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

丙型肝炎病毒是黄病毒科的一种正链RNA病毒，是引起严重肝病的重要人类病原体。丙型肝炎病毒是变异最大的病毒之一，在每个患者中都会产生高度多样化的准种，但到目前为止，人们对病毒分离株对感染结局或疾病进展的影响知之甚少。随着一例肝移植(LTX)患者的病毒进化，我们发现LTX后病毒多样性显著减少，同时RNA复制适合性显著增加。我们在丙型肝炎病毒非结构蛋白(NS)5A中发现了一个高度可变区，我们现在称之为RFDR(复制适合性决定区)，通过突变的积累大大提高了RNA复制效率。到目前为止，这种表型似乎更多地与突变的数量有关，而不是与突变的性质有关，就像之前在干扰素治疗中发现的那样。我们进一步发现，LTX后高复制RFDR突变体的出现与由于快速和严重的疾病进展而发展成纤维淤胆性肝炎(FCH)的患者之间存在明显的相关性，表明丙型肝炎病毒复制适合性对直接病毒发病有重大贡献，至少在缺乏适应性免疫反应的情况下是如此。本项目旨在了解RFDR变异体的一般临床意义，决定RNA复制适合性的序列模式的定义，以及RFDR如何调控RNA复制的机制，重点是文献中提出的聚合酶活性调节假说。目的1将根据疾病进展对LTX后丙型肝炎病毒变异进行更广泛的研究，以获得复制适合性在发病机制中作用的进一步证据，并解决进入和组装能力对病毒适合性的影响。目标2将研究在LTX前和LTX后分离株中RFDR的进化，以研究高复制子RFDR是主要选择在LTX上还是已经流行的LTX。我们将进一步遵循文献中的提示，即高复制子RFDRs对肝细胞癌的发生、直接作用抗病毒治疗失败和一般高病毒滴度的贡献。此外，我们还将评估RFDR调节活性在丙型肝炎病毒1b以外的可能意义。目标3将识别和验证控制复制效率的不同序列决定因素，以实现基于序列的预测，并支持机械性理解。AIM 4将在结构分析的支持下，阐明RFDR调控RNA复制的潜在机制。在第一步中，基于纯化的聚合酶和NS5A变体的体外分析将被用来寻找RFDR变体的不同调节活性，与RNA复制适合性相关。基于细胞的研究将以更公正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.