Launching HBV with RNA to assess antiviral resistance and explore fundamental aspects of virus-host biology

项目摘要 超过2.5亿人慢性感染乙肝病毒(乙肝病毒)，生活在日益增长的 有发生肝功能衰竭、癌症和过早死亡的风险。虽然有预防乙肝病毒感染的疫苗，但它不能。 阻止垂直传播，也不能帮助已经感染的数百万人。虽然有治疗方法(例如 核苷类似物)抑制病毒复制，治疗是终生的，很少能治愈。实现 因此，慢性乙肝病毒感染的功能性治疗是全球尚未得到满足的主要医疗需求。 幸运的是，新的乙肝病毒抑制剂正在开发中，一些已经进入临床。 试验，但许多基本的(和临床相关的)乙肝生物学方面仍然没有答案。《博大》 该项目的长期目标是利用一种以细胞培养为基础的新方法来解决该领域的知识差距 方法我们建立了用RNA启动乙肝病毒复制的方法。具体地说，我们利用最独特的 这项技术的特点是(I)解决药物开发的一个实际问题--抗病毒耐药性，(Ii) 填补我们在乙肝病毒蛋白翻译和基因组复制基础知识方面的空白，以及(Iii)作为 发现工具，用于识别限制或促进乙肝病毒生命周期的宿主因素。 目前还没有办法用现有的基于乙肝病毒细胞培养的系统来评估抗病毒耐药性。 在我们的RNA方法中，我们利用了这样一个事实，即噬菌体聚合酶(例如T7)通常用于 试管转录的RNA很容易出错。因此，使用RNA而不是DNA(像大多数 系统)允许对不同的病毒变种进行采样。正如我们展示的令人信服的初步结果 数据，这种序列多样性与深度测序技术相结合，使选择和 检测罕见的乙肝病毒耐药病毒变异株。这项提议的目标1是进一步开发这项技术。 用RNA启动乙肝病毒复制在很大程度上消除了污染乙肝病毒的背景信号 QPCR反应，由于并不是所有的病毒RNA都需要启动复制，所以一些病毒蛋白 只有在病毒生命周期进展和病毒DNA模板-共价闭合环状DNA(CccDNA)-是 已经成立了。我们在目标2中利用这一点来研究乙肝病毒蛋白翻译的基本方面和 基因组复制，并在目标3中发现病毒与宿主的相互作用。具体地说，我们使用目标2中的方法 研究在慢性感染者中经常出现的突变和乙肝病毒基因类型的差异。我们 使用AIM 3中的方法作为全基因组CRISPR基因敲除筛查的基础，以确定乙肝病毒宿主因子。 直到我们有了消除慢性乙肝的解决方案，对乙肝病毒的持续和新的基础研究 需要生物学来确定可以探索用于治疗干预的新策略。新的RNA- 基于我们开发的研究乙肝病毒生物学和评估抗病毒耐药性的系统，与NIH的系统保持良好的一致性 治愈慢性乙肝的战略计划和我们提出的工作将对这一持续努力做出重大贡献。

Project Summary More than 250 million people are chronically infected with hepatitis B virus (HBV), living at increased risk of liver failure, cancer, and early death. Though there is a vaccine to prevent HBV infection, it does not prevent vertical transfer, nor does it help the millions of people already infected. While there are therapies (e.g. nucleoside analogs) to suppress virus replication, treatment is life-long and rarely leads to cure. Achieving a functional cure for chronic HBV infection therefore represents a major global unmet medical need. Fortunately, new classes of HBV inhibitors are being developed and some have already entered clinical trials, but many fundamental (and clinically-relevant) aspects of HBV biology remain unanswered. The broad long-term objectives of this project are to address knowledge gaps in the field using a new cell culture-based method we developed to initiate HBV replication with RNA. Specifically, we capitalize on the most unique features of this technology to (i) address a practical problem of drug development—antiviral resistance, (ii) to fill gaps in our fundamental knowledge of HBV protein translation and genome replication, and (iii) as a discovery tool to identify host factors that restrict or promote the HBV lifecycle. There is currently no way to assess antiviral resistance with existing HBV cell culture-based systems. With our RNA approach, we take advantage of the fact that phage polymerases (e.g. T7) commonly used to in vitro-transcribe RNA are error-prone. As such, initiating HBV replication with RNA rather than DNA (like most systems) allows a diverse population of viral variants to be sampled. As we show with compelling preliminary data, this sequence diversity coupled with deep sequencing technology makes it possible to select for and detect rare HBV drug-resistant viral variants. Aim 1 of this proposal is to further develop this technology. Initiating HBV replication with RNA largely eliminates the background signal that contaminates HBV qPCR reactions, and since not all viral RNAs are required to initiate replication, some viral proteins are made only if the viral lifecycle progresses and the viral DNA template—covalently closed circular DNA (cccDNA)—is established. We capitalize on this in Aim 2 to study fundamental aspects of HBV protein translation and genome replication, and in Aim 3 to discover virus-host interactions. Specifically we use the method in Aim 2 to study mutations that frequently arise in chronically infected individuals and differences in HBV genotypes. We use the method in Aim 3 as the basis for a genome-wide CRISPR knockout screen to identify HBV host factors. Until we have a solution for eliminating chronic HBV, continuing and new fundamental studies of HBV biology are needed to identify new strategies that can be explored for therapeutic intervention. The new RNA- based system we developed to study HBV biology and assess antiviral resistance align well with the NIH's strategic plan to cure chronic HBV and the work we propose will contribute significantly to this ongoing effort.