蝦白點症病毒結構性蛋白質基因wssv396轉錄及轉譯機制之分析

Abstract

由蝦類白點症病毒 (white spot syndrome virus, WSSV) 引起之白點症為目前嚴重威脅全世界養蝦產業之病毒性疾病。基於白點症病毒蛋白質體學及基因體序列分析之研究，在其532個預測之開放譯讀區中，有部分功能已知之非結構性蛋白質及結構性蛋白質基因，在其轉譯終點前後不具有聚腺嘌呤訊號 (AATAAA)，推測可能與其下游之開放譯讀區共用加聚腺嘌呤位置 (poly A addition site) 有關。白點症病毒結構性蛋白質基因wssv396、wssv395及wssv394為同向轉錄基因群 (cluster)，三者之加聚腺嘌呤位置皆位於wssv394基因下游之聚腺嘌呤訊號後第17個核苷酸處，符合真核細胞聚腺嘌呤訊號規則。根據北方雜合法分析結果，此基因群利用wssv396之5’端啟動子 (promoter) 轉錄出包含wssv396、wssv395及wssv394之三基因mRNA (3.4 kb)；wssv394基因之riboprobe尚可偵測到利用其5’端啟動子轉錄之單基因mRNA (1.6 kb)。實驗結果證明wssv396及wssv394基因主要利用帽依賴型轉譯作用 (cap-dependent translation) 合成蛋白質，而位於第二個基因之wssv395係以非帽依賴型轉譯作用即內部核醣體進入位置 (Internal Ribosome Entry Site, IRES)合成蛋白質，推測IRES序列可能位於wssv396基因轉錄區內 (coding region)，關於其確切位置猶待進一步之實驗分析以確認之。

WSSV396, WSSV395 and WSSV394 are identified in pervious research as the three of thirty-nine structural proteins of shrimp white spot syndrome virus (WSSV). This study focuses on the transcriptional and translational analysis of wssv396, wssv395 and wssv394. The 3’RACE results reveal that wssv396, wssv395, and wssv394 share the same polyA tail. A common ~3.4-kb transcript, which is consistent with the predicted size of a polycistronic transcript encoding these three genes, was detected by using gene-specific probes respectively in northern blot analysis. Two primer sets were designed to generate two overlapping RT-PCR products to confirm the existence of the ~3.4-kb transcript, and the sequence data also indicated that the ~3.4-kb transcript is entire and intron-less. It reveals that wssv396, wssv395 and wssv394 are transcribed from the same promoter of wssv396. In the northern blot analysis, another ~1.6-kb transcript was recognized by wssv394-specific probe. According to the in vitro transcription and translation data, it could be concluded that WSSV396 and WSSV394 are translated from the ~3.4-kb and ~1.6-kb transcript by cap-dependent translation mechanism; the wssv395 is translated from the ~3.4-kb transcript by cap-independent translational mechanism. Based on the in vitro transcription and translation analysis of bicistronic constructs encoding wssv396 and wssv395, it suggests that the translational mechanism of wssv395 from the polycistronic transcript might occur by a mechanism using the internal ribosome entry site (IRES) on the upstream of the wssv396 ORF.