Phenotypic analysis of Hepatitis C virus evolution in the post-transplant setting: Understanding mechanisms of rapid fitness adaptation to a new environment

丙型肝炎病毒（Hepatitis C virus，HCV）是黄病毒科的一种正链RNA病毒，是引起人类严重肝病的重要病原体。HCV是变异最大的病毒之一，在每个患者中产生高度多样的准种，但到目前为止，对病毒分离株对感染结果或疾病进展的影响知之甚少。随着肝移植（LTX）患者的病毒进化，我们发现LTX后病毒多样性显着减少，沿着RNA复制适应性的强烈增加。我们在HCV非结构蛋白（NS）5A中发现了一个高度可变的区域，我们现在称之为RFDR（复制适合度决定区），通过突变的积累大大提高了RNA复制效率。到目前为止，表型似乎与突变的数量有关，而不是与突变的性质有关，如以前在干扰素治疗中发现的那样。我们进一步发现，由于快速和严重的疾病进展，LTX后发生纤维化胆汁淤积性肝炎（FCH）的患者中出现高度复制的RFDR突变体之间存在明显的相关性，这表明HCV复制适应性对直接病毒发病机制的重大贡献，至少在缺乏适应性免疫应答的情况下。该项目现在旨在了解RFDR变体的一般临床意义，控制RNA复制适合度的序列模式的定义以及RFDR如何调节RNA复制的机制，重点关注文献中提出的聚合酶活性调节的假设。目的1将研究更广泛的LTX后HCV变异体，根据疾病进展分层，以获得进一步的证据，复制健身在发病机制中的作用，并解决进入和组装能力对病毒健身的影响。目标2将解决前和后LTX分离株的RFDR的演变，以研究是否高复制RFDR主要选择LTX或已经普遍的LTX之前。我们将进一步遵循文献中关于高复制因子RFDR对肝细胞癌发展、直接作用抗病毒治疗失败和一般高病毒滴度的贡献的提示。此外，我们将评估RFDR调控活性在HCV基因型1b以外的可能意义。目标3将确定和验证不同的序列决定因素控制复制效率，允许基于序列的预测，并支持一个机械的理解。目的4将阐明RFDR调控RNA复制的机制，并得到结构分析的支持。在第一步中，建立在纯化的聚合酶和NS5A变体上的体外测定将用于发现RFDR变体的不同调节活性，与RNA复制适应性相关。基于细胞的研究将用更无偏倚的方法补充体外分析。

Hepatitis C virus (HCV) is a positive strand RNA virus belonging to the family of Flaviviridae and an important human pathogen causing severe liver disease. HCV is among the most variable viruses, creating a highly diverse quasispecies in every patient, but so far very little is known about the impact of the viral isolate on infection outcome or disease progression. Following the virus evolution in a liver transplant (LTX) patient, we identified a dramatically reduced virus diversity after LTX, along with a strong increase in RNA replication fitness. We identified a highly variable region in HCV nonstructural protein (NS)5A, which we now term RFDR (replication fitness determining region), substantially increasing RNA replication efficiency by accumulation of mutations. So far, the phenotype appears rather related to the number than to the nature of mutations, as previously found in the context of interferon therapy. We further found a clear correlation between the appearance of highly replicating RFDR mutants after LTX in patients developing a fibrosing cholestatic hepatitis (FCH) due to a fast and severe disease progression, pointing to substantial contribution of HCV replication fitness to direct viral pathogenesis, at least in absence of adaptive immune responses. This project now aims at understanding the general clinical significance of RFDR variants, the definition of sequence patterns governing RNA replication fitness and the mechanism how the RFDR regulates RNA replication, focusing on the hypothesis of a regulation of polymerase activity, which is suggested by literature. Aim 1 will study a wider range of post LTX HCV variants stratified according to disease progression to obtain further evidence for a role of replication fitness in pathogenesis and address the impact of entry and assembly competence to viral fitness. Aim 2 will address the evolution of the RFDR in pre- and post-LTX isolates to study whether high replicator RFDRs are primarily selected upon LTX or already prevalent prior LTX. We will further follow hints in literature on the contribution of high replicator RFDRs to development of hepatocellular carcinoma, direct-acting-antiviral treatment failure and generally high viral titers. In addition, we will assess the possible significance of RFDR regulatory activity beyond HCV genotype 1b. Aim 3 will identify and validate distinct sequence determinants governing replication efficiency, to allow sequence-based predictions and to support a mechanistic understanding. Aim 4 will clarify the mechanism underlying the regulation of RNA replication by the RFDR, supported by structural analyses. In a first step, in vitro assays building on purified polymerase and NS5A variants will be used to find different regulatory activities of RFDR variants, correlating with RNA replication fitness. Cell based studies will complement the in vitro analysis with more unbiased approaches.