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.

项目摘要/摘要 几种哺乳动物Arena病毒会导致人类严重和致命的人畜共患病，针对这些疾病的疫苗和 治疗方法非常有限。拉萨病毒(LASV)是拉沙热(LF)的病原体，目前 非洲西部的地方病。尽管它对公共卫生很重要，但重要的知识差距仍然存在于 LASV和其他高致病性阿雷诺病毒的基础生物学，部分原因是高度遏制的限制 BSL4感染实验所需的设施。RNA病毒RNA合成通常容易出错，因为 依赖病毒RNA的RNA聚合酶缺乏校对活性。作为负义RNA病毒，阿雷诺病毒如何 确保适当的RNA合成在很大程度上是不清楚的。LASV核蛋白具有DEDDH3‘至5’外切核糖核酸酶 Motif(外显子)，其在病毒生命周期中的作用仍是一个谜。目前该领域的范例是 NP外显子活性有效降解病毒来源的双链RNA，是LASV逃避的关键 先天免疫力。有趣的是，外显子基序在所有ArenaVirus的NP中高度保守，无论 致病性。因此，NP外显子很可能具有重要但尚未确定的功能(S)。 除了免疫逃避之外，禽流感病毒的复制。在这个项目中，我们的目标是定义S的重要角色 病毒复制中的ArenaVirus NP外显子。 初步数据表明：1)。NP外显子活性丧失导致基因组RNA产生异常 以及干扰素缺陷细胞中LASV RNA水平的急剧下降；NP外显子活性丧失增加 LASV对诱变核苷类似物治疗的敏感性。我们认为LASV NP外显子促进适当的 病毒RNA合成，控制病毒基因组RNA的异常形成和/或确保病毒RNA的保真度 复制。为了确定NP外显子对病毒基因组RNA的完整性和功能的影响，我们将 探索利用创新的长读测序技术来系统地研究序列和 在单分子水平上基因组RNA的丰度。已知阿雷纳病毒形成有缺陷的干扰 颗粒，它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。长读测序技术可能使我们能够识别DI候选对象和 NP外显子在调节DI形成中的潜在作用。我们还将调查ArenaVirus NP外显子是否有 确保病毒RNA复制的保真度的校对活动。 在这个项目的最后，我们可能会发现病毒NP外显子在病毒生命中的新的重要功能(S) 循环并将字段向前移动。以LASV为模型，我们可以更好地理解禽流感病毒是如何 确保适当的病毒RNA合成。有了新的长读测序技术，这项研究可能会克服 技术障碍，增加我们对致病性禽流感病毒的基础病毒学知识。数据可能会打开 朝着新的方向前进。例如，未来对NP外显子重要作用的机制的研究 都是有根据的。该项目还可能通过靶向NP外显子活性来促进抗病毒的开发。