Cyclosporine, Cyclophilins and HCV Replication

描述（由申请人提供）：我们计划的广泛，长期目标是通过表征丙型肝炎病毒（HCV）感染所必需的细胞辅因子来促进病毒-宿主细胞相互作用的知识。HCV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，培养的肝癌细胞的HCV感染是严重依赖于细胞蛋白，亲环素A（CyPA），和消融这种蛋白质的功能，不仅可以防止新的感染，但也抑制现有的感染。此外，CyPA是HCV对环孢菌素A（CsA）及其衍生物的耐药性的主要介导物，其以不确定的机制抑制HCV复制，并且目前正在临床试验中作为候选抗HCV药物进行评估。这里提出的实验将调查的分子机制，确定的重要辅因子功能的CyPA，CsA的行动，和相关的CsA耐药。将进行以下实验：（1）使用CsA和针对CyPA mRNA的小干扰RNA来鉴定需要CyPA作为辅因子的HCV复制酶的特定功能。详细了解病毒为什么以及在哪里需要这种细胞伴侣才能生存，将为HCV的复制策略提供新的视角。(2)生化测定和诱变将用于表征CyPA和病毒复制酶之间的关键相互作用，这代表了病毒-宿主细胞相互作用的新型结构界面，其可以被破坏用于治疗目的。(3)将采用大分子相互作用和反向遗传学来剖析CsA的作用模式和CsA抗性的分子基础，最终目标是预测和规避该抗性。拟议研究的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制以有效复制提供重要见解，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（HCV）感染世界人口的3%，并引起纤维化、肝硬化和肝细胞癌（HCC）。没有预防性疫苗存在，目前的治疗和正在开发的药物都面临严重的耐药性问题。拟议的研究将说明新候选药物耐药性的分子基础，也可能确定病毒-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.