Iron metabolism and hepatitis C virus (HCV) replication

丙型肝炎病毒感染是世界范围内严重的卫生保健问题，是血液传播的慢性肝炎、肝硬变和肝细胞癌的主要原因。丙型肝炎病毒的感染过程受到多种因素的影响，包括身体的铁状态。我们打算在丙型肝炎病毒亚基因组复制的模型细胞培养系统中研究丙型肝炎病毒病毒蛋白影响细胞铁代谢的机制(复制子)。我们以前利用过这个系统，并证明了给复制子细胞注射铁可以显著降低丙型肝炎病毒的复制。其机制涉及铁介导的对病毒RNA聚合酶的抑制(Fillebe等人(2005)J.Biol。化学。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丙型肝炎病毒病毒蛋白如何影响细胞铁代谢(特定目标I)。根据我们的发现，干扰素对丙型肝炎病毒复制的抑制作用，以及铁代谢和干扰素信号之间公认的串扰，我们还将探索铁和细胞因子在抑制病毒复制方面的潜在协同作用(特定目标II)。传统的生化和分子生物学技术，如Western和Northern blotting，将被用来分析铁代谢基因在亲本细胞和复制子细胞中的表达，包括铁蛋白、转铁蛋白受体、Irp1、IRP2、铁端口蛋白、DMT1和海普西丁。此外，高通量筛选，如微阵列分析和蛋白质组学方法，将被用来评估复制子的存在如何干扰对铁干扰和干扰素信号的稳态反应。

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.