神經壞死病毒RNA2非轉譯區對轉譯的調控

Abstract

病毒為了有效複製新病毒顆粒，已演化出各種不同機制來劫持細胞的轉譯系統，用於製造自身蛋白質。尤其位於病毒基因體的非轉譯區(Untranslated regions, UTRs)常窩藏許多順式作用元件(Cis-acting elements)，能調控病毒基因體的轉譯，例如18S rRNA結合位與microRNA結合位。我們經由序列分析發現神經壞死病毒(Nervous necrosis virus, NNV)的RNA2 UTRs包含幾個可能的順式作用元件，但這些序列能否影響NNV的轉譯仍須研究。本研究透過針對這些序列建構刪除與替換突變質體，利用西方墨點法、北方墨點法、RT-qPCR與螢光酵素基因報導系統，探討RNA2 UTRs之順式作用元件對轉譯的影響。結果顯示，位於NNV RNA2 5’-UTR中的Kozak序列對於外套蛋白的轉譯十分重要。完全刪除5’-UTR後，RNA2就無法轉譯出完整的外套蛋白，但加上病毒的Kozak序列後，質體便能轉譯出完整的外套蛋白。只保留單一個18S rRNA結合位的質體也能轉譯出完整的外套蛋白，更發現NNV RNA2 5’-UTR並無內部核醣體進入位點(Internal ribosomal entry sites, IRESs)。另一方面，NNV RNA2可以透過3’-UTR與外套蛋白AUG起始子附近的18個核苷酸序列互補配對，使RNA2形成環型結構，有助於核醣體快速進入下一輪轉譯，促進外套蛋白的轉譯效率。此外，3’-UTR上也含有多個可能可與石斑魚腦細胞microRNA結合的位置。實驗結果顯示RNA2的表現量會隨著刪除越多的3’-UTR序列而增多，顯示3’-UTR不僅能影響RNA2穩定，也可能參與外套蛋白轉譯的調控。以上結果顯示NNV以不同順式作用元件，有效利用細胞資源來調控自身RNA的穩定性和蛋白質的產量。藉由了解NNV的轉譯機制，不僅對病毒防治的研究有所幫助，甚至有助於解析病毒的演化路徑。

In order to translate itself efficiently, virus has evolved many mechanisms to utilize the resources from it host cell. The cis-acting elements located untranslated region (UTR), including rRNA binding site and microRNA binding site, allow virus to take advantage of cellular translation systems. The bioinformatic data shows that nervous necrosis virus (NNV) RNA2 UTR harbors several 18S rRNA and miRNA binding sites in silico. To evaluate the effect of these elements on translation of NNV coat protein, we designed a series of deleted and substituted mutation RNA2 plasmids. By transfecting plasmid into grouper brain (GB) cells, to estimate its translation efficiency by Western blotting, luciferase reporter assay, Northern blotting and RT-qPCR. The result shows that the 5’-UTR of RNA2 is important for translation of coat protein. Once deleted the 5’-UTR of only 26 nt, RNA2 cannot translate whole coat protein. However, even only 5 nt of Kozak sequence in the front of AUG start codon of RNA2 can translate the whole coat protein well. Remaining each 18S rRNA binding site can also translate the whole coat protein. Furthermore, 5’-UTR of NNV RNA2 has no IRESs activity that indicates the translation abolishment by hairpin structure blocking the screening. On the other hand, NNV RNA2 can form a circular structure through the 3’-UTR, which complements the 18 nucleotides near the coat protein AUG start codon, facilitating ribosome recycling and enhancing translation efficiency. Besides, the microRNA of GB cell has several possible binding sites on 3’ UTR of NNV RNA2. The more 3’-UTR were deleted, the greater number of RNA2 it remains, shows that these sites not only unstabilize the viral RNA but could also possibly regulate the translation of coat protein. The above data indicate that NNV employ these cis-acting elements to manipulate the generation of its coat protein. These detailed results may provide deep insight in the translation mechanisms of NNV and its regulation, which will surely help the development of therapeutic for controlling NNV and understanding its evolution.