Towards Developing type 1a HCV cell culture model

丙型肝炎病毒(丙型肝炎病毒)在很大一部分感染者中引起慢性感染，导致肝炎、肝硬变和肝细胞癌。1a型(H77C)丙型肝炎病毒是北美最流行的毒株，迄今为止还没有关于这种毒株的组织培养的报道。这项研究的目标是开发一种强大的H77C(1a型)丙型肝炎病毒的细胞培养系统，它使用一种独特的方法制造传染性病毒。通过操纵丙型肝炎病毒内部核糖体进入位点(IRES)的序列(或结构)或细胞因子(否则会减弱病毒翻译)来提高丙型肝炎病毒内部核糖体进入位点(IRES)的翻译效率的假设可能会导致丙型肝炎病毒基因组的复制增加，从而在细胞培养中获得更高的病毒滴度。使用这一策略的理由有两个：第一，病毒RNA聚合酶和其他病毒非结构蛋白合成的增加应该直接影响病毒RNA合成的速度和程度。其次，较高数量的病毒蛋白应能更彻底地阻止丙型肝炎病毒宿主逃避反应，从而实现有效的病毒复制。一种新的基于体内荧光蛋白(EBFP/EGFP)的筛选将用于筛选随机的H77C丙型肝炎病毒IRES文库，以确定在不影响病毒RNA复制的情况下显著增强病毒蛋白质合成的突变。筛选出的突变较强的IRES元件将用于替代全长感染性克隆H77C中较弱的wt H77C IRES元件。这些突变的全长结构在指导病毒蛋白质和RNA合成以及产生传染性H77C病毒颗粒方面的效率将在Huh-7.5细胞中通过生化和免疫学技术进行测试。抑制病毒RNA翻译的细胞因素的操纵将涉及针对抗病毒反应蛋白PKR的siRNA沉默PKR(dsRNA激活的蛋白激酶)。PKR基因敲除对病毒蛋白质和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H77C RNA产生感染性病毒的影响。这些研究将导致开发一种强大的1a型丙型肝炎病毒组织培养系统，该系统适用于分子遗传分析和病毒-宿主相互作用，并为未来开发针对1型a型病毒的抗病毒治疗药物改进策略。

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.