Hepatitis C virus polyprotein processing efficiency regulates infectious virus production

 描述(申请人提供)：在过去十年中，随着丙型肝炎病毒细胞培养传染性病毒的出现，实验室中研究丙型肝炎病毒的模型有了显著的改进，但这些系统仍然产生相对较低的病毒滴度。我们试图通过在日益严格的条件下连续传代广泛使用的实验室适应病毒J6/JFH-1 Jc1(Jc1)来提高病毒滴度，结果得到的病毒产生的滴度比Jc1高10倍。我们将该病毒命名为J6/JFH-1 JC1.1(JC1.1)。JC1.1的序列分析和功能定位表明，病毒多蛋白中NS5A/NS5B裂解位点的P1‘位置NS5B的氨基末端需要两个氨基酸的替换和一个氨基酸的改变，滴度增加。由于这些结果提示JC1.1可能表现出改变的多蛋白切割动力学，我们使用脉冲追逐标记策略观察到JC1.1而不是JC1在感染过程中产生了少量相对稳定的未切割的NS5A-NS5B。我们开发了一个基于质粒的过表达系统，在感染期间提供JC1.1突变体NS5A-NS5B的反式，认为更多未切割的NS5A-NS5B的存在可能会导致比我们在JC1.1感染中观察到的更高的病毒滴度。令人惊讶的是，当JC1.1的NS5A-NS5B以反式方式供应给JC1和JC1.1感染时，我们观察到比最初的JC1亲本病毒的滴度增加了100倍，这表明未切割的NS5A-NS5B对传染性病毒的产生有戏剧性的影响。我们推测，为了在病毒生产中发挥作用，NS5A-NS5B应该定位于病毒粒子组装的位置，并以一种依赖于组件蛋白NS5A和NS5B的已知组装和膜结合活性的方式增加病毒粒子的产生和分泌。为了验证这一假设，我们开展了一系列实验，研究NS5A-NS5B对病毒组装、RNA复制、颗粒分泌、特异性传染性、病毒成熟和低密度脂蛋白结合以及病毒粒子稳定性的影响。我们还想了解NS5A-NS5B处理效率的变化与病毒生产的关系，所以我们在NS5A-NS5B的P1和P1‘位置产生了一系列突变，产生了一系列未切割的NS5A-NS5B数量，我们建议测试它们的切割效率和传染性释放。我们还在NS5A和NS5B中产生了一组特征良好的突变，这些突变已知会改变这些成熟、完全加工形式的蛋白质的组装、复制和膜结合功能，我们将评估它们在感染中未切割的NS5A-NS5B的背景下提供时对病毒生产的影响，希望这将为NS5A-NS5B如何提高病毒生产提供一些见解。我们还设计了一些方法来确定NS5A-NS5B在感染细胞中的定位，重点放在病毒RNA复制和病毒粒子生物发生的位置。完成我们的目标将定义NS5A-NS5B在病毒粒子生产中如何以及在哪里发挥作用，突出未切割的多蛋白中间体在丙型肝炎病毒生命周期中的新角色，并潜在地定义一个新的抗病毒靶点。

 DESCRIPTION (provided by applicant): Models to study hepatitis C virus (HCV) in the laboratory have improved dramatically over the last decade with the advent of HCV cell culture infectious viruses, but these systems still produce relatively low viral titers. We attempted to improve viral titers by serially passaging the widely used laboratory adapted virus, J6/JFH-1 JC1 (JC1), under increasingly stringent conditions for 20 weeks, and the resulting virus produced titers 10 times higher than JC1. We designated this virus J6/JFH-1 JC1.1 (JC1.1). Sequence analysis and functional mapping of JC1.1 indicated the increase in titer required two amino acid substitutions in the NS3 protease and a single amino acid change at the amino-terminus of NS5B, the P1' position of the NS5A/NS5B cleavage site in the viral polyprotein. As these results hinted that JC1.1 might exhibit altered polyprotein cleavage kinetics, we used a pulse chase labeling strategy and observed that JC1.1, but not JC1, produced a small amount of relatively stable uncleaved NS5A-NS5B during infections. We developed a plasmid based overexpression system to supply the JC1.1 mutant NS5A-NS5B in trans during infections, thinking that the presence of more uncleaved NS5A-NS5B might result in even greater virus titers than we observed in JC1.1 infections. Surprisingly, we observed a 100-fold increase in titer over the original JC1 parental virus when the NS5A-NS5B from JC1.1 was supplied in trans to both JC1 and JC1.1 infections, indicating that uncleaved NS5A-NS5B has a dramatic impact on infectious virus production. We hypothesized that for a role in virus production, NS5A-NS5B should localize to the site of virion assembly and increases virion production and secretion in a manner dependent on the known assembly and membrane binding activities of the component proteins NS5A and NS5B. To test this hypothesis we developed a series of experiments to investigate the impact of NS5A-NS5B on virus assembly, RNA replication, particle secretion, specific infectivity, virus maturation and LDL association, and virion stability. We also wanted to understand how varying the processing efficiency of NS5A-NS5B correlated with virus production, so we generated a series of mutations in the P1 and P1' positions of NS5A-NS5B that produce a range of uncleaved NS5A-NS5B amounts and we propose to test them for cleavage efficiency and infectivity release. We also generated a panel of well-characterized mutations in NS5A and NS5B known to alter the assembly, replication, and membrane association functions of these proteins in their mature, fully processed forms, and we will assess how they impact virus production when supplied in the context of uncleaved NS5A-NS5B in trans in infections, with the hope that this will provide some insights into how NS5A- NS5B enhances virus production. We also have designed approaches to determine where NS5A-NS5B localizes in infected cells with a focus on sites of viral RNA replication and virion biogenesis. Completing our aims will define how and where NS5A-NS5B functions in virion production, highlight the novel role of uncleaved polyprotein intermediates in the HCV lifecycle, and potentially define a novel antiviral target.