Cyclophilins and the IFN Response

项目总结。亲环素(Cyp)抑制剂(CypI)在PASSE I期丙型肝炎患者中的临床效力很高 和II研究。最近的一项I期研究表明，每天服用Cypi可以减少病毒血症和 迅速增加血浆中干扰素、1和3以及2‘5’OAS-1的浓度。血浆的变化 所有标志物的浓度与CypI的血浆浓度变化一致。这些小说 吸引人的数据揭示了细胞色素P450和干扰素反应之间的第一个联系。我们能够部分地 在体外复制患者数据。具体地说，CypI可促进人外周血中干扰素和2‘5’OAS-1的分泌 复制细胞。这些数据表明，干扰素反应的激活可能代表着一种机制，通过 其中Cypi发挥其临床抗病毒活性。我们调查了细胞内靶标的可能性 CypI，CypA，与干扰素反应的组件结合。值得注意的是，我们发现CypA与干扰素结合 调节因子9(IRF9)通过其异构酶口袋。CypI阻止IRF9-CypA相互作用。CypA还绑定到 IRF3、5和7，表明CypA与所有IRF成员绑定。我们的工作首次证明了 CypA与干扰素反应的一类主要元件--IRF--特异性结合。它还揭示了一部小说 有机会调节干扰素的反应。在本应用程序中，我们建议通过执行 一系列实验旨在剖析CypA在干扰素反应中的作用。此应用程序应该 阐明CypA中和如何触发干扰素的产生，并确定CypI介导的干扰素 产物代表了CypI抗病毒作用的一种新机制。此应用程序还应确定 CypA对IRF施加了什么动作或功能。有趣的是，我们最新的研究表明，CypA，通过 与红外线分子相互作用，极大地影响其泛素化程度。我们提出了一套新的实验 目的确定CypA介导的IRF泛素化效应和CypI介导的IRF泛素化效应之间的联系 激活干扰素反应。我们发现CypI能刺激丙型肝炎病毒分离的PBMC释放干扰素 患者开启了令人兴奋的新问询路线。这一体外实验可能会让我们确定CypI 应答者比CypI非应答者产生更多的干扰素，以及CypI反应是基因型还是病毒 滴度特异值。如果发生这种情况，我们将利用这些信息为受试者开发一个经验性的预筛选， 他们很可能对包含Cypi的方案有反应。我们最近发现Cypi对小鼠的管理 显著抑制与丙型肝炎病毒无关但对丙型肝炎病毒高度敏感的病毒的传染性 干扰素。此外，我们发现，与cypi治疗相比，感染小鼠的干扰素血浆水平更高。 在未经治疗的感染小鼠中，提示CypI干扰素的诱导是一种更广泛使用的机制。 这些了不起的初步发现开启了令人兴奋的调查的新线索。这样做的最终目的是 应用是为了提高我们对Cyps在干扰素应答病毒感染中的作用的理解。

PROJECT SUMMARY. Cyclophilin (Cyp) inhibitors (CypI) are clinically highly potent in HCV patients in pahse I and II studies. A recent phase I study showed that the daily administration of a CypI reduces viremia and rapidly increases plasma concentrations of IFN¿, ¿1 and ¿3, and 2'5'OAS-1. Changes in plasma concentrations for all markers were coincident with changes in plasma concentration of the CypI. These novel and attractive data revealed the first link between Cyps and the IFN response. We were able to partly reproduce the patient data in vitro. Specifically, CypI enhance secretion of IFN¿, ¿ and 2'5'OAS-1 from replicon cells. These data suggest that activation of the IFN response may represent a mechanism through which CypI exert their clinical antiviral activity. We investigated the possibility that the intracellular target for CypI, CypA, binds to components of the IFN response. Remarkably, we found that CypA binds to the IFN regulatory factor 9 (IRF9) via its isomerase pocket. CypI prevent IRF9-CypA interactions. CypA also binds to IRF3, 5 and 7, suggesting that CypA binds to all IRF members. Our work demonstrates for the first time that CypA binds specifically to a major class of elements of the IFN response - IRFs. It also reveals a novel opportunity to modulate the IFN response. In this application, we propose to extend this work by conducting a series of experiments aimed at dissecting the roles of CypA in the IFN response. This application should elucidate how CypA neutralization triggers IFN production and determine whether the CypI-mediated IFN production represents a new mechanism of antiviral action of CypI. This application should also determine what action or function CypA exerts on IRFs. Interestingly, our most recent work suggests that CypA, by interacting with IRFs, greatly impacts their degree of ubiquitination. We propose a new set of experiments aimed at identifying the link between the CypA-mediated IRF ubiquitination effect and the CypI-mediated activation of the IFN response. Our finding that CypI triggers IFN release from PBMCs isolated from HCV patients opens exciting new lines of enquiries. This ex vivo assay may allow us to determine whether CypI responders produce more IFN than CypI non-responders and whether the CypI response is genotype- or virus titer-specific. If this occurs, we then would use this information to develop an empiric pre-screen for subjects, who are likely to respond to a regimen comprising a CypI. We recently found that CypI administration to mice significantly inhibits the infectivity of a virus, which is unrelated to HCV, but which is also highly sensitive to IFN. Moreover, we found that the IFN plasma levels were superior in infected mice treated with CypI compared to those in untreated infected mice, suggesting that the CypI IFN induction is a more widely used mechanism. These remarkable preliminary finding opened new lines of exciting enquiries. The ultimate goal of this application is to improve our understanding of the role of Cyps in the IFN response to viral infection.