Structural and biochemical characterization of paramyxovirus fusion complexes

描述（由申请方提供）：副粘病毒家族和非pH依赖性进入的病毒是主要的人类发病率和死亡率的原因。包膜病毒的成功进入需要病毒和细胞膜的融合。病毒已经进化出多种机制来实现融合，包括pH和蛋白质-蛋白质相互作用诱导的病毒糖蛋白的构象变化，其最终导致膜融合和孔形成。虽然这一过程的广泛机制是了解大多数包膜病毒，具体的变化和构象状态只有少数病毒已被彻底阐明。为了更好地理解中性pH下蛋白质介导的融合，这些实验将研究副粘病毒家族的原型麻疹病毒（MV）的结构重排。具体目的1 -以生物化学方式确定融合前和融合后副粘病毒糖蛋白异源寡聚体，并使用天然电泳、碳水化合物屏蔽、定点诱变和功能互补确定受体结合对融合前复合物的影响。具体目标2 -通过纯化病毒粒子的冷冻电子断层扫描（cryo-ET）原位确定副粘病毒糖蛋白的几何形状。糖蛋白的晶体结构数据将叠加到从冷冻ET中识别的电子密度上，以产生高分辨率的异源寡聚体伪原子结构。这项工作将进一步了解一个家庭的主要人类和动物病原体感染的分子机制。了解融合的分子机制将为新型抗病毒治疗药物的结构导向设计提供基础。 公共卫生相关性：尽管有有效的疫苗，麻疹病毒感染在美国和世界各地仍在上升。了解麻疹病毒如何感染细胞将为开发针对麻疹病毒和其他密切相关的人类病原体的新型抗病毒疗法奠定框架。

DESCRIPTION (provided by applicant): The paramyxovirus family and viruses that undergo pH independent entry are responsible for major human morbidity and mortality. Successful entry of enveloped viruses requires the fusion of viral and cellular membranes. Viruses have evolved a variety of mechanisms to accomplish fusion including pH and protein-protein interaction induced conformational changes in the viral glycoprotein that ultimately lead to membrane fusion and pore formation. Although the broad mechanism of this process is understood for most enveloped viruses, the specific changes and conformational states have only been thoroughly elucidated for a handful of viruses. To better understand protein-mediated fusion at neutral pH, these experiments will investigate the structural rearrangements of an archetype of the paramyxovirus family, measles virus (MV). Specific Aim 1 - to biochemically define the pre- and post-fusion paramyxovirus glycoprotein hetero- oligomers and determine the effect of receptor binding on the pre-fusion complex using native- electrophoresis, carbohydrate shielding, site-directed mutagenesis, and functional complementation. Specific Aim 2 - to determine the geometry of paramyxovirus glycoproteins in situ by cryo-electron tomography (cryo-ET) of purified virions. Crystal structure data of the glycoproteins will be superimposed onto the electron densities identified from the cryo-ET to produce a high resolution hetero-oligomer pseudo-atomic structure. This work will further the understanding of the molecular mechanism of infection of a family of major human and animal pathogens. Understanding the molecular mechanism of fusion will provide a basis for structure-guided design of novel antiviral therapeutics. PUBLIC HEALTH RELEVANCE: Measles virus infections are on the rise in the US and around the world despite an effective vaccine. Understanding how the measles virus infects cells will lay the framework for the development of novel antiviral therapies against measles virus and other closely related human pathogens.