Iron metabolism and hepatitis C virus (HCV) replication

丙型肝炎病毒（HCV）感染在世界范围内构成严重的卫生保健问题，并且是血液传播的慢性肝炎、肝硬化和肝细胞癌的主要原因。HCV感染的过程受各种因素的影响，包括身体的铁状态。我们打算在亚基因组HCV复制（“复制子”）的模型细胞培养系统中研究HCV病毒蛋白影响细胞铁代谢的机制。我们以前利用这个系统，并证明了铁复制子细胞的管理显着降低HCV复制。该机制涉及铁介导的病毒RNA聚合酶的抑制（Fillebeen等（2005）J.Biol.Chem.280，9049-9057）。在这里，我们计划扩展这些研究，并利用复制子模型系统来解决HCV病毒蛋白如何影响细胞铁代谢（具体目标I）。基于我们的研究结果，干扰素在HCV复制中的抑制作用以及铁代谢和干扰素信号传导之间公认的串扰，我们还将探索铁和细胞因子在抑制病毒复制中的潜在协同作用（具体目标II）。常规的生物化学和分子生物学技术，如Western和北方印迹，将用于分析亲本和复制子细胞中铁代谢基因的表达，包括铁蛋白、转铁蛋白受体、IRP 1、IRP 2、膜铁转运蛋白、DMT 1和铁调素。此外，高通量筛选，如微阵列分析和蛋白质组学方法将用于评估复制子的存在如何干扰对铁扰动和干扰素信号传导的稳态应答。

Infection with hepatitis C virus (HCV) poses a serious health care problem worldwide and is the leading cause of blood-transmitted chronic hepatitis, liver cirrhosis and hepatocellular cancer. The course of HCV infection is affected by various factors, including the body's iron status. We intend to study the mechanisms by which HCV viral proteins affect cellular iron metabolism in a model cell culture system for subgenomic HCV replication ("replicon"). We have previously utilized this system and demonstrated that administration of iron to replicon cells dramatically decreases HCV replication. The mechanism involves iron-mediated inhibition of the viral RNA polymerase (Fillebeen et al (2005) J. Biol. Chem. 280, 9049-9057). Here we plan to extend these studies and utilize the replicon model system to address how HCV viral proteins affect cellular iron metabolism (specific aim I). On the basis of our findings, the inhibitory effects of interferons in HCV replication and the well-established cross talk between iron metabolism and interferon signaling, we will also explore potential synergistic effects of iron and cytokines in the inhibition of viral replication (specific aim II). Conventional biochemical and molecular biology techniques, such as Western and Northern blotting, will be used to analyze the expression of iron metabolism genes, including ferritin, transferrin receptor, IRP1, IRP2, ferroportin, DMT1 and hepcidin in parent and replicon cells. In addition, high throughput screens, such as microarray analysis and proteomics approaches will be employed to assess how the presence of the replicon interferes with homeostatic responses to iron perturbation and interferon signaling.