Structure and Function of Flaviviruses

描述(申请人提供)：黄病毒是人类的主要病原体。它们包括西尼罗河病毒、黄热病和登革热病毒。这些病毒每年在地球温带和热带地区造成数百万人死亡。在过去的十年里，西尼罗河病毒在美国各地蔓延，每年造成100多人死亡。同样，1960年至2010年间，全球登革热发病率增加了30倍，这可能是由于缺乏适当的杀虫剂来阻止蚊子媒介的传播。我们打算继续我们对病毒生命周期的结构研究，以便绘制出当病毒组装成未成熟颗粒时，当未成熟颗粒变成传染性病毒时，以及最后当这些颗粒变成融合子时，为了合成新的病毒成分而入侵新宿主时发生的结构变化。未成熟的颗粒含有60个三聚体PrM：E，其中PrM是膜前蛋白，E是包膜糖蛋白。成熟病毒含有90个M：E二聚体，融合后的颗粒至少含有一些M：E的三聚体，由于未成熟和成熟颗粒二十面体骨架内三个亚基所处的环境不同，60个二十面体不对称单元中每一个内的三种亚基可以被不同地标记。因此，在成熟(30个三聚体到90个二聚体)或融合(90个二聚体到至少一些三聚体)期间亚基的重新分类可以通过标记来遵循。目的是通过使用冷冻电子显微镜检查标记的亚基在运动的开始和结束时的各种路径来研究亚基运动的各种路径，并用冷冻电子断层扫描来研究多形性的中间产物。此外，标签已被证明可以阻止(至少在一个实例中)完整的转化并在各种中间步骤捕捉粒子。我们计划使用三种类型的标记：抗体的FAB片段、重金属簇；以及去除E蛋白表面暴露更多的多糖。不对称标记(对每个二十面体不对称单元中的三个不同亚基进行不同标记)的初步结果已经成功。我们已经学会了以毫克量生产纯化的登革热病毒和西尼罗河病毒，这些病毒的质量足以进行结构研究。西尼罗河病毒特别适合，因为它更稳定，但需要生物安全3级设施和预防措施。我们还计划继续对病毒最初识别和随后感染合适宿主的结构进行研究。我们将通过将潜在受体分子的重组产生的片段与病毒结合来检测许多细胞表面分子中的一些，这些分子已被初步证明是黄病毒受体。这应该会在内吞作用过程中启动与宿主细胞内质膜的融合，最终触发病毒基因组释放到宿主细胞的细胞质中。

DESCRIPTION (provided by applicant): Flaviviruses are major human pathogens. They include West Nile, yellow fever and dengue viruses. These viruses cause millions of deaths each year in temperate and tropical regions of the Earth. West Nile virus has spread throughout the US in the last decade, causing more than 100 deaths annually. Similarly the incidence of dengue increased 30-fold worldwide between 1960 and 2010, probably due to lack of suitable insecticide to stop the spread of the mosquito vector. We intend to continue our structural studies of the viral life cycle in order to map the structural changes that occur when the virus assembles into immature particles, when the immature particles change to infectious virus and, finally, when these become fusogenic in order to invade a new host for synthesis of new viral components. The immature particles contain 60 trimers of prM:E heterodimer, where prM is the pre-membrane protein and E is the envelope glycoprotein. The mature virus contains 90 dimers of M:E heterodimers and the post-fusion particles contain at least some trimers of M:E. Because of the different environment of the three subunits within the icosahedral framework of the immature and mature particles, the three types of subunits within each of the 60 icosahedral asymmetric units can be labeled differently. Thus, the re-assortment of the subunits during maturation (30 trimers to 90 dimers) or fusion (90 dimers to at least some trimers) can be followed by virtue of the labels. The purpose will be to study the various pathways of the subunit motions by inspecting the labeled subunits at the beginning and end of their motions using cryo-electron microscopy, and also to study the pleomorphic intermediates with cryo-electron tomography. Furthermore, the labels have been shown to block (at least in one instance) the full transformation and catch the particles at various intermediate steps. We plan to use three types of labels: Fab fragments of antibodies, heavy metal clusters; and the removal of the more exposed glycans on the E protein surface. Preliminary results of asymmetric labeling (different labels to the three different subunits in each icosahedral asymmetric unit) have been successful. We have learned to produce purified dengue virus and West Nile virus in milligram quantities of sufficient quality for structural studies. West Nile virus is especially suitable because of its greater stability, but requires biosafety level 3 facilities and precautions. We also plan to continue structural studies of the initial recognition and subsequent infection of a suitable host by the virus. We will examine some of the many cell surface molecules that have been tentatively shown to be flavivirus receptors by binding recombinantly produced fragments of potential receptor molecule to the virus. This should initiate fusion with the host cell endoplasmi membrane during endocytosis and, eventually, trigger release of the viral genome into the host cell's cytoplasm.