Structure and Function of Flaviviruses

描述（由申请人提供）：黄病毒是主要的人类病原体。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的温带和热带地区造成数百万人死亡。西尼罗河病毒在过去十年中在美国蔓延，每年造成100多人死亡。同样，1960年至2010年期间，全世界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发生的结构变化，当未成熟颗粒变成传染性病毒时，最后，当这些病毒变成融合性病毒以入侵新宿主以合成新的病毒成分时。未成熟颗粒含有60个三聚体prM:E异二聚体，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异源二聚体，融合后颗粒含有至少一些M:E三聚体。由于未成熟和成熟颗粒的二十面体框架内的三种亚基环境不同，因此60个二十面体不对称单元内的三种亚基可以进行不同的标记。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凭借标签进行。目的是通过低温电子显微镜观察亚基运动的开始和结束，研究亚基运动的各种途径，并利用低温电子断层扫描研究多形性中间体。此外，这些标签已经被证明可以阻止（至少在一个实例中）整个转化过程，并在各种中间步骤中捕获粒子。我们计划使用三种类型的标签：抗体Fab片段，重金属簇；以及去除E蛋白表面更多暴露的聚糖。不对称标记（对每个二十面体不对称单元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为单位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用于结构研究。西尼罗河病毒因其更大的稳定性而特别适用，但需要生物安全3级设施和预防措施。我们还计划继续进行病毒对合适宿主的初始识别和随后感染的结构研究。通过将重组产生的潜在受体分子片段与病毒结合，我们将研究一些已初步证明是黄病毒受体的许多细胞表面分子。这将在内吞过程中启动与宿主细胞内质膜的融合，并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.