Towards Developing type 1a HCV cell culture model

致力于开发 1a 型 HCV 细胞培养模型

• 批准号: 7178904
• 负责人: ASIM DASGUPTA
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7178904
• 关键词: 5' Untranslated Regions; Achievement; Address; Affect; Antiviral Agents; Antiviral Response; Attenuated; Biochemical; Cell Culture System; Cell Line; Cells; Cellular Structures; Chronic; Cirrhosis; Complementary DNA; Cultured Cells; DNA-Directed RNA Polymerase; Data; Development; Double-Stranded RNA; Elements; Encephalomyocarditis virus; Future; Genome; Genomics; Genotype; Goals; Hepatitis; Hepatitis C virus; Immunologic Techniques; Indium; Individual; Infection; Infectious hepatitides; Interferons; Internal Ribosome Entry Site; Laboratories; Lead; Length; Libraries; Liver; Liver Cirrhosis; Mediating; Modeling; Molecular Genetics; Mutation; North America; Patients; Peptide Initiation Factors; Primary carcinoma of the liver cells; Production; Protein Biosynthesis; Protein Kinase; Proteins; RNA; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Randomized; Rate; Reporting; Research; Research Personnel; Structural Protein; Structure; System; Techniques; Testing; Therapeutic; Transfection; Translations; Vaccines; Viral; Viral Genome; Viral Nonstructural Proteins; Viral Proteins; Virion; Virus; attenuation; base; cell type; eIF-2 Kinase; genetic analysis; improved; in vivo; mutant; novel; particle; programs; response; tissue culture; tissue/cell culture; viral RNA; virus host interaction

DESCRIPTION: Hepatitis C virus (HCV) causes chronic infection in a large proportion of infected individuals leading to hepatitis, liver cirrhosis and hepatocellular carcinoma. The type 1a (H77C) HCV is the most prevalent strain in North America, for which no tissue culture has been reported to date. The goal of this research is to develop a robust cell culture system for the H77C (type 1a) HCV, which makes infectious virus using a unique approach. The hypothesis that increasing the translation efficiency of the HCV internal ribosome entry site (IRES) by manipulating either the HCV IRES sequence (or structure) or cellular factors (that otherwise attenuate viral translation) may lead to increased replication of the HCV genome and consequently higher viral titers in cell culture will be tested. The rationale for utilizing this strategy is two fold: First, increased synthesis of viral RNA polymerase and other viral non-structural proteins should directly affect the rate and extent of viral RNA synthesis. Secondly, higher quantities of viral proteins should block HCV host evasion response more completely allowing efficient viral replication to occur. A novel in vivo fluorescent protein (EBFP/EGFP)-based screen will be used to screen a randomized H77C HCV IRES library to identify mutations that significantly enhance viral protein synthesis without compromising viral RNA replication. The stronger mutant IRES elements identified by the screen will be used to substitute the weaker wt H77C IRES element in the full-length infectious H77C clone. The efficiency of these mutant full-length constructs in directing viral protein and RNA synthesis as well as producing infectious H77C virus particle will be tested in Huh-7.5 cells by using biochemical and immunological techniques. The manipulation of cellular factors that attenuate viral RNA translation will involve silencing of the antiviral response protein, PKR (dsRNA-activated protein kinase) by SiRNA against PKR. The effect of PKR knockdown on viral protein and RNA synthesis will be determined by established biochemical techniques. Finally, the effect of PKR silencing on production of infectious virus by H77C RNA containing stronger IRES mutants will be determined. These studies should lead to development of a robust type 1a HCV tissue culture system amenable to molecular genetic analysis and virus-host interaction and improved strategies for future development of antiviral therapeutics against the type 1 a virus.

产品说明：丙型肝炎病毒（HCV）在大部分感染个体中引起慢性感染，导致肝炎、肝硬化和肝细胞癌。1a型（H77 C）HCV是北美最流行的病毒株，迄今为止尚未报道其组织培养。本研究的目标是开发一种用于H77 C（1a型）HCV的强大细胞培养系统，该系统使用独特的方法制备感染性病毒。通过操纵HCV内部核糖体进入位点（IRES）序列（或结构）或细胞因子（以其他方式减弱病毒翻译）增加HCV内部核糖体进入位点（IRES）的翻译效率可能导致HCV基因组复制增加，从而导致细胞培养物中病毒滴度升高的假设将被检验。利用这种策略的基本原理有两个方面：首先，病毒RNA聚合酶和其他病毒非结构蛋白的合成增加应直接影响病毒RNA合成的速率和程度。其次，更高数量的病毒蛋白应该更完全地阻断HCV宿主逃避应答，从而允许发生有效的病毒复制。一种新的基于体内荧光蛋白（EBFP/EGFP）的筛选将用于筛选随机化的H77 C HCV IRES文库，以鉴定显著增强病毒蛋白合成而不影响病毒RNA复制的突变。通过筛选鉴定的较强突变IRES元件将用于替代全长感染性H77 C克隆中较弱的wt H77 C IRES元件。将通过使用生物化学和免疫学技术在Huh-7.5细胞中测试这些突变全长构建体在指导病毒蛋白和RNA合成以及产生感染性H77 C病毒颗粒中的效率。减弱病毒RNA翻译的细胞因子的操纵将涉及通过针对PKR的SiRNA使抗病毒应答蛋白PKR（dsRNA活化蛋白激酶）沉默。PKR敲低对病毒蛋白质和RNA合成的影响将通过已建立的生物化学技术来确定。最后，将确定PKR沉默对含有更强IRES突变体的H77 C RNA产生感染性病毒的影响。这些研究应导致一个强大的1a型HCV组织培养系统的发展，适合分子遗传学分析和病毒-宿主相互作用，并为未来发展抗病毒治疗对1a型病毒的改进策略。