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

描述（由申请人提供）：丙型肝炎病毒（HCV）在自然界中作为遗传多样性准种存在。在全球范围内，该病毒由七种主要基因型（gt 1-7）代表，其核苷酸序列差异高达30-35%。在理解HCV生物学和导致病毒持续存在或根除的病毒-宿主相互作用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巨大挑战。迄今为止，只有单个gt 2a克隆pJFH 1及其嵌合衍生物在体外和体内有效复制。这也阻碍了根除不同病毒基因型的有效治疗剂和治疗策略的开发。本申请旨在通过采用在我们的实验室中基于单基因组测序（SGS）开发的新的实验策略来克服该科学障碍，以鉴定和克隆负责人类中生产性临床感染的实际传播/创始者（T/F）全长HCV基因组。我们的假设是，T/F基因组，根据定义包含所有的遗传元件，是必要的和足够的生产性病毒感染和复制在人类中，将复制在组织培养和小动物模型中，概括了HCV的生物学，并允许详细的分子评估这些病毒的敏感性干扰素（IFN），直接作用的抗病毒剂（DAA）和其他新的治疗干预措施。我们的实验方法改编自我们最近对HIV-1的研究，其中我们证明了全长的、有复制能力的、传播的HIV-1基因组可以被鉴定、克隆、表达和分析传播和生产性感染所必需的生物学特性（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鉴定T/F HCV基因组的可行性（Li et al.，PLoS Pathog，出版中）。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）中分子鉴定对应于gt 1a、1b、2b和3a的全长T/F HCV基因组;（ii）化学合成和克隆这些基因组，并在人肝嵌合小鼠（R21）中开始复制;（iii）在体外生物测定的层级中分析表达的T/F基因组的复制能力、病毒体产生、感染性和对I、II和III型IFN和DAA的敏感性（R33）;和 扩大HCV T/F克隆的数量和基因型代表性，以包括gt 1a、1b、2b、3a和4a（每种n=3），鉴定IFN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价实现导致病毒根除的治疗方式之间协同作用的策略（R33）。这项工作将为HCV复制和持续性的分子生物学提供新的见解，并促进消除全球传播的HCV基因型的策略。

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.