CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China
CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai, China.
J Virol. 2020 Aug 17;94(17). doi: 10.1128/JVI.00622-20.
Hepatitis C virus (HCV) infection remains a major worldwide health problem despite development of highly effective direct-acting antivirals. HCV rapidly evolves upon acute infection and generates multiple viral variants (quasispecies), leading to immune evasion and persistent viral infection. Identification of epitopes of broadly neutralizing anti-HCV antibodies (nAbs) is critical to guide HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on -complementation of viral structural proteins. The HCV genome (JFH1 strain) lacking the structural protein-coding sequence can be efficiently rescued by ectopic expression of core-E1-E2-p7-NS2 (core-NS2) or core-E1-E2-p7 (core-p7) in , leading to production of single-round infectious virions designated HCVΔS. JFH1-based HCVΔS can be also rescued by expressing core-NS2 of other HCV genotypes, rendering it an efficient tool to display the structural proteins of HCV strains of interests. Furthermore, we successfully rescued HCVΔS with structural proteins from clinical isolates. Multiple viral structural proteins with different sensitivities to nAbs were identified from a same patient serum, demonstrating the genetic diversity of HCV quasispecies Interestingly, the structural protein-coding sequences of highly divergent viral quasispecies from the same patient can be clustered based on their hypervariable region 1 (HVR1) in viral envelope protein E2, which critically dictates the sensitivity to neutralizing antibodies. In summary, we developed a novel reverse genetics system that efficiently displays viral structural proteins from HCV clinical isolates, and analysis of quasispecies from the same patient using this system demonstrated that E2 HVR1 is the major determinant of viral evolution A cell culture model that can recapitulate the diversity of HCV quasispecies in patients is important for analysis of neutralizing epitopes and HCV vaccine development. In this study, we developed a new reverse genetics system for HCV infection based on -complementation of viral structural proteins (HCVΔS). This system can be used to display structural proteins of HCV strains of multiple genotypes as well as clinical isolates. By using this system, we showed that multiple different HCV structural proteins from a same patient were displayed on HCVΔS. Interestingly, these variant structural proteins within the same patient can be classified according to the sequence of HVR1in E2, which dictates viral sensitivity to nAbs and viral evolution Our work provided a new tool to study highly divergent HCV quasispecies and shed light on underlying mechanisms driving HCV evolution.
丙型肝炎病毒 (HCV) 感染仍然是一个全球性的重大健康问题，尽管已经开发出了高度有效的直接作用抗病毒药物。HCV 在急性感染后迅速进化，并产生多种病毒变异体（准种），导致免疫逃逸和持续性病毒感染。鉴定广泛中和抗 HCV 抗体（nAb）的表位对于指导 HCV 疫苗的开发至关重要。在这项研究中，我们开发了一种基于 HCV 病毒结构蛋白 -互补的 HCV 感染的新的反向遗传学系统。缺乏结构蛋白编码序列的 HCV 基因组（JFH1 株）可以通过在细胞中转异位表达核心-E1-E2-p7-NS2（核心-NS2）或核心-E1-E2-p7（核心-p7）来有效地挽救，从而产生单轮感染性病毒粒子，称为 HCVΔS。基于 JFH1 的 HCVΔS 也可以通过表达其他 HCV 基因型的核心-NS2 来挽救，使其成为展示感兴趣的 HCV 株结构蛋白的有效工具。此外，我们还成功地从临床分离株中挽救了 HCVΔS。从同一患者血清中鉴定出具有不同 nAb 敏感性的多种病毒结构蛋白，表明 HCV 准种具有遗传多样性。有趣的是，同一患者高度分化的病毒准种的结构蛋白编码序列可以根据其包膜蛋白 E2 中的高变区 1（HVR1）聚类，这对于中和抗体的敏感性至关重要。总之，我们开发了一种新的反向遗传学系统，能够有效地展示来自 HCV 临床分离株的病毒结构蛋白，并且使用该系统分析同一患者的准种表明，E2 HVR1 是病毒进化的主要决定因素。能够重现患者 HCV 准种多样性的细胞培养模型对于中和表位分析和 HCV 疫苗的开发非常重要。在这项研究中，我们开发了一种基于 HCV 病毒结构蛋白 -互补的 HCV 感染的新的反向遗传学系统（HCVΔS）。该系统可用于展示多种基因型和临床分离株的 HCV 株的结构蛋白。通过使用该系统，我们表明同一患者的多种不同 HCV 结构蛋白可以在 HCVΔS 上显示。有趣的是，这些来自同一患者的变异结构蛋白可以根据 E2 中的 HVR1 序列进行分类，这决定了病毒对 nAb 的敏感性和病毒进化。我们的工作提供了一种新的工具来研究高度分化的 HCV 准种，并揭示了驱动 HCV 进化的潜在机制。
