Structure and Function of Flaviviruses

性状（由申请方提供）：黄病毒属是主要的人类病原体。它们包括西尼罗河病毒、黄热病病毒和登革热病毒。这些病毒每年在地球的温带和热带地区导致显著的发病率和死亡率。在过去的十年中，西尼罗河病毒在美国各地蔓延，仅在2006年就造成了4052例感染和146例死亡。我们打算继续我们的病毒生命周期的结构研究，以阐明的过程中，不同的病毒成分组装成不成熟的颗粒，然后变形成成熟的感染性病毒粒子。我们还计划继续对这些病毒感染宿主的途径进行结构研究，包括最初的宿主细胞识别，与宿主细胞质膜融合引发内吞作用，最后将病毒基因组释放到宿主细胞的细胞质中。大多数感染哺乳动物的病毒都需要一个成熟过程，这一过程发生在感染性病毒粒子从细胞释放之前的最后时刻。未成熟的病毒体在被释放出来感染其他细胞之前，必须保护自己不与细胞膜过早相互作用。最后，但并非最不重要的是，我们打算扩大我们的抗体和黄病毒之间的相互作用的研究，以建立各种机制的中和作为一个援助的疫苗，不引起抗体依赖性增强感染的发展。在过去的几年里，我们已经学会了生产纯化的登革热病毒（各种毒株）和西尼罗河病毒，其质量足以进行结构研究。西尼罗河病毒由于其更大的稳定性而特别适合，但需要生物安全3级设施和预防措施。这些病毒将用于产生冷冻电子显微镜三维重建，以研究与各种中和抗体和细胞受体分子复合的未成熟和成熟黄病毒。一些抗体在病毒生命周期的特定中间步骤抑制病毒成熟或融合。我们计划利用我们最近的成功，确定晶体结构的不成熟病毒的异源二聚体胞外域组成的前体膜蛋白（prM）和包膜（E）糖蛋白。这种结构使得有可能建立假原子模型的未成熟的登革病毒和低pH值的中间体之前的pr多肽的释放和成熟成感染性颗粒。突变和结构研究现在将允许确定控制病毒成熟为感染性颗粒时发生的非常大的构象变化的氨基酸。这些研究对于开发抗病毒和疫苗策略以建立针对自然流行病或基于使用黄病毒作为武器的生物恐怖袭击的可行防御至关重要。公共卫生相关性：黄病毒，包括西尼罗河病毒、黄热病病毒和登革热病毒，是重要的人类病原体，引起世界各地人类健康的重大关注。仅登革热病毒每年就在全世界造成5 000万或更多的感染病例，导致24 000人死亡。我们建议深入研究黄病毒，特别是西尼罗河病毒和登革热病毒的生命周期中组装途径和感染过程中发生的结构变化。这些信息对于开发抗病毒药物和疫苗以及确定在发生任何可能的流行病时的最佳应对措施至关重要。

DESCRIPTION (provided by applicant): Flaviviruses are major human pathogens. They include West Nile, yellow fever and dengue viruses. These viruses result in significant morbidity and mortality each year in temperate and tropical regions of the Earth. West Nile virus has spread throughout the US in the last decade, having caused 4052 recorded infections and 146 deaths in 2006 alone. We intend to continue our structural studies of the viral life cycle in order to elucidate the processes by which the different viral components are assembled first into immature particles and then metamorphose into mature infectious virions. We also plan to continue our structural studies of the path by which these viruses infect their hosts, including the initial host cell recognition, fusion with the host cell plasma membrane initiating endocytosis and, finally, the release of the viral genome into the host cell's cytoplasm. A maturation process that occurs in the final moments before release of infectious virions from a cell is required by most viruses that infect mammals. The immature virions must protect themselves against premature interaction with the cell's own membranes before being released to infect other cells. Last, but not least, we intend to extend our studies of the interaction between antibodies and flaviviruses in order to establish the various mechanisms of neutralization as an aid to the development of vaccines that do not cause antibody-dependent enhancement of infection. Over the past few years we have learned to produce purified dengue (various strains) 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 bio-safety level 3 facilities and precautions. These viruses will be used to produce cryo-electron microscopy three-dimensional reconstructions to study immature and mature flaviviruses complexed with various neutralizing antibodies and with cellular receptor molecules. Some of the antibodies inhibit virus maturation or fusion at specific intermediate steps in the viral life cycle. We plan to exploit our recent success in determining the crystal structure of the immature virus' heterodimer ectodomain consisting of the precursor membrane protein (prM) and envelope (E) glycoprotein. This structure has made it possible to build pseudo-atomic models of immature dengue virus and of a low pH intermediate prior to the release of the pr polypeptide and maturation into infectious particles. Mutational and structural studies will now permit determination of the amino acids that control the very large conformational changes that occur when the virus matures into infectious particles. These studies are essential for developing anti-viral and vaccine strategies to establish viable defenses against natural epidemics or bio-terrorist attacks based on using flaviviruses as a weapon. PUBLIC HEALTH RELEVANCE: Flaviviruses, which include West Nile, yellow fever and dengue viruses, are significant human pathogens that give rise to major concerns for human health the World over. Dengue virus alone causes 50 million or more cases of infection worldwide each year, resulting in 24,000 deaths. We propose in-depth studies of the structural changes that occur in the assembly pathway and infection process during the life cycle of flaviviruses, in particular West Nile and dengue viruses. Such information is essential for the development of antiviral drugs and vaccines and for determining the best response in the event of any possible epidemic.