Transmitted/Founder HCV clones as targets for treatment and eradication.

描述(申请人提供)：丙型肝炎病毒(丙型肝炎病毒)在自然界中作为一种遗传多样性的准种存在。在全球范围内，该病毒由七种主要基因类型(GT1-7)代表，它们的核苷酸序列差异高达30%-35%。在了解丙型肝炎病毒生物学和病毒-宿主相互作用导致病毒持久或根除方面取得进展的一个关键障碍是在产生在体外和体内有效复制的全长分子病毒克隆方面面临的特殊挑战。到目前为止，只有一个GT 2a克隆pJFH1及其嵌合衍生物在体外和体内都能有效复制。这也阻碍了开发有效的治疗剂和治疗战略来根除不同的病毒基因类型。本申请旨在通过采用我们实验室基于单基因组测序(SGS)开发的新的实验策略来克服这一科学障碍，以识别和克隆导致人类生产性临床感染的实际传播的/创始(T/F)的丙型肝炎病毒全长基因组。我们的假设是，T/F基因组根据定义包含了在人类中产生病毒感染和复制所必需和充分的所有遗传元素，将在组织培养和小动物模型中复制，概括了丙型肝炎病毒的生物学，并允许对这些病毒对干扰素(IFN)、直接作用抗病毒药物(DAA)和其他新型治疗干预措施的敏感性进行详细的分子评估。我们的实验方法改编自我们最近对HIV-1的工作，其中我们证明了可以识别、克隆、表达全长、具有复制能力的传播的HIV-1基因组，并分析对传播和生产性感染至关重要的生物学特性(J Exp Med 206：1273,2009；PLoS pathog 6：e1000890,2010；PLoS pathog 8：e1002686,2012)。最近，我们通过分析17名急性感染的人类受试者的亚基因组(5‘半基因组)血浆vRNA序列，证明了这种方法用于识别T/F丙型肝炎病毒基因组的可行性(Li等人，PLoS pathog，在出版社)。在目前的项目中，具体目标是：(I)通过SGS从急性感染的人类受试者(R21)中鉴定与GT 1a、1b、2b和3a相对应的全长T/F丙型肝炎病毒基因组；(Ii)化学合成和克隆这些基因组并启动它们在人肝嵌合小鼠(R21)中的复制；(Iii)分析表达的T/F基因组在体外生物测试中的复制能力、病毒子产量、感染性以及对I、II和III型IFN和DAAs的敏感性(R33)；和(Iv) 扩大丙型肝炎病毒T/F克隆的数量和基因类型表示，包括GT 1a、1b、2b、3a和4a(每个克隆n=3)，确定干扰素和DAA敏感性的基因型相关决定因素，并在组织培养和小动物模型中评估实现导致病毒根除的治疗方法之间的协同作用的策略(R33)。这项拟议的工作将为丙型肝炎病毒复制和持久性的分子生物学提供新的见解，并促进根除全球流行的丙型肝炎病毒基因型的战略。

DESCRIPTION (provided by applicant): Hepatitis C virus (HCV) exists in nature as a genetically diverse quasispecies. Globally, the virus is represented by seven major genotypes (gt 1-7) that differ by as much as 30-35% in nucleotide sequence. A critical obstacle to progress in understanding HCV biology and virus-host interactions leading to virus persistence or eradication has been the extraordinary challenges faced in generating full-length molecular viral clones that replicate efficiently in vitro and in vivo. To date, there is only a single gt 2a clone pJFH1, and chimeric derivatives thereof, that replicate efficiently in vitro and in vivo. This has also hindered the development of effective therapeutic agents and treatment strategies for eradication of different viral genotypes. The present application aims to overcome this scientific roadblock by employing a novel experimental strategy developed in our laboratory based on single-genome sequencing (SGS), to identify and clone actual transmitted/founder (T/F) full-length HCV genomes responsible for productive clinical infection in humans. Our hypothesis is that T/F genomes, which by definition contain all of the genetic elements that are necessary and sufficient for productive virus infection and replication in humans, will replicate in tissue cultue and in small animal models, recapitulating the biology of HCV, and allowing for a detailed molecular assessment of the sensitivity of these viruses to interferons (IFNs), direct-acting antiviral agents (DAA) and other novel therapeutic interventions. Our experimental approach is adapted from our recent work with HIV-1 where we demonstrated that full-length, replication-competent, transmitted HIV-1 genomes could be identified, cloned, expressed and analyzed for biological properties that are essential for transmission and productive infection (J Exp Med 206:1273, 2009; PLoS Pathog 6:e1000890, 2010; PLoS Pathog 8:e1002686, 2012). More recently, we demonstrated the feasibility of this approach for identifying T/F HCV genomes by analyzing subgenomic (5' half-genome) plasma vRNA sequences in 17 acutely infected human subjects (Li et al., PLoS Pathog, in press). In the current project, specific aims are to: (i) molecularly identify by SGS full-length T/F HCV genomes corresponding to gt 1a, 1b, 2b and 3a from acutely-infected human subjects (R21); (ii) chemically synthesize and clone these genomes and launch their replication in human liver chimeric mice (R21); (iii) analyze the expressed T/F genomes in a hierarchy of in vitro biological assays for replication competence, virion production, infectivity, and sensitivity to type I, II and III IFNs and DAAs (R33); and (iv) expand the number and genotypic representation of HCV T/F clones to include gt 1a, 1b, 2b, 3a and 4a (n=3 for each), identify genotype-associated determinants of IFN and DAA sensitivity, and evaluate in tissue culture and small animal models strategies for achieving synergy between therapeutic modalities that lead to virus eradication (R33). The proposed work will provide new insights into the molecular biology of HCV replication and persistence and facilitate strategies for eradicating globally circulating HCV genotypes.