【作者】 彭辂；

【导师】 袁正宏； 童文燕； 易志刚；

【作者基本信息】 复旦大学 ， 病原生物学， 2010， 博士

【摘要】 丙型肝炎病毒(hepatitis C virus, HCV)是引起急、慢性肝炎的重要肝嗜性病毒之一。然而,目前对HCV感染慢性化和免疫逃逸的致病机理仍然缺乏全面深入的理解。要建立持续性感染,病毒必须调控参与细胞存活、生长、生物大分子合成和代谢的关键信号通路。哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin, mTOR)信号通路即为这样的信号通路。已发现多种DNA病毒调控mTOR通路以促进持续性感染和致病。同时,凋亡作为维持细胞稳定性的重要生理机制,其功能失调常常见于持续性病毒感染。我们前期发表的结果表明HCV非结构蛋白5A (nonstructural protein 5A, NS5A)结合细胞蛋白FKBP38从而抑制细胞凋亡,但其机理不详。近期发现FKBP38为mTOR通路新成员,在营养或生长因子控制下,FKBP38结合并抑制mTOR激酶活性。为了进一步理解NS5A的生物学活性以及HCV持续性感染的致病机理,本实验研究了HCV NS5A对mTOR信号通路的调节及其对细胞存活的影响。在人肝癌细胞系Huh7中过表达HCV NS5A的结果表明,血清饥饿条件下NS5A以时间和剂量依赖方式增加mTOR下游靶点S6K1和4EBP1磷酸化水平。而在10%血清存在条件下,NS5A不能提高S6K1和4EBP1磷酸化水平。同样,在血清饥饿条件下,NS5A稳定表达的Huh7细胞(NS5A-Huh7)和HCV亚基因组复制子细胞中,S6K1和4EBP1磷酸化水平明显高于对照细胞(neo-Huh7)。mTOR特异性抑制剂雷帕霉素(rapamycin)或NS5A特异性的siRNA (siNS5A)可阻断NS5A对S6K1和4EBP1的磷酸化作用,提示NS5A特异性的活化mTOR信号通路。但是,在NS5A-Huh7细胞中,PI3K特异性抑制剂LY294002不能阻断NS5A对S6K1和4EBP1的磷酸化作用,表明NS5A活化mTOR通路不依赖于其上游组分PI3K。进一步运用NS5A突变体(NS5A-AI,不与FKBP38结合)、FKBP38突变体(FKBP38-Δ3×TPR,不与NS5A结合)以及FKBP38特异性siRNA (siFKBP38)研究发现,NS5A对mTOR通路的活化作用依赖于NS5A与FKBP38的结合。为了探索NS5A活化mTOR信号通路的机制,我们考察了NS5A、FKBP38、mTOR三者的结合情况。在过表达NS5A的Hela和Huh7细胞中,运用GST-pull down方法证明,在血清饥饿条件下,GST-FKBP38与mTOR的结合消失,而GST-FKBP38仍与NS5A结合。这些结果在NS5A-Huh7和HCV亚基因组复制子细胞中得到了一致的验证。更重要的是,在NS5A-Huh7和HCV亚基因组复制子细胞中,GST-FKBP38-Δ3×TPR可回复性结合mTOR。这些结果提示NS5A可破坏FKBP38与：mTOR的结合。与GST-pull down结果相同,在同时过表达FKBP38与NS5A的Huh7细胞中,免疫共沉淀实验表明,血清饥饿条件下FKBP38与mTOR的结合消失,FKBP38仍与NS5A结合。同时,有无血清并不影响NS5A-FKBP38的结合。单独过表达FKBP38且无血清存在时,FKBP38与mTOR结合,而有血清存在时两者结合消失。NS5A与mTOR之间不存在直接或间接结合。这些结果在neo-Huh7、NS5A-Huh7及HCV亚基因组复制子细胞中得到了进一步验证。另外,同时过表达NS5A-ΔI和FKBP38、或NS5A和FKBP38-Δ3×TPR时,可恢复mTOR与FKBP38的结合。共定位分析发现,血清饥饿条件下,NS5A-Huh7、HCV Replicon细胞中FKBP38与mTOR的共定位消失。这些结果提示NS5A是通过与mTOR竞争结合后者内源性抑制剂FKBP38,从而活化mTOR信号通路。以往研究表明mTOR信号通路活化可促进细胞存活。因此,我们进一步在无血清存在条件下利用凋亡诱导剂星孢菌素(stauroporine)考察了NS5A活化mTOR信号通路对细胞凋亡的影响。结果发现,无rapamycin预处理的NS5A-Huh7中,caspase3和PARP活化水平明显低于rapamycin预处理的NS5A-Huh7细胞、rapamycin处理或不处理的对照neo-Huh7细胞。Hoechst33342染色结果也表明,rapamycin未处理的NS5A-Huh7凋亡数量明显低于rapamycin处理的NS5A-Huh7细胞、以及rapamycin处理或不处理的对照neo-Huh7细胞。而且,NS5A特异性siRNA可明显恢复NS5A-Huh7细胞对staurosporine诱导凋亡的敏感性,表现为caspase3和PARP活化水平明显升高。这些结果提示NS5A通过mTOR信号通路特异性抑制细胞凋亡。进一步实验表明,同时过表达NS5A和FKBP38且无rapamycin预处理的Huh7细胞中,caspase3、PARP活化水平低于同时过表达NS5A和FKBP38-Δ3×TPR、或NS5A-ΔI和FKBP38且rapamycin预处理或不预处理的Huh7细胞。与此一致,用FKBP38特异性siRNA (siFKBP38)单独处理NS5A-Huh7、HCV亚基因组复制子细胞发现,其caspase3、PARP活化水平明显低于rapamycin联合siFKBP38处理或rapamycin单独处理的细胞。Hoechst33342染色也得到一致的结果。这些结果提示,NS5A通过mTOR信号通路抑制细胞凋亡依赖于NS5A-FKBP38结合。上述结果表明,HCV NS5A通过与mTOR竞争结合后者内源性抑制剂FKBP38,活化mTOR信号通路,从而抑制细胞凋亡、促进细胞存活。提示HCV编码的病毒蛋白可通过调控细胞关键信号通路,从而促进HCV持续性感染、参与HCV致病机制。更多还原

