海水魚類細胞腫大病毒腫瘤壞死因子受體相關因子(TRAF)基因特性分析

Abstract

歸屬虹彩病毒科(Iridoviridae)中細胞腫大病毒屬(Megalocytivirus)的病毒為導致亞洲國家淡水和海水魚類大量死亡的主要因子之一。傳染性脾腎壞死病毒(Infectious spleen and kidney necrosis virus； ISKNV)為細胞腫大病毒屬(Megalocytivirus)之典型種(type species)。ISKNV ORF111L轉譯一個296胺基酸蛋白質，具部分類似於人(Homo sapiens)和家鼷鼠(Mus musculus)的腫瘤壞死因子受體相關因子(TRAF)蛋白質。本篇歸類一株持續感染嘉鱲魚鰭細胞株(PI-RSBF-2)之病毒分離株為ISKNV，並藉由選殖ISKNV ORF111L基因及功能分析來探討病毒蛋白所涉及之細胞凋亡過程或細胞增殖調控。選殖的基因序列與ISKNV ORF111L序列有100% 相似度，其推演之胺基酸片段與其他細胞腫大病毒(Megalocytiviruses) TRAF homolog有高相似度(60%-92%)。ISKNV ORF111L胺基酸序列含有一個TRAF superfamily domain及一個RING finger domain但不具有穿膜片段。系統發生分析結果顯示ISKNV ORF111L與其他細胞腫大病毒TRAF homologs位於同一單元類群且接近魚類TRAF homologs之類群。此外，ISKNV ORF111L的轉錄作用最早於ISKNV感染嘉鱲魚鰭細胞(RSBF-2) 2小時後可偵測到，而RSBF-2細胞過度表現ISKNV ORF111L蛋白質導致星型孢菌素(Staurosporine)誘導之細胞凋亡現象提前產生。細胞生長實驗結果顯示過度表現ISKNV ORF111L蛋白質促使細胞增殖能力提高。

Viruses belonging to the genus Megalocytivirus in the family Iridoviridae are one of the major agents causing mass mortalities in marine and freshwater fish in Asian countries. Infectious spleen and kidney necrosis virus (ISKNV) is the type species of the Megalocytivirus genus. ISKNV ORF111L encodes a 296-residue protein that has some similarity to the tumor necrosis factor receptor-associated factor (TRAF) proteins of Homo sapiens and Mus musculus. In this study, a virus isolated from a persistently infected red sea bream fin cell line (PI-RSBF-2) was classified to ISKNV, and we elucidate the viral protein which involved in apoptosis process or cell proliferarion regulation by cloning ISKNV ORF111L and functional assays. Cloned gene sequence revealed 100% identity to ISKNV ORF111L sequence and the deduced amino acid sequences showed high similarity (60-92%) to other Megalocytiviruses TRAF homolog. ISKNV ORF111L contains a TRAF superfamily domain and a RING finger domain but has no transmembrane fragments. The results from phylogenetic analysis shows that ISKNV ORF111L and TRAF homologs from other Megalocytiviruses are in a monophyletic group and this group is relative close to that from fish TRAF homologs. Furthermore, the transcription of ISKNV ORF111L was detected as early as 2 h post infection in ISKNV infected red sea bream fin cells (RSBF-2). Whereas overexpression of ISKNV ORF111L resulted in advanced apoptosis induced by Staurosporine. In cell growth experiment showed that overexpression of ISKNV ORF111L can promote the proliferation RSBF-2 cells.