The role of hepatitis C virus 5' untranslated region in virus morphogenesis

描述（由申请方提供）：丙型肝炎病毒（HCV）引起人类感染，导致慢性肝炎、肝硬化和肝细胞癌。目前没有可用的HCV疫苗，并且目前的利巴韦林-干扰素α组合治疗仅对一部分感染个体有效。我们实验室的最新结果导致了一个高分辨率，定量，基因组规模的突变分析系统的发展，以研究HCV的顺式作用元件，是必不可少的JFH 1（2型）HCV在细胞培养中复制。发现在本研究中鉴定的HCV IRES（5 'UTR）内的许多插入显著增加了Huh-7.5.1细胞中感染性病毒的产量（相对于wt约50倍）。我们实验室的其他研究已经鉴定了HCV 5 'UTR内的RNA二级结构/序列，其似乎在感染性病毒的产生中起重要作用。与野生型相比，具有该区域的小插入导致高达100-500倍的感染性病毒的产生。初步研究表明，突变体病毒的蛋白质合成、蛋白质加工和RNA合成与野生型病毒相似。与野生型5 'UTR相比，突变型5' UTR的IRES活性也保持不变。考虑到突变的位置和背景（在高度结构化的5 'UTR内）以及许多病毒的包装信号位于5' UTR内的事实，合乎逻辑的是假设这些突变导致病毒RNA的有效包装/组装。该提案将利用遗传学、生物化学和免疫学方法来检验JFH 1 HCV 5 'UTR的限定区域内的突变增强/减少RNA包装/组装/释放的假设。成功完成拟议的实验应确定HCV RNA结构/序列所需的有效组装和释放的感染性病毒颗粒，以及提供基础的治疗性抗病毒药的开发，干扰病毒组装。 公共卫生相关性：丙型肝炎病毒（HCV）引起人类感染，导致慢性肝炎、肝硬化和肝细胞癌（肝癌）。目前没有可用的HCV疫苗，并且目前的利巴韦林-干扰素α组合治疗仅对一部分感染个体有效。该提案将研究单个病毒组分（病毒遗传物质和病毒蛋白）如何在培养的肝细胞中组装形成感染性病毒颗粒。从这项研究中获得的知识应该有助于开发新的抗病毒治疗策略来干扰病毒组装过程。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) causes infection of humans leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Currently there is no available vaccine for HCV and current treatment of ribavarin-interferon alpha combination is only effective in a fraction of infected individuals. Recent results from our laboratory have led to development of a high resolution, quantitative, genome scale mutational analysis system to study HCV cis-acting elements that are essential for JFH1 (type 2) HCV replication in cell culture. A number of insertions within the HCV IRES (5'UTR) identified in this study were found to increase significantly (~50 fold over wt) the yield of infectious virus in Huh-7.5.1 cells. Additional studies from our laboratory have identified RNA secondary structures/sequences within the HCV 5'UTR that appear to play important role in production of infectious virus. Small insertions with this area lead to production of infectious virus by as much as 100-500 fold compared to the wt. Preliminary studies showed that viral protein synthesis, protein processing and RNA synthesis were similar between the mutants and wt virus. The IRES activity of the mutant 5'UTRs also remained unchanged compared with the wt 5'UTR. Considering the location and context of the mutation (within the highly structured 5'UTR) and the fact that the packaging signal for many viruses are located within the 5' UTR, it is logical to assume that these mutations lead to efficient packaging/assembly of viral RNA. This proposal will utilize genetic, biochemical, and immunological approaches to examine the hypothesis that mutations within defined regions of the JFH1 HCV 5'UTR enhance/reduce RNA packaging/assembly/release. Successful completion of the proposed experiments should identify HCV RNA structures/sequences required for efficient assembly and release of infectious virus particles as well as provide the basis for development of therapeutic antiviral agents that interfere with virus assembly. PUBLIC HEALTH RELEVANCE: Hepatitis C virus (HCV) causes infection of humans leading to chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (liver cancer). Currently there is no available vaccine for HCV and current treatment of ribavarin-interferon alpha combination is only effective in a fraction of infected individuals. This proposal will examine how the individual viral components (viral genetic material and viral proteins) assemble to form infectious virus particles in cultured liver cells. The knowledge gained from this study should help develop novel antiviral therapeutic strategies to interfere with the virus assembly process.