【Abstract】 Hepatitis C virus (HCV) is one of important hepatotropic viruses that cause acute, chronic hepatitis. However, the mechanisms underlying HCV persistence, immune evasion and pathogenesis have been incompletely understood. To establish persistent infection, a virus must manipulate key cellular signaling pathways that control cellular survival, growth, macromolecular biosynthesis and metabolism. The mammalian target of rapamycin (mTOR) pathway is one such pathway. It has been found that many DNA viruses evolved strategies to regulate the mTOR pathway for persistent infections and pathogenesis. Meanwhile, apoptosis is a critically physiological mechanism for maintaining cellular homeostasis, its dysfunction is usually found in persistent viral infections. Our previously published results have proved that HCV nonstructural protein 5A (NS5A) bound to cellular FKBP38 and led to inhibiting apoptosis, but the detailed mechanism was not defined. Recent researches have discovered that as a new member of the mTOR pathway, FKBP38 interacted with mTOR and suppressed the latter’s kinase activity dependent on nutrition and growth factors availability. Therefore, in order to further understand the biological activity of NS5A and the mechanism of HCV persistent infection and pathogenesis, we investigated the effect of hepatitis C virus nonstructural protein 5A on the mTOR Pathway and its corresponding role in cellular survival.Initially, we overexpressed HCV NS5A in Huh7 cells in the absence of serum, the results showed that NS5A significantly increased phosphorylation levels of two mTOR-controlled substrates, S6K1 and 4EBP1, in a time-and dose-dependent manner; but under serum-supplemented condition, NS5A was unable to promote S6K1 and 4EBP1 phosphorylations. Similarly, the phosphorylation levels of S6K1 and 4EBP1 in NS5A-Huh7 or HCV subgenomic replicon cells were obviously higher than those of control neo-Huh7 cells. siRNA special for NS5A or mTOR kinase inhibitor rapamycin blocked the increased S6K1 and 4EBP1 phosphorylations mediated by NS5A, suggesting that NS5A specifically activates the mTOR pathway. However, PI3K inhibitor LY294002 could not abolish NS5A-mediated phosphorylations of S6K1 and 4EBP1 in NS5A-Huh7 cells in the absence of serum, indicating that NS5A-upregulated mTOR activity is independent of PI3K, a component upstream of mTOR. Additionally, overexpression of deleted mutants (NS5A-AI with FKBP38-binding region deleted, FKBP38-Δ3×TPR with NS5A-binding region deleted) in Huh7, and knockdown of FKBP38 in NS5A-Huh7 and HCV replicon cells showed that NS5A-activated mTOR pathway was dependent on NS5A-FKBP38 binding.To exploit the mechanism of NS5A-activated mTOR pathway, we further investigated the binding among NS5A, FKBP38 and mTOR. GST pull-down experiments showed that in NS5A-overexpressed Hela and Huh7 cellls in the absence of serum, GST-FKBP38 was unable to pull down mTOR, while GST-FKBP38 could still pull down NS5A. These results were confirmed in NS5A-Huh7 and HCV subgenomic replicon cells. More importantly, GST-FKBP38-Δ3×TPR reversibly pulled down mTOR in NS5A-Huh7 and HCV subgenomic replicon cells. These results suggested that NS5A impairs mTOR-FKBP38 binding. In accordance with GST pull-down results, coimmunoprecipitation experiments showed that in Huh7 cells cotransfected with NS5A and FKBP38 with no serum culture, the mTOR-FKBP38 binding disappeared, while the