Characterization of the arenavirus glycoprotein complex and mechanism of fusion

描述（由申请人提供）：本项目将开始研究沙粒病毒糖蛋白复合物的结构变化和功能域。融合孔的形成机制对所有包膜病毒都是必要的。对经典i类融合蛋白的详细研究揭示了流感病毒、艾滋病病毒和副粘病毒的融合机制。2007年沙粒病毒糖蛋白被归类为i类融合蛋白，但关于这些融合蛋白的结构、构象变化和生化特性的细节是有限的。沙病毒科被认为是一种新出现的病毒，因为尽管该病毒在啮齿动物种群中是地方性的，但不断发现新的毒株感染人类。一些菌株，包括拉沙病毒和朱宁病毒，可引起具有高发病率和死亡率的毁灭性出血热。此外，新的证据表明，美国和世界其他地区流行的淋巴细胞性脉络丛脑膜炎病毒可能是导致临床显著疾病的原因。目前还没有获得许可的有效治疗方法或疫苗来治疗这些感染，仅拉沙热每年就影响50万人，并在西非造成5000人死亡。了解进入机制，特别是确定糖蛋白复合物（GPC）内的结构/功能域，将为未来的治疗提供新的靶点，并提供对未被研究的i类融合蛋白的基本了解。为此，本提案有两个具体目标：1)确定GP1亚基中的受体结合位点；2)原位确定拉沙热糖蛋白复合物的几何形状。利用生化分析，以及细胞间融合分析，原生电泳和最先进的低温电子断层扫描，糖蛋白复合物诱导融合所需的构象变化模型将出现。未来使用基于结构设计的研究将能够针对GPC中的特定构象来生产有效的治疗方法和疫苗。该项目为我提供了充分的机会，利用创新的电子显微镜技术，并将结构数据与先进的生化方法相结合，阐明沙粒病毒在自然状态下融合的机制。这种结合的专业知识将为我未来在分子病毒学，计算生物学，生物化学和结构分析交叉领域的独立研究生涯提供坚实的基础。以协作、跨学科的方式实现我的项目目标，将成功地为我未来领导研究团队和解决未来日益复杂和多方面的生物学挑战所需的多样化专业知识做好准备。

DESCRIPTION (provided by applicant): This project will begin to examine the structural changes and the functional domains of the arenavirus glycoprotein complex. The mechanism of fusion pore formation is necessary for all enveloped viruses. Detailed examination of classical class-I fusion proteins have illuminated the fusion mechanisms of influenza, HIV and paramyxoviruses. As evidenced by the 2007 classification of the arenavirus glycoprotein as a class-I fusion protein, details about the structure, conformational changes, and biochemical characterization of these fusion proteins is limited. The arenavirus family in considered an emerging virus, because although the viruses are endemic in the rodent population, new strains continually are identified to infect humans. Some strains, including Lassa and Junin, cause devastating hemorrhagic fevers with high morbidity and mortality. In addition new evidence suggests that Lymphocytic Choriomeningitis virus, endemic to the US and the rest of the world may be responsible for clinically significant disease. There are no effective therapeutics or vaccines currently licensed to treat the infections, and Lassa fever alone affects half a million people each year and is responsible for 5000 deaths in West Africa. Understanding the mechanism of entry, specifically identifying the structural/functional domains within the glycoprotein complex (GPC) will provide new targets for future therapeutics, as well as provide a basic understanding of an understudied class-I fusion protein. Towards this end, this proposal has two Specific Aims: 1) Determine the receptor binding site in the GP1 subunit; 2) Define the geometry of the Lassa fever glycoprotein complex in situ. Utilizing biochemical analysis, as well as cell-to-cell fusion assays, native-electrophoresis, and state-of-the-art cryo-electron tomography, a model of the conformational changes required for the glycoprotein complex to induce fusion will emerge. Future studies using structure based designs will be able to target specific conformations in the GPC to produce effective therapeutics and vaccines. The project offers ample opportunity for me to utilize innovative electron microscopy techniques, and combine the structural data with advanced biochemical approaches to elucidate the mechanism of arenavirus fusion in their natural state. This combined expertise will provide a solid foundatio for my future independent research career at the intersection of molecular virology, computational biology, biochemistry and structural analysis. Reaching my project goals in a collaborative, interdisciplinary approach will successfully prepare me for future leadership of research teams and diverse expertise that will be required to solve increasingly complex and multifaceted biological challenges of the future.