Mechanisms of Hepatitis C Virus Evolution

项目描述（由申请人提供）：本项目的主要目标是通过研究丙型肝炎病毒（HCV）从急性感染到慢性感染转变过程中的序列，并研究驱动和限制病毒变异的潜在机制，促进对体内病毒进化的理解。我们将测量体液和细胞宿主免疫反应，以及免疫驱动变异对必需病毒酶活性的功能影响。该结果将促进我们对宿主-病原体关系、免疫保护相关因素和病毒逃避的理解。我们假设HCV的进化在很大程度上是决定性的。具体来说，随机（离散的和随机产生的）突变是通过选择过滤的，这是由驱动（积极选择）和约束（消极选择）结果变化的可测量因素介导的。了解这些因素将有助于合理设计疫苗，并指导HCV复制小分子抑制剂的使用。我们提出以下目标：(1)确定细胞免疫反应在HCV从急性感染向慢性感染转变过程中驱动HCV进化的作用；(2)为了研究慢性感染HCV包膜基因的进化机制，以及HIV合并感染对HCV进化的影响，我们将研究HIV感染前后HCV的体液反应，并精心匹配对照受试者；(3)为了研究免疫逃逸对病毒适应性的影响和负选择的机制，我们将利用新型细胞培养和HCV复制的单轮亚基因组复制子模型，测量免疫驱动变异对病毒复制适应性的影响。我们已经证明，我们可以获得这项研究所需的关键组织，对从急性感染过渡到确定感染的人进行独特的纵向研究，测量细胞和体液免疫反应，并使用体外模型来阐明HCV发病机制。因此，我们期望在这些独特的研究成功的积极和消极的选择作用于传染因子在人类感染。

DESCRIPTION (provided by applicant): The primary objective of this project is to advance understanding of viral evolution in vivo by studying hepatitis C virus (HCV) sequences during the transition from acute to chronic infection, and examining the underlying mechanisms that drive and constrain viral variation. We will measure humoral and cellular host immune responses, as well as the functional impact of immune-driven variation on essential viral enzyme activities. The results will advance our understanding of the host-pathogen relationship, correlates of immune protection, and viral evasion. We hypothesize that HCV evolution is largely deterministic. Specifically, stochastic (discrete and randomly-generated) mutations are filtered by selection, which is mediated by measurable factors that both drive (positive selection) and constrain (negative selection) the resulting variation. Understanding these factors will facilitate rational vaccine design, and guide the use of small-molecule inhibitors of HCV replication. We propose the following aims: (1) to determine the role of the cellular immune response in driving HCV evolution during the transition from acute to chronic infection; (2) to investigate the mechanisms for evolution in the envelope genes of HCV during chronic infection, and the effects of HIV co-infection on HCV evolution, we will study humoral responses to HCV before and after HIV infection, with carefully-matched control subjects; and (3) to investigate the impact of immune escape on viral fitness and the mechanisms of negative selection, we will measure the impact of immune-driven variation on viral replicative fitness, using novel cell culture and single-round subgenomic replicons models of HCV replication. We have already demonstrated that we can obtain the critical tissues required in this investigation, conduct unique longitudinal studies of persons transitioning from acute to established infection, measure cellular and humoral immune responses, and use in vitro models to elucidate HCV pathogenesis. Therefore, we anticipate success in these unique studies of positive and negative selection acting on an infectious agent during human infection.