Functional regulation of an arenavirus polymerase by the viral matrix protein and RNA ligands

病毒基质蛋白和 RNA 配体对沙粒病毒聚合酶的功能调节

• 批准号: 9981617
• 负责人: Jesse Dylan Pyle
• 金额: $ 3.12万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-08-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9981617
• 关键词: Active Sites; Address; Africa; Antiviral Agents; Architecture; Arenavirus; Binding; Biochemical; Biological Assay; Biology; Bioterrorism; Bolivian Hemorrhagic Fever; Bolivian Hemorrhagic Fever Virus; Bunyaviridae; Catalytic Domain; Category A pathogen; Cells; Comparative Biology; Complex; Crimean-Congo Hemorrhagic Fever Virus; Cryoelectron Microscopy; Crystallization; Development; Disease; Disease Outbreaks; Enzymes; Event; Family; Gene Expression; Gene Expression Regulation; Genetic Transcription; Genome; Human; In Vitro; Infection; Influenza; La Crosse virus; Lassa Fever; Lassa virus; Length; Ligands; Lymphocytic Choriomeningitis; Mediating; Messenger RNA; Modeling; Molecular; Molecular Machines; National Institute of Allergy and Infectious Disease; Nucleoproteins; Phosphoproteins; Play; Polymerase; Positioning Attribute; Proteins; Public Health; RNA; RNA Binding; RNA Caps; RNA Sequences; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Regulation; Resolution; Ribonucleoproteins; Role; Site; South America; Structural Models; Structure; System; Testing; Therapeutic; Viral; Viral Genome; Viral Hemorrhagic Fevers; Viral Matrix Proteins; Virus; Virus Diseases; Virus Replication; Western Africa; Work; Zoonoses; base; cofactor; comparative; design; endonuclease; experimental study; fascinate; functional outcomes; genomic RNA; inhibitor/antagonist; insight; mortality; novel; plasma protein Z; promoter; structural biology; structured data; vesicular stomatitis virus L protein; viral RNA; viral genomics; weapons

Arenaviruses are responsible for significant zoonotic human illnesses and mortality across the globe, inducing Lassa fever in West Africa, lymphocytic choriomeningitis, and numerous regional hemorrhagic fever diseases in South America. These viruses are unable to infect their host cells without the delivery of a large transcriptionally primed replication and gene expression complex, consisting of the viral polymerase (L) directly bound to the nucleoprotein-encapsidated negative-sense genomic RNA (vRNA) segments. Arenavirus L proteins play essential roles in nearly every step of the virus infection cycle, including gene expression, genome replication, and formation of the infectious viral ribonucleoprotein. An accurate mechanistic understanding of L proteins has been hindered by a lack of available biochemical systems and structural models for these multifunctional enzymes. Machupo virus (MACV) is an arenavirus responsible for localized outbreaks of Bolivian hemorrhagic fever. Our lab has pioneered the use of MACV L as a model for arenavirus replication studies, developing multiple experimental systems for the purification and functional analyses of a full-length MACV L. Previous work in our lab has revealed that the catalytic activity of the MACV L protein is completely inhibited due to direct binding by the viral matrix protein (Z). As a result, the L-Z complex is locked in an inactive state on the viral RNA promoter. Moreover, a recently proposed model has suggested that the interaction of L with the 5' termini of the genome segments (5' vRNA) mediates enhanced RNA synthesis activity. The central hypothesis for my proposal is that the RNA synthesis and cap-snatching domains of MACV L are under opposing regulatory influences by the viral matrix protein (Z) and the terminal 5' genomic RNA sequences. The experiments outlined for this project will address the following gaps in our understanding of the MACV system and arenavirus biology: i) the structural mechanisms underlying the regulation of arenavirus L RNA synthesis by its protein and RNA ligands, ii) the biochemical role of the 5' vRNA in modulating L activity, and iii) the catalytic activity and regulation of the MACV L cap-snatching machinery Findings from this project will offer novel insight into the unanswered questions of arenavirus biology, and will certainly be applicable in downstream endeavors for the development of targeted antiviral therapeutic strategies. Our lab has recently made significant progress in the field of polymerase structural biology with the resolved atomic model of the vesicular stomatitis virus (VSV) L protein in complex with its phosphoprotein cofactor. Moreover, our lab has developed the only available in vitro and cell-based systems for the study of MACV L biochemical functions and regulation by its unique cofactors (Z and the 5' vRNA). These experimental systems make MACV the ideal candidate for L structure-function studies. Collectively, these efforts will significantly advance our understanding of arenavirus replication and gene expression, and the regulation of these activities via small viral ligands.

禽流感病毒是导致全球人畜共患疾病和死亡的主要原因， 在西非引发拉沙热、淋巴细胞性脉络膜脑膜炎和大量区域性出血热 南美的疾病。这些病毒不能感染宿主细胞，除非 转录启动复制和基因表达复合体，由病毒聚合酶(L)直接组成 结合核蛋白包裹的负义基因组RNA(VRNA)片段。阿丽娜病毒L 蛋白质在病毒感染周期的几乎每一个步骤中都发挥着至关重要的作用，包括基因表达， 基因组复制和传染性病毒核糖核蛋白的形成。精准的机械师 由于缺乏可用的生化系统和结构，对L蛋白质的理解一直受到阻碍 这些多功能酶的模型。马丘波病毒(Machupo Virus，MACV)是一种引起本地化的广谱病毒。 玻利维亚出血热暴发。我们实验室率先将巨型空泡病毒L作为禽流感病毒的模型 复制研究，开发用于纯化和功能分析的多个实验系统 我们实验室以前的工作表明，巨细胞病毒L蛋白的催化活性是 由于与病毒基质蛋白(Z)的直接结合，完全被抑制。因此，L-Z综合体被锁定 在病毒RNA启动子上处于非活性状态。此外，最近提出的一个模型表明， L与基因组片段5‘端(5’vRNA)相互作用促进核糖核酸合成 活动。我的建议的中心假设是，RNA合成和帽子抢夺结构域 L的基因受到病毒基质蛋白(Z)和末端5‘的相反调控作用 基因组RNA序列。本项目概述的实验将解决我们在以下方面的差距 对MACV系统和ArenaVirus生物学的理解：I)潜在的结构机制 L病毒蛋白质和核糖核酸配体对核糖核酸合成的调节作用--II)5‘端的生化作用 VRNA在调节L活性中的作用，以及III)巨细胞病毒L的催化活性及其调控 这个项目的机械发现将为阿雷诺病毒尚未解答的问题提供新的洞察力 生物学，并将肯定适用于下游努力开发靶向抗病毒药物 治疗策略。我们实验室最近在聚合酶结构领域取得了重大进展。 水疱性口炎病毒L蛋白及其复合体的可分辨原子模型生物学 磷蛋白辅因子。此外，我们的实验室还开发了唯一可用的体外和基于细胞的系统 为研究巨细胞病毒L的生化功能及其独特的辅因子(Z和5‘vRNA)的调控作用。 这些实验系统使MACV成为L结构-功能研究的理想候选者。总而言之， 这些努力将极大地促进我们对阿拉伯病毒复制和基因表达的理解，以及 通过小病毒配体对这些活动的调节。