Structure-function of Junin virus envelope glycoproteins

描述（由申请人提供）：沙粒病毒是人类高死亡率急性出血热的病原。旧大陆沙粒病毒（如拉沙病毒）和新世界沙粒病毒（如朱宁病毒）通过偶然接触或气溶胶接触传播。迫切需要预防性疫苗和有效的抗病毒治疗，以遏制世界各地反复发生和新出现的疫情，并在发生生物袭击时保护公众。沙粒病毒是包膜病毒，其包膜糖蛋白GP-1和GP-2介导受体结合和膜融合。我们的初步分析表明，GP-2外域的七重重复区域可能形成一个六螺旋束结构，代表了蛋白质的融合活性构象。该应用程序的长期目标是详细了解Junin病毒包膜糖蛋白的结构-功能关系，并利用这种理解来靶向病毒进入过程，用于疫苗设计和抗病毒药物开发。本研究的具体目的是：1)表征Junin病毒包膜糖蛋白的生物发生和生化特性，并确定ph依赖性膜融合活性的遗传决定因素。扫描诱变将用于定义在融合活性六螺旋束形成的基础上的七重重复区域内的氨基酸残基。2)利用生物化学和免疫化学方法表征GP-1/GP-2复合物的ph依赖性构象变化，以了解酸诱导的Junin病毒膜融合在核内体中的激活。3)检验包膜糖蛋白介导的细胞-细胞融合实验是否真实再现了Junin病毒感染的早期事件，旨在阐明Junin病毒的入侵机制，促进沙粒病毒感染保护策略的制定。本申请是根据生物防御和新发传染病研究的PA-03-080提交的。

DESCRIPTION (provided by applicant): Arenaviruses are the etiological agents of acute hemorrhagic fevers with high mortality in humans. Old World arenaviruses, such as Lassa virus, and New World arenaviruses, such as Junin virus, are transmitted through casual or aerosol contact. Prophylactic vaccines and effective antiviral treatments are urgently needed to curb recurring and emerging outbreaks throughout the world, and to protect the public in the event of a biological attack. Arenaviruses are enveloped viruses, and their envelope glycoproteins, GP-1 and GP-2, mediate receptor binding and membrane fusion. Our preliminary analyses suggest that the heptad-repeat regions of the GP-2 ectodomain may form a six-helix bundle structure that represents the fusion-active conformation of the protein. The long-term goal of this application is to develop a detailed understanding of the structure-function relationships of the Junin virus envelope glycoproteins, and to use this understanding to target the viral entry process for vaccine design and antiviral drug development. Specific aims of this developmental research application are: 1) To characterize the biogenesis and biochemical properties of the Junin virus envelope glycoproteins and to identify the genetic determinants of pH-dependent membrane fusion activity. Scanning mutagenesis will be used to define amino acid residues within the heptad-repeat regions that underlie formation of the fusion-active six-helix bundle. 2) To characterize pH-dependent conformational changes in the GP-1/GP-2 complex using biochemical and immunochemical approaches, in order to understand the acid-induced activation of Junin virus membrane fusion in endosomes. 3) To test whether the envelope glycoprotein-mediated cell-cell fusion assay faithfully recapitulates the early events of Junin virus infection, in an effort to elucidate the mechanism of Junin virus entry and to facilitate the development of protective strategies against arenavirus infection. This application is submitted in response to PA-03-080 for Biodefense and Emerging Infectious Disease Research.