Cyclosporine, Cyclophilins and HCV Replication

描述(由申请人提供)：我们计划的广泛的、长期的目标是通过表征丙型肝炎病毒(丙型肝炎病毒)感染所必需的细胞辅助因素来促进病毒与宿主细胞相互作用的知识。丙型肝炎病毒感染全球1.7亿人，是肝细胞癌的主要原因。了解这种病毒如何与宿主相互作用对于诊断、抗病毒药物和预防性疫苗的开发至关重要。我们以前的研究表明，丙型肝炎病毒对培养的肝癌细胞的感染严重依赖于一种细胞蛋白-亲环素A(CyPA)，而去除该蛋白的功能不仅可以防止新的感染，而且可以抑制现有的感染。此外，CyPA是丙型肝炎病毒对环孢素A(CsA)及其衍生物耐药的主要媒介，环孢素A及其衍生物抑制丙型肝炎病毒复制的机制尚不明确，目前正作为候选抗丙型肝炎病毒药物进行临床试验。这些实验将研究决定CyPA的基本辅因子功能、CsA的作用以及相关的CsA耐药性的分子机制。将进行以下实验：(1)CsA和针对CyPA mRNA的小干扰RNA将被用来鉴定需要CyPA作为辅因子的丙型肝炎病毒复制酶的特异性功能。对病毒为什么以及在哪里需要这种细胞伴侣才能生存的详细了解，将为丙型肝炎病毒的复制策略提供新的视角。(2)生化分析和诱变将被用来表征CyPA和病毒复制酶之间的关键相互作用，这代表了一种新的病毒-宿主细胞相互作用的结构界面，可能被破坏用于治疗目的。(3)利用大分子相互作用和反向遗传学，剖析CsA的作用方式和CsA抗性的分子基础，最终达到预测和规避CsA抗性的目的。拟议的研究结果不仅将为人类病毒等高度成功的寄生虫如何劫持宿主细胞机器进行有效复制提供重要的见解，而且还将揭示抗病毒干预的新治疗靶点。公共卫生相关性：丙型肝炎病毒(丙型肝炎病毒)感染了世界3%的人口，并导致纤维化、肝硬变和肝细胞癌。目前尚不存在预防性疫苗，目前的治疗方法和正在开发的药物都面临严重的耐药性问题。这项拟议的研究将阐明新候选药物耐药的分子基础，并可能识别病毒-宿主细胞相互作用的一个新方面，而不是作为一类新药物的靶点。

DESCRIPTION (provided by applicant): The broad, long-term goal of our program is to advance the knowledge of virus-host cell interactions by characterizing cellular cofactors essential for hepatitis C virus (HCV) infection. HCV infects 170 million people worldwide and is major cause of hepatocellular carcinoma. Understanding how this virus interacts with the host is of critical importance to the development of diagnostics, antiviral drugs, and a prophylactic vaccine. Our previous studies demonstrated that HCV infection of cultured hepatoma cells is critically dependent on a cellular protein, cyclophilin A (CyPA), and that ablating the function of this protein can not only prevent new infections but also suppress an existing infection. In addition, CyPA is a principal mediator of HCV resistance to cyclosporine A (CsA) and its derivatives, which inhibit HCV replication with an undefined mechanism and are currently being evaluated in clinical trials as candidate anti-HCV drugs. The experiments proposed here will investigate the molecular mechanisms that determine the essential cofactor function of CyPA, the action of CsA, and the related CsA resistance. The following experiments will be performed: (1) CsA and small interfering RNA directed at CyPA mRNA will be used to identify the specific function of HCV replicase that requires CyPA as a cofactor. A detailed understanding of why and where the virus needs this cellular chaperone to survive will offer new perspectives on the replication strategy of HCV. (2) Biochemical assay and mutagenesis will be used to characterize the critical interaction between CyPA and the viral replicase, which represents a novel structural interface of virus-host cell interaction that may be disrupted for therapeutic purposes. (3) Macromolecular interaction and reverse genetics will be employed to dissect the mode of action for CsA and molecular basis of CsA resistance with the ultimate goal of predicting and circumventing that resistance. The results of the proposed studies will not only provide significant insights into how highly successful parasites such as human viruses hijack host cell machineries to replicate efficiently but also reveal new therapeutic targets for antiviral intervention. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) infects 3% of the world's population and cause fibrosis, liver cirrhosis and hepatocellular carcinoma (HCC). No prophylactic vaccine exists, and both the current treatment and the drugs in the development pipeline face serious drug-resistance issues. The proposed research will illustrate the molecular basis of drug resistance for a new drug candidate and may also identify a novel aspect of virus-host cell interaction than can serve as target for a new class of drugs.