錄反應 (in vitro translation) 之分析結果，顯示vp39b之轉譯作用與vp31並無直接關係，也排除vp39b蛋白合成是經由轉譯作用再起始 (translation reinitiation) 所調控之可能性。本研究以雙冷光酵素報導基因分析系統 (dual luciferase reporter assay) 證實在vp31/vp39b 編碼區內 (coding region) 具有一特別之IRES序列，負責調控雙冷光酵素報導基因載體中位於其下游之螢火蟲冷光酵素 (firefly luciferase) 的表現。vp31/vp39b/vp11之研究成果不僅在探討WSSV特殊轉譯及轉錄機制的研究領域具有代表性，據此成果我們亦可進一步推測其他會產生多順反子mRNA的結構性/非結構性蛋白基因群也有可能利用IRES序列調控下游基因之轉譯作用。
蝦白點症病毒 (White spot syndrome virus, WSSV) 為一大型dsDNA病毒，其基因體大小約為300 kbp。先前研究證實WSSV經由轉錄作用可產生許多多順反子mRNA (polycistronic mRNA)，並且可利用內部核醣體進入位置 (internal ribosome entry site; IRES) 進行轉譯作用。非結構性蛋白基因icp35為一高表現量基因，且可與其他基因共同被轉錄成一個多順反子mRNA，因此我們利用雙冷光酵素報導基因分析系統 (dual luciferase reporter assay) 證實icp35之5' UTR具有一個IRES序列可調控該蛋白之轉譯作用。根據刪除法 (deletion analysis) 之分析結果，發現莖環結構 (stem loop) VII及VIII主要負責icp35 IRES之活性。利用無啟動子(promoterless) 實驗、RT-PCR以及置入一穩定莖環結構於水母冷光酵素 (Renilla luciferase) ATG上游等分析，已排除此IRES活性是來自於潛在啟動子 (cryptic promoter)、異常剪接作用 (abnormal splicing) 或是核醣體連讀轉譯 (ribosomal read-through) 之可能性。利用icp35的雙股RNA干擾 (dsRNAi) 技術進行基因靜默化後，會導致WSSV攻毒實驗之死亡率以及病毒複製量呈現顯著性下降，因此我們推論ICP35對於WSSV之複製增殖具有重要性。利用tunicamycin處理細胞，eIF2α會受到磷酸化進而導致宿主之帽依賴型轉譯作用受到抑制，同時我們也發現icp35 IRES活性亦會受到影響而下降，顯示此IRES活性需要eIF2之參與。本研究證實一個核酸嵌入劑 (nucleic acid intercalating drug) quinacrine (QC) 在細胞實驗中對於icp35 IRES具有顯著性的抑制作用；此外，WSSV攻毒試驗結果也顯示若同時施打WSSV病毒液與QC會降低死亡率以及病毒複製量。

關鍵字

多順反子mRNA ； 內部核醣體進入位置 ； 白點症病毒 ； 基因群 ； 蝦 ； ICP35

並列摘要

The genome of the white spot syndrome virus (WSSV) Taiwan isolate has many structural and non-structural genes that are arranged in clusters. Screening with Northern blots showed that at least four of these clusters produce polycistronic mRNA, and one of these (vp31/vp39b/vp11) was studied in detail. The vp31/vp39b/vp11 cluster produces two transcripts, including a large 3.4-kb polycistronic transcript of all three genes. No monocistronic vp39b mRNA was detected. TNT (coupled transcription and translation) and in vitro translation assays showed that vp39b translation was independent of vp31 translation, and that ribosomal reinitiation was not a possible mechanism for vp39b. An unusually located IRES (internal ribosome entry site) element was identified in the vp31/vp39b coding region, and this region was able to promote the expression of a downstream firefly luciferase reporter. We show that vp31/vp39b/vp11 is representative of many other WSSV structural/non-structural gene clusters, and argue that these are also likely to produce polycistronic mRNAs and that use an IRES mechanism to regulate their translation.
Although shrimp white spot syndrome virus (WSSV) is a large dsDNA virus (~300 kbp), it expresses many polycistronic mRNAs that are likely to use internal ribosome entry site (IRES) elements for translation. The gene of the highly expressed non-structural protein ICP35 is encoded by a polycistronic mRNA, and here we use a dual luciferase assay to demonstrate that this protein is translated cap-independently by an IRES element located in the 5' UTR of icp35. A deletion analysis of this region showed that IRES activity was due to stem loops VII and VIII. A promoterless assay, an RT-PCR analysis and a stable stem-loop insertion upstream of Renilla luciferase ORF were respectively used to rule out the possibility that cryptic promoter activity, abnormal splicing or read-through were contributing to the IRES activity. The importance of ICP35 to viral replication was demonstrated in a dsRNAi knockdown experiment in which the mortality of the icp35 dsRNA group was significantly reduced. Using tunicamycin, we further show that eIF2α is required for icp35 IRES activity. We also found that the intercalating drug quinacrine significantly inhibited icp35 IRES activity in vitro, and reduced the mortality rate and viral copy number in WSSV-challenged shrimp.

並列關鍵字

polycistronic mRNA ； internal ribosome entry site (IRES) ； white spot syndrome virus (WSSV) ； gene cluster ； shrimp ； ICP35

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