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温带和热带地区造成大量发病率和死亡率。西尼罗河病毒在过去十年中在美国蔓延，仅在2006年就造成4052例感染和146例死亡。我们打算继续我们对病毒生命周期的结构研究，以阐明不同的病毒成分首先组装成未成熟的颗粒，然后蜕变成成熟的感染性病毒粒子的过程。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，开始内吞作用，最后将病毒基因组释放到宿主细胞质中。大多数感染哺乳动物的病毒都需要在感染性病毒粒子从细胞释放之前的最后时刻发生成熟过程。未成熟的病毒粒子在被释放感染其他细胞之前，必须保护自己不过早地与细胞膜相互作用。最后，但并非最不重要的是，我们打算扩展我们对抗体和黄病毒之间相互作用的研究，以建立各种中和机制，以帮助开发不会引起抗体依赖性增强感染的疫苗。在过去几年中，我们已经学会了以毫克为单位生产纯化登革热病毒（各种毒株）和西尼罗河病毒，这些病毒的质量足以用于结构研究。西尼罗河病毒特别适合，因为它的稳定性更强，但需要生物安全3级设施和预防措施。这些病毒将用于制作低温电子显微镜三维重建，以研究未成熟和成熟的黄病毒与各种中和抗体和细胞受体分子络合。有些抗体在病毒生命周期的特定中间阶段抑制病毒成熟或融合。我们计划利用我们最近的成功来确定由前体膜蛋白（prM）和包膜糖蛋白(E)组成的未成熟病毒异二聚体外畴的晶体结构。这种结构使得建立未成熟登革热病毒和pr多肽释放和成熟进入感染性颗粒之前的低pH中间物的伪原子模型成为可能。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。这些研究对于制定抗病毒和疫苗战略至关重要，以便建立可行的防御措施，以抵御以黄病毒为武器的自然流行病或生物恐怖袭击。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，在全世界引起对人类健康的重大关切。仅登革热病毒每年就在全世界造成5 000万或更多感染病例，造成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。这些信息对于研制抗病毒药物和疫苗以及在发生任何可能的流行病时确定最佳对策至关重要。

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.