NS5A-FKBP38 binding kept intact, in which presence or absence of serum did not affect the binding of NS5A to FKBP38. In FKBP38 alone overexpressed Huh7 cells, mTOR kept binding to FKBP38 under serum-deprived condition, while this interaction disappeared under serum-supplemented condition. These results were confirmed in neo-Huh7, NS5A-Huh7 and HCV subgenomic replicon cells. Furthermore, in Huh7 cells cotransfected with NS5A-AI and FKBP38, or NS5A and FKBP38-Δ3×TPR in the absence of serum, the association between mTOR and FKBP38 was recovered. Colocalization analyses showed that the colocalization of mTOR and FKBP38 disappeared in NS5A-Huh7 and HCV replicon cells in the absence of serum. Collectively, these results indicated that NS5 A activates the mTOR pathway via competing with mTOR for binding to its intrinsic antagonist FKBP38.Previous reports have showed that the activation of the mTOR pathway contributed to cellular survival. Based on these data, we further examined the effect of NS5A-activated mTOR pathway on apoptosis induced by staurosporine in the absence of serum. The results showed that the levels of cleaved caspase 3 and cleaved PARP in NS5A-Huh7 cells without rapamycin pretreatment were much lower than those in NS5A-Huh7 cells with rapamycin pretreatment and those in neo-Huh7 cells with or without rapamycin pretreatment. Consistently, Hoechst33342 staining showed that the numbers of apoptotic cells in NS5A-Huh7 cells without rapamycin pretreatment were fewer than those in NS5A-Huh7 cells with rapamycin pretreatment and those in neo-Huh7 cells with or without rapamycin pretreatment. Furthermore, siRNA special for NS5A recovered the sensitivity of NS5A-Huh7 cells to apoptosis induced by staurosporine, characterized by remarkable increase of cleaved caspase 3 and cleaved PARP. These results indicated that NS5A specifically represses apoptosis via the mTOR pathway. Finally, the levels of cleaved caspase 3 and cleaved PARP in the Huh7 cells cotransfected with wild type NS5A and FKBP38 without rapamycin pretreatment were lower than those in the Huh7 cells cotransfected with wild type NS5A and FKBP38-Δ3×TPR, or NS5A-ΔI and wild type FKBP38 with or without rapamycin pretreatment. Consistantly, FKBP38 knockdown dramatically decreased the leveles of cleaved caspase 3 and cleaved PARP in NS5A-Huh7 and HCV replicon cells, which were much lower than those in cells with a combined treatment of rapamycin and siFKBP38, or rapamycin treatment alone; also, Hoechst33342 staining produced similar results. These results suggested that the apoptotic repression via the NS5A-activated mTOR pathway is dependent on NS5A-FKBP38 binding.Taken together, HCV NS5A activates the mTOR pathway to inhibit apoptosis and contribute to cellular survival via competing with mTOR for binding to the latter’s intrinsic antagonist FKBP38, which suggests that HCV-encoded viral protein can manipulate cellular key signaling pathway-the mTOR pathway-for HCV persistent infection and pathogenesis.更多还原