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

使用 RNA 启动 HBV 来评估抗病毒耐药性并探索病毒宿主生物学的基本方面

• 批准号: 10555333
• 负责人: Charles M Rice
• 金额: $ 61.11万
• 依托单位: ROCKEFELLER UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-02-10 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10555333
• 关键词: Address; Advocate; Affect; Antiviral Agents; Antiviral resistance; Attitude; Bacteriophages; Belief; Biological Assay; Biology; Cell Culture System; Cell Culture Techniques; Cessation of life; Chromosomes; Chronic; Chronic Hepatitis B; Chronic Hepatitis C; Circular DNA; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Complex; Core Protein; Coupled; DNA; DNA Integration; DNA Viruses; Data; Dedications; Development; Drug resistance; Exhibits; Fostering; Foundations; Funding; Genetic Transcription; Genome; Genotype; HBV Genotype; Hepatitis B Infection; Hepatitis B Surface Antigens; Hepatitis B Virus; Human; In Vitro; Individual; Infection; Integration Host Factors; Knock-out; Knowledge; Life Cycle Stages; Liver; Liver Cirrhosis; Liver Failure; Malignant Neoplasms; Measures; Medical; Methods; Mus; Mutation; Nucleotides; Open Reading Frames; Patients; Persons; Pharmaceutical Preparations; Polymerase; Population Heterogeneity; Primary carcinoma of the liver cells; Proteins; Protocols documentation; RNA; Reaction; Replication Initiation; Request for Applications; Research; Research Personnel; Resistance; Reverse Transcriptase Inhibitors; Reverse Transcription; Risk; Sampling; Signal Transduction; Strategic Planning; Structure; Surface Antigens; System; Technology; Testing; Therapeutic; Therapeutic Intervention; Transcriptional Regulation; Translating; Translational Regulation; Translations; United States National Institutes of Health; Vaccines; Variant; Viral; Viral Drug Resistance; Viral Genome; Viral Proteins; Viral Reverse Transcription; Viral reservoir; Virus; Virus Diseases; Virus Inhibitors; Virus Replication; Work; anti-hepatitis B; antiviral drug development; clinically relevant; deep sequencing; detection assay; drug development; drug resistance development; drug resistant virus; genome-wide; high risk; improved; in vivo; inhibitor; innovation; insight; new therapeutic target; next generation; novel strategies; nucleoside analog; optimism; plasmid DNA; preference; prevent; resistance mutation; success; tool; vaccine access; viral DNA; viral RNA; viral fitness; virus core; virus host interaction

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.

项目摘要 超过2.5亿人慢性感染B型肝炎病毒（HBV），生活在不断增加的 肝衰竭、癌症和早死的风险。虽然有一种疫苗可以预防乙肝病毒感染，但它不能 它既不能防止垂直转移，也不能帮助数百万已感染者。虽然有一些治疗方法（例如， 核苷类似物）抑制病毒复制，治疗是终身的，很少导致治愈。实现 因此，慢性HBV感染的功能性治愈代表了全球未满足的主要医疗需求。 幸运的是，新的HBV抑制剂正在开发中，有些已经进入临床 试验，但HBV生物学的许多基本（和临床相关）方面仍然没有答案。广大 该项目的长期目标是利用新的基于细胞培养的 我们开发了一种用RNA启动HBV复制的方法。具体来说，我们利用最独特的 该技术的特点是（i）解决药物开发的实际问题-抗病毒耐药性，（ii） 填补了我们对HBV蛋白翻译和基因组复制的基础知识的空白，以及（iii）作为一种 发现工具，以确定限制或促进HBV生命周期的宿主因素。 目前还没有办法用现有的基于HBV细胞培养的系统评估抗病毒耐药性。 通过我们的RNA方法，我们利用了噬菌体聚合酶（例如T7）通常用于在细胞中表达的事实。 体外转录RNA是容易出错的。因此，启动HBV复制与RNA，而不是DNA（像大多数 系统）允许对不同的病毒变体群体进行采样。正如我们所展示的令人信服的初步 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HBV耐药病毒变异。本提案的目标1是进一步发展这项技术。 用RNA启动HBV复制在很大程度上消除了污染HBV的背景信号 qPCR反应，并且由于并非所有病毒RNA都需要启动复制，因此产生一些病毒蛋白质。 只有当病毒生命周期进展并且病毒DNA模板-共价闭合环状DNA（cccDNA）- 确立了习我们在目标2中利用这一点来研究HBV蛋白翻译的基本方面， 基因组复制，目的3是发现病毒-宿主相互作用。具体来说，我们使用目标2中的方法来 研究在慢性感染个体中经常出现的突变和HBV基因型的差异。我们 使用目标3中的方法作为全基因组CRISPR敲除筛选的基础，以识别HBV宿主因子。 直到我们有一个解决方案，消除慢性乙肝病毒，继续和新的基础研究乙肝病毒 需要生物学来确定可用于治疗干预的新策略。新的RNA- 我们开发的用于研究HBV生物学和评估抗病毒耐药性的基于系统与NIH的 治疗慢性乙型肝炎的战略计划和我们提出的工作将为这一持续的努力做出重大贡献。