Molecular determinants of host-cell interactions required for arenavirus replicat

描述(申请人提供)：ARENAV可引起全球高死亡率的急性出血热。Jun病毒被认为是美国人口的A类病原体和物质威胁。迫切需要针对ARENAV出血热的有效疗法，以解决持续的公共卫生和生物防御问题。我们的长期目标是阐明Junén病毒利用特定细胞功能在宿主细胞质中实现其基于RNA的复制周期的分子机制。我们最近发现了独特的细胞质结构，它们作为ArenaVirus复制和转录(RTCs)的隔间，也可能参与病毒的翻译。我们的总体假设是，病毒核蛋白的新活性对于这些膜相关RTCs的诱导和核糖体向病毒mRNAs的优先募集是必不可少的。在这项提案中，我们将定义多功能核蛋白中的决定因素，这些决定因素与细胞基础设施相连接，以组织一个允许病毒复制的环境。这些知识将增加我们对ARENAV生物学的理解，并为治疗急性出血热提出新的抗病毒策略。我们的具体目标是：(1)通过系统地鉴定共定位的细胞成分来研究RTCs的来源、结构和功能。我们的初步结果表明，RTCs包括来自膜重塑和翻译控制途径的独特的细胞成分组合。对增选蛋白和途径的显微和生化分析将建立一个框架，以了解阿拉伯病毒复制所需的基本病毒-细胞相互作用。(2)鉴定核蛋白在核糖体募集和病毒翻译中的功能作用。共聚焦显微镜研究表明，RTCs含有各种翻译起始因子和核糖体，但特异性地缺乏帽结合蛋白eIF4E。我们假设N作为代理结合m7G封顶的病毒mRNA并招募eIF4G支架蛋白，以获得对核糖体的特权访问。我们将使用生化和药理学方法来确定病毒翻译中核蛋白的功能需求。从这些研究中获得的知识对于指导小分子化合物的开发以抑制这种新的活性将是重要的。我们预计，基本的病毒-细胞相互作用可以被靶向地产生高度特异的抗病毒效果，从而抵抗遗传逃逸。 公共卫生相关性：禽流感病毒可引起急性出血热，在全球范围内死亡率很高。迫切需要有效的治疗方法来解决持续的公共卫生和生物防御问题。病毒在很大程度上依赖于与宿主细胞的相互作用，该项目将表征病毒复制、转录和翻译所需的病毒核蛋白的新功能。这些研究的结果将增加我们对ArenaVirus生物学的理解，并为治疗ArenaVirus出血热提出新的抗病毒策略。

DESCRIPTION (provided by applicant): Arenaviruses cause acute hemorrhagic fevers with high mortality worldwide. Jun¿n virus is recognized as a category A pathogen and Material Threat to the US population. Effective therapies against arenaviral hemorrhagic fevers are urgently needed to address ongoing public health and biodefense concerns. Our long- term goal is to elucidate the molecular mechanisms by which Jun¿n virus appropriates specific cellular functions to implement its RNA-based replicative cycle in the host-cell cytoplasm. We have recently identified unique cytoplasmic structures that serve as compartments for arenavirus replication and transcription (RTCs) and may also be involved in viral translation. Our overarching hypothesis is that novel activities of the virus nucleoprotein are essential for induction of these membrane-associated RTCs and for preferential recruitment of ribosomes to viral mRNAs. In this proposal, we will define determinants in the multifunctional nucleoprotein that interface with the cellular infrastructure to organize an environment permissive for viral replication. This knowledge will increase our understanding of arenavirus biology and suggest novel antiviral strategies for the treatment of acute hemorrhagic fevers. Our Specific Aims are to: (1) Investigate the origins, structure and function of RTCs by systematically identifying co-localizing cellular components. Our preliminary results suggest that RTCs comprise a unique combination of cellular elements from both membrane-remodeling and translational-control pathways. Microscopic and biochemical analysis of the co-opted proteins and pathways will establish a framework for understanding fundamental virus- cell interactions required for arenavirus replication. (2) Characterize the functional role of nucleoprotein in ribosome recruitment and viral translation. Confocal microscopy studies reveal that RTCs contain various translation initiation factors and ribosomes, but are specifically lacking the cap-binding protein eIF4E. We posit that N acts as a surrogate to bind m7G-capped viral mRNA and recruit the eIF4G scaffold protein, in order to gain privileged access to ribosomes. We will employ biochemical and pharmacological approaches to determine the functional requirement for nucleoprotein in viral translation. Knowledge gained from these studies will be important in guiding the development of small-molecule compounds to inhibit this novel activity. We anticipate that essential virus-cell interactions can be targeted to generate highly specific antiviral effects that are resistant to genetic escape. PUBLIC HEALTH RELEVANCE: Arenaviruses cause acute hemorrhagic fevers with high mortality worldwide. Effective therapies are urgently needed to address ongoing public health and biodefense concerns. Viruses depend extensively on interactions with the host cell, and this project will characterize novel functions of the viral nucleoprotein required for viral replication transcription and translation. The results from these studies will increase our understanding of arenavirus biology and suggest novel antiviral strategies for the treatment of arenavirus hemorrhagic fevers.