ISKNV通过PI3K-Akt-mTOR通路调控鳜脑细胞自噬机制研究

Infectious spleen and kidney necrosis virus (ISKNV) is a causative agent which causes high mortality and great economic losses to fish farming industry. Uncovering molecular mechanisms of interaction between ISKNV and hosts is meaningful for understanding its pathogenic mechanism. We have reported that ISKNV infection induced autophagy and increased the levels of autophagic flux in CPB cells. At same time, ISKNV also hijacked autophagy to facilitate itself replication and proliferation. However, ISKNV regulated autophagy by which pathway, and what is the mechanism? In cancer cells, the regulation of autophagy is very closely related to the phosphatidylinositol 3-kinase-Akt-mTOR pathway. Our previous experiment results showed that ISKNV infection up-regulated gene expression and protein synthesis in PI3K-Akt-mTOR pathway. So we postulate that ISKNV induce autophagy by activating the PI3K-Akt-mTOR pathway. The following three issues will be studied in this proposal: 1) influences of ISKNV infection to PI3K-Akt-mTOR pathway; 2) influences of interfering PI3K-Akt-mTOR pathway to autophagy and ISKNV proliferation; 3) The signal transduction pathway of ISKNV activating the PI3K-Akt-mTOR pathway. Based above experiments, we want to elucidate the molecular mechanism of ISKNV regulated autophagy. And these results will provide new insights into viral pathogenesis of ISKNV and novel targets for antiviral treatment strategies.

传染性脾肾坏死病毒（ISKNV）是危害养殖鱼类重要病原，揭示其与宿主细胞互作机制对阐明其致病机理具有重要意义。前期研究表明ISKNV可诱导CPB细胞自噬并利用自噬促进病毒的增殖，但ISKNV通过何种信号通路调控细胞自噬？其机制是什么？已有研究表明肿瘤细胞自噬调控与PI3K-Akt-mTOR通路关系密切，预实验结果显示ISKNV感染显著上调PI3K/Akt/mTOR相关基因转录及蛋白表达。因此假设ISKNV通过PI3K-Akt-mTOR通路调控细胞自噬并促进自身增殖。本项目拟开展以下三方面研究： 1）ISKNV感染对细胞PI3K-Akt-mTOR通路的影响；2）调控细胞PI3K-Akt-mTOR通路对自噬和病毒增殖的影响；3）病毒调控PI3K-Akt-mTOR通路信号及其传导途径研究。从而解析ISKNV调控细胞自噬的分子机制，为阐明ISKNV致病机制和找寻传染性脾肾坏死病防控新靶点提供指导。

传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus, ISKNV)是危害养殖鱼类重要病原，给鳜养殖业带来巨大经济损失。细胞自噬是真核生物抵抗外界干扰、维持正常能量代谢的保守机制，是病毒与宿主互作的重要方式之一。因此，开展ISKNV与宿主细胞互作研究对该病防控具有重要意义。针对ISKNV如何调控自噬的信号通路这一科学问题，本项目开展了ISKNV通过PI3K-AKT-p53-mTOR通路调控自噬研究，揭示了自噬、PI3K-AKT-p53-mTOR通路、 ISKNV复制增殖之间的关系，阐明了ISKNV调控宿主细胞自噬的分子机制。取得的主要成果如下：.1、发现ISKNV感染显著上调细胞PI3K-Akt-mTOR通路。.ISKNV感染早期，PI3Kr2、AKT、p53、mTOR在mRNA水平显著上调，LC3在mRNA水平显著下降；P-PI3K、P-AKT、p53、P-mTOR/mTOR在蛋白水平显著上调，LC3II/LC3I比值下降。说明ISKNV通过PI3K-AKT-p53-mTOR通路调控细胞自噬。.2、揭示了调控PI3K-Akt-mTOR通路可调节细胞自噬和ISKNV增殖。.激活PI3K-Akt-mTOR通路，LC3II/LC3I的比值下降，自噬被抑制，ISKNV产量增加；抑制PI3K-Akt-mTOR通路，LC3II/LC3I的比值上升，自噬被激活，ISKNV产量降低。证明调控PI3K-Akt-mTOR通路可调节细胞自噬和ISKNV增殖。.3、解析了ISKNV通过PI3K-Akt-p53-mTOR通路介导细胞自噬的信号传导机制。.ISKNV感染细胞后，刺激或过表达PI3K、AKT、p53，可激活PI3K-AKT-mTOR通路，抑制细胞自噬，上调先天免疫因子的表达如IRF3、IRF7、Mx，抑制ISKNV病毒增殖；抑制或敲降PI3K、AKT、p53，可抑制PI3K-AKT-mTOR通路，促进细胞自噬，下调先天免疫因子的表达，促进 ISKNV的胞内增殖。证明ISKNV通过PI3K-Akt-p53-mTOR通路介导细胞自噬的信号传导。.综上，本研究率先揭示了ISKNV通过PI3K-AKT-p53-mTOR通路调控鳜脑细胞自噬的分子机制，为ISKNV抗病毒治疗以及药物靶标的筛选提供了新的视角。