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

描述（由申请人提供）：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。目前没有可用的丙肝病毒疫苗，目前利巴韦林-干扰素联合治疗仅对一小部分感染者有效。我们实验室最近的研究结果导致了高分辨率、定量、基因组规模突变分析系统的发展，以研究HCV顺式作用元件，这些元件在细胞培养中对JFH1（2型）HCV复制至关重要。在本研究中发现的HCV IRES （5'UTR）内的许多插入被发现可以显著增加Huh-7.5.1细胞中感染性病毒的产量（约50倍于wt）。我们实验室的其他研究已经确定HCV 5'UTR内的RNA二级结构/序列似乎在感染性病毒的产生中起重要作用。与wt病毒相比，这个区域的小插入导致感染性病毒的产生高达100-500倍。初步研究表明，突变体与wt病毒之间的病毒蛋白质合成、蛋白质加工和RNA合成相似。突变体5'UTR的IRES活性与wt 5'UTR相比也保持不变。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，假设这些突变导致病毒RNA的有效包装/组装是合乎逻辑的。本提案将利用遗传、生化和免疫学方法来检验JFH1 HCV 5'UTR定义区域内突变增强/减少RNA包装/组装/释放的假设。上述实验的成功完成将确定有效组装和释放感染性病毒颗粒所需的HCV RNA结构/序列，并为开发干扰病毒组装的治疗性抗病毒药物提供基础。

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.