Novel Function(s) of Arenavirus NP Exoribonuclease

Arenavirus NP 核糖核酸外切酶的新功能

基本信息

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
项目类别：
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&apos -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

项目摘要

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/摘要几种哺乳动物的舞台病毒引起人类严重和致命的人畜共患病，为此，疫苗和疫苗治疗非常有限。 LASSA病毒（LASV）是LASSA热（LF）的病因，目前是西非地方病。尽管对公共卫生的重要性很重要，但重要的知识差距仍然存在于 LASV和其他高度致病性舞台病毒的基本生物学，部分是由于高遏制的限制感染实验所需的BSL4设施。 RNA病毒的RNA合成通常易于误解病毒RNA依赖性RNA聚合酶缺乏校对活性。作为阴性的RNA病毒，如何体育症病毒确保正确的RNA合成在很大程度上不清楚。 LASV核蛋白具有3'至5'的驱虫核酸酶主题（外显子），其在病毒生命周期中的功能仍然是一个难题。该领域的当前范式是 NP外显子活性有效地降解病毒衍生的双链RNA，是LASV逃避的关键先天免疫。有趣的是，外显子基序在所有体育疾病病毒的NP中都高度保守致病性。因此，NP外显子很有可能具有重要但尚未确定的功能除了免疫逃避外，体育病毒复制。在这个项目中，我们旨在定义病毒复制中的体育症病毒NP外显子。我们的初步数据表明：1）。 NP外显子活性的丧失导致基因组RNA异常以及IFN缺陷型细胞中LASV RNA水平的急剧降低；和2）。 NP外显子活性的损失增加 LASV对诱变核苷类似物的敏感性。我们建议LASV NP外显子促进适当病毒RNA合成，控制异常的病毒基因组RNA形成和/或确保病毒RNA的保真度复制。为了定义NP外显子对病毒基因组RNA的完整性和功能的影响，我们将探索以利用创新的长阅读测序技术来系统地研究顺序和基因组RNA在单分子水平上的丰富性。已知体育症病毒会形成有缺陷的干扰调节体内和体外病毒感染的颗粒。 DI基因组的分子基础仍然难以捉摸由于技术障碍。长阅读的测序技术可能使我们能够确定DI候选人和 NP外显子在调节DI形成中的潜在作用。我们还将调查Arenavirus NP外显子是否具有确保病毒RNA复制的保真度的校对活动。在该项目的结尾，我们可能会发现病毒life中体育病毒NP外显子的新颖和重要功能循环并向前进。使用LASV作为模型，我们可以更好地了解体育症病毒病毒如何确保正确的病毒RNA合成。通过新颖的长阅读测序技术，这项研究可能会克服技术障碍并提高我们对病原病毒基本病毒学的知识。数据可能打开启动新的方向。例如，关于NP外显子重要作用的机制的未来研究有必要。该项目还可以通过靶向NP外显子活性来促进抗病毒发育。