Novel Function(s) of Arenavirus NP Exoribonuclease

Arenavirus NP 核糖核酸外切酶的新功能

• 批准号: 10525101
• 负责人: Cheng Huang
• 金额: $ 24万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-05-18 至 2024-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10525101
• 关键词: 5' -exoribonuclease; Address; Affect; Africa; Arenavirus; Arenavirus Infections; Biochemical; Biological; Biological Assay; Biology; Bolivian Hemorrhagic Fever Virus; Category A pathogen; Cells; Containment; Cultured Cells; Data; Defective Viruses; Disease; Double-Stranded RNA; Ensure; Exoribonucleases; Family; Future; Genes; Genetic Transcription; Genome; Human; Immune Evasion; Impairment; In Vitro; Infection; Intercistronic Region; Interferons; Junin virus; Knowledge; Lassa Fever; Lassa virus; Life Cycle Stages; Lymphocytic choriomeningitis virus; Mediating; Messenger RNA; Modeling; Molecular; Mutation; Natural Immunity; Northern Blotting; Nucleoproteins; Pathogenicity; Production; Public Health; RNA; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Replicon; Role; Signal Transduction; Structure; Techniques; Technology; Time; Vaccines; Variant; Viral; Viral Packaging; Viral Physiology; Virion; Virus; Virus Diseases; Virus Replication; Western Africa; Zoonoses; antiviral drug development; biosafety level 4 facility; deep sequencing; experimental study; genomic RNA; high risk; in vivo; innovation; insight; mutant; novel; nucleoside analog; particle; prevent; single molecule; transcription termination; viral RNA; viral genomics; virology

PROJECT SUMMARY/ABSTRACT Several mammalian arenaviruses cause severe and fatal zoonotic diseases in humans, for which vaccines and treatments are very limited. Lassa virus (LASV) is the causative agent for Lassa fever (LF) that is currently endemic in Western Africa. Despite its importance to public health, important knowledge gaps still exist in the basic biology for LASV and other highly pathogenic arenaviruses, partly due to the limitation of high containment BSL4 facilities required for infection experiments. The RNA synthesis of RNA virus is generally error prone as viral RNA-dependent RNA polymerase lacks proofreading activity. As negative-sense RNA virus, how arenavirus ensures proper RNA synthesis is largely unclear. The LASV nucleoprotein has a DEDDH 3' to 5' exoribonuclease motif (ExoN), of which its function in virus life cycle is still a puzzle. The current paradigm in the field is that the NP ExoN activity efficaciously degrades virus-derived double-stranded RNA and is the key to LASV evasion of innate immunity. Intriguingly, the ExoN motif is highly conserved in NPs of all arenaviruses, regardless of pathogenicity. Therefore, it is very likely that the NP ExoN has important but yet-to-be-identified function(s) in arenavirus replication in addition to immune evasion. In this project, we aim to define the important role(s) of arenavirus NP ExoN in virus replication. Our preliminary data indicated that: 1). Loss of NP ExoN activity resulted in aberrant genomic RNA production and a drastic reduction in LASV RNA level in IFN-deficient cells; and 2). Loss of NP ExoN activity increased LASV sensitivity to mutagenic nucleoside analogue treatment. We propose that LASV NP ExoN promotes proper viral RNA synthesis, controls aberrant viral genomic RNA formation and/or ensures the fidelity of viral RNA replication. To define the impact of NP ExoN on the integrity and functionality of viral genomic RNA, we will explore to utilize an innovative long-read sequencing technology to systematically investigate the sequence and abundancy of genomic RNAs at single molecule level. Arenavirus has been known to form defective interfering particles, which regulates virus infection in vivo and in vitro. The molecular basis for DI genome remains elusive due to technical obstacle. The long-read sequencing technology may enable us to identify DI candidates and the potential role of NP ExoN in regulating DI formation. We will also investigate whether arenavirus NP ExoN has proofreading activity that ensures the fidelity of viral RNA replication. At the end of this project, we may discover novel and important function(s) of arenavirus NP ExoN in virus life cycle and move the field forward. Using LASV as a model, we may better understand how arenavirus virus ensures proper viral RNA synthesis. With the novel long-read sequencing technology, this study may overcome technical barrier and increase our knowledge on basic virology of pathogenic arenaviruses. The data may open up new directions. For instance, future studies on the mechanisms underlying the important roles of NP ExoN are warranted. This project may also facilitate antiviral development by targeting NP ExoN activity.

项目概要/摘要 几种哺乳动物沙粒病毒会引起人类严重和致命的人畜共患疾病，针对这些疾病的疫苗和疫苗 治疗方法非常有限。拉沙病毒（LASV）是目前拉沙热（LF）的病原体 西非流行。尽管它对公共卫生很重要，但在这方面仍然存在重要的知识差距 LASV 和其他高致病性沙粒病毒的基础生物学，部分原因是高遏制的限制 感染实验所需的 BSL4 设施。 RNA病毒的RNA合成通常容易出错，如下所示 病毒RNA依赖性RNA聚合酶缺乏校对活性。作为负义RNA病毒，沙粒病毒如何 确保正确的 RNA 合成在很大程度上尚不清楚。 LASV 核蛋白具有 DEDDH 3' 至 5' 核糖核酸外切酶 基序（ExoN），其在病毒生命周期中的功能仍然是一个谜。该领域当前的范式是 NP ExoN 活性可有效降解病毒来源的双链 RNA，是 LASV 逃避的关键 先天免疫。有趣的是，ExoN 基序在所有沙粒病毒的 NP 中都高度保守，无论 致病性。因此，NP ExoN 很可能在以下方面具有重要但尚未确定的功能： 除了免疫逃避之外，还有沙粒病毒的复制。在这个项目中，我们的目标是定义以下人员的重要角色： 病毒复制中的沙粒病毒 NP ExoN。 我们的初步数据表明：1）。 NP ExoN 活性丧失导致基因组 RNA 产生异常 IFN 缺陷细胞中 LASV RNA 水平急剧下降；和2）。 NP ExoN 活性丧失增加 LASV 对诱变核苷类似物治疗的敏感性。我们建议 LASV NP ExoN 促进适当的 病毒RNA合成，控制异常病毒基因组RNA的形成和/或确保病毒RNA的保真度 复制。为了定义 NP ExoN 对病毒基因组 RNA 完整性和功能的影响，我们将 探索利用创新的长读长测序技术来系统地研究序列和 单分子水平的基因组RNA丰度。已知沙粒病毒会形成有缺陷的干扰 颗粒，调节体内和体外的病毒感染。 DI 基因组的分子基础仍然难以捉摸 由于技术障碍。长读长测序技术可能使我们能够识别 DI 候选者和 NP ExoN 在调节 DI 形成中的潜在作用。我们还将调查沙粒病毒 NP ExoN 是否具有 确保病毒 RNA 复制保真度的校对活动。 在这个项目结束时，我们可能会发现沙粒病毒 NP ExoN 在病毒生命中新颖且重要的功能 循环并推动该领域向前发展。使用LASV作为模型，我们可以更好地理解沙粒病毒是如何 确保正确的病毒RNA合成。凭借新颖的长读长测序技术，这项研究可能会克服 技术障碍并增加我们对致病性沙粒病毒的基础病毒学知识。数据可能会打开 上升新的方向。例如，未来对 NP ExoN 重要作用机制的研究 是有保证的。该项目还可以通过靶向 NP ExoN 活性来促进抗病毒药物的开发。