Molecular determinants of host-cell interactions required for arenavirus replicat

描述（由申请人提供）：沙粒病毒引起急性出血热，在世界范围内具有高死亡率。埃博拉病毒被认定为a类病原体和对美国人口的物质威胁。目前迫切需要有效治疗沙粒病毒性出血热，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Jun¿n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，作为沙粒病毒复制和转录（rtc）的区室，也可能参与病毒翻译。我们的首要假设是，病毒核蛋白的新活性对于诱导这些膜相关rtc和优先招募核糖体到病毒mrna是必不可少的。在本提案中，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，以组织允许病毒复制的环境。这一知识将增加我们对沙粒病毒生物学的理解，并为急性出血热的治疗提出新的抗病毒策略。我们的具体目标是：(1)通过系统地识别共定位的细胞成分来研究rtc的起源、结构和功能。我们的初步结果表明，rtc包括来自膜重塑和翻译控制途径的细胞元件的独特组合。对增选蛋白和途径的显微和生化分析将为理解沙粒病毒复制所需的基本病毒-细胞相互作用建立一个框架。(2)描述核蛋白在核糖体募集和病毒转译中的功能作用。共聚焦显微镜研究显示，rtc含有多种翻译起始因子和核糖体，但特别缺乏帽结合蛋白eIF4E。我们假设N作为替代物结合m7g -帽状病毒mRNA并募集eIF4G支架蛋白，从而获得进入核糖体的特权。我们将采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抑制这种新活性的小分子化合物的发展将是重要的。我们预计基本的病毒-细胞相互作用可以靶向产生高度特异性的抗病毒作用，从而抵抗遗传逃逸。

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.