Assembly of enveloped viruses

我们建议研究包膜病毒的组装和结构。我们提议的研究将主要检查黄病毒。黄病毒包括一类正链RNA病毒，其中许多病毒会导致人类重大疾病。这个家族的成员包括黄热病病毒、登革热病毒和西尼罗河病毒。我们将研究这些病毒利用衣壳蛋白进行基因组RNA募集、组装和与跨膜糖蛋白结合的过程。我们在甲型病毒和黄病毒结构方面的经验将支持进一步的研究，以检查其他球形、囊膜RNA病毒的结构特征，如风疹病毒、牛病毒性腹泻病毒和冠状病毒。 将采用多学科方法来研究这些病毒的结构和组装。这一方法将需要使用分子遗传学、生物化学和结构技术来探索病毒组装的机制及其与病毒粒子结构的关系。与迈克尔·罗斯曼和蒂姆·贝克的实验室合作，我们将使用冷冻电子显微镜和X射线结晶学来研究核衣壳和整个病毒粒子的结构。我们将与Carol Post合作，利用核磁共振技术扩展我们对黄病毒和鼠疫病毒衣壳蛋白的结构研究。同时，我们将对纯化的衣壳蛋白进行生化研究，这与结构结果无关。 在大肠杆菌中。这些研究将与包括复制子技术在内的分子遗传学方法相结合，以检查这些衣壳蛋白的结构-功能关系。这项研究将促进我们对病毒组装、大分子相互作用的了解，并有助于阐明病毒和细胞萌发的分子机制。对病毒组装的了解有望被证明有助于设计针对这些不同人类和动物病原体的抗病毒化合物。

We propose to investigate the assembly and structure of enveloped viruses. Our proposed research will primarily examine flaviviruses. The flaviviruses comprise a genus of plus-strand RNA viruses, many of which cause significant disease in humans. Members of this family include yellow fever virus, dengue virus, and West Nile virus. We will investigate the process by which these viruses utilize their capsid protein for genome RNA recruitment, assembly, and association with the transmembrane glycoproteins. Our experience with alphavirus and flavivirus structure will support additional studies to examine the structural features of other spherical, enveloped, RNA viruses, such as rubella virus, bovine viral diarrhea virus, and coronaviruses. A multi-disciplinary approach will be employed to investigate the structure and assembly of these viruses. This approach will entail the use molecular genetics, biochemistry, and structural techniques to probe the mechanism of virus assembly and its relation to virion structure. In collaboration with the laboratories of Michael Rossmann and Tim Baker, we will use cryo-electron microscopy and X-ray crystallography to examine the structure of nucleocapsids and whole virions. We will collaborate with Carol Post to extend our structural studies on the flavivirus and pestivirus capsid proteins using NMR techniques. In parallel and independent of structural results, we will carry out biochemical studies on purified capsid proteins produced in E. coli. These studies will be combined with molecular genetic approaches including replicon technology to examine structure-function relationships in these capsid proteins. The proposed research will advance our knowledge of virus assembly, macromolecular interactions, and serve to elucidate the molecular mechanisms of virus and cellular budding. An understanding of virus assembly is expected to prove useful in the design of antiviral compounds against these various human and animal pathogens.