Studies On The Effect Of Hepatitis C Virus Nonstructural Protein 5A On The Mammalian Target Of Rapamycin Pathway And Its Corresponding Function

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.