Structure-function studies of the tripartite Junin arenavirus GP-C

描述（由申请人提供）：这是一个重新提交的申请。我们根据重要的新成果并针对审查者的批评，修订并加强了该提案。沙粒病毒是世界范围内啮齿动物种群的地方病，可传播给人类引起急性出血热。预防性疫苗和有效的治疗方法缺乏，迫切需要解决公共卫生和生物防御问题。因此，靶向病毒包膜糖蛋白（GP-C）和病毒进入靶细胞的干预策略是有吸引力的。沙粒病毒GP-C的不寻常之处在于成熟复合物保留其裂解的信号肽（SSP）作为与常规受体结合（G1）和跨膜融合（G2）蛋白相关的必需亚基。我们的初步研究结果表明，SSP与G2的胞外域相互作用，调节GP-C复合物的pH依赖性膜融合活性，并形成一类新发现的小分子融合抑制剂的目标。SSP还与G2的胞质结构域结合，以调节GP-C复合物向细胞表面的细胞内转运，用于病毒体组装和出芽。我们最近的证据表明，SSP和G2之间的细胞质相互作用是通过一个新的亚基间锌指基序介导的。该项目的长期目标是确定促进病毒体组装和病毒进入的三重GP-C复合物的结构-功能关系，以利用GP-C的独特功能设计有效的抗病毒治疗药物。为了实现这一目标，我们提出了三个具体的目标：i）在分子上详细描述SSP和G2胞外结构域之间的相互作用，并了解新发现的小分子沙粒病毒融合抑制剂的作用机制; ii）为了鉴定在不寻常的桥接SSP和G2的胞质结构域的锌指基序中锌结合的分子要求，并表征其在GP-C复合物中的作用;和iii）研究SSP和亚基间锌指在病毒体形态发生中的作用。公共卫生相关性：沙粒病毒是导致高死亡率出血热的原因。迫切需要针对拉沙热、阿根廷出血热和其他疾病的有效疗法，以解决持续的公共卫生和A类生物防御问题。该项目的目标是在分子细节上表征不寻常的沙粒病毒包膜糖蛋白，以指导预防和治疗药物的开发策略。

DESCRIPTION (provided by applicant): This is a resubmitted application. We have revised and strengthened the proposal in light of significant new results and in response to the reviewers' critiques. Arenaviruses are endemic in rodent populations worldwide and can be transmitted to humans to cause acute hemorrhagic fevers. Prophylactic vaccines and effective therapies are lacking and urgently needed to address public health and biodefense concerns. Intervention strategies that target the viral envelope glycoprotein (GP-C) and virus entry into target cells are thus attractive. The arenavirus GP-C is unusual in that the mature complex retains its cleaved signal peptide (SSP) as an essential subunit in association with the conventional receptor-binding (G1) and transmembrane fusion (G2) proteins. Our preliminary results have shown that SSP interacts with the ectodomain of G2 to modulate pH-dependent membrane-fusion activity of the GP-C complex, and to form the target for a class of newly discovered small-molecule fusion inhibitors. SSP also associates with the cytoplasmic domain of G2 to regulate intracellular transport of the GP-C complex to the cell surface for virion assembly and budding. Our recent evidence indicates that the cytoplasmic interaction between SSP and G2 is mediated through a novel intersubunit zinc-finger motif. The broad, long-term goal of this project is to define the structure-function relationships in the tripartite GP-C complex that promote virion assembly and virus entry, in order to exploit the unique features of GP-C in the design of effective antiviral therapeutics. Towards this goal, we propose three specific aims: i) To characterize in molecular detail the interaction between SSP and the ectodomain of G2, and to understand the mechanism of action of the newly discovered small-molecule arenavirus fusion inhibitors; ii) To identify the molecular requirements for zinc binding in an unusual zinc-finger motif bridging SSP and the cytoplasmic domain of G2, and to characterize its role in the GP-C complex; and iii) To investigate the role of SSP and the intersubunit zinc finger in virion morphogenesis. PUBLIC HEALTH RELEVANCE: Arenaviruses are responsible for hemorrhagic fevers with high mortality. Effective therapies against Lassa fever, Argentine hemorrhagic fever and others are urgently needed to address ongoing public health and Category A biodefense concerns. The goal of this project is to characterize the unusual arenavirus envelope glycoprotein in molecular detail to guide strategies towards the development of prophylactic and therapeutic agents.