STRUCTURAL STUDIES OF VIRUSES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们研究病毒的结构及其组成部分。我们感兴趣的是病毒的装配途径、病毒与宿主的相互作用和进入机制。我们广泛使用电子显微镜来研究不能结晶的病毒或病毒片段。这些相对低分辨率的地图可以通过蛋白质组分的晶体学研究来增强。 1.黄病毒[1]：西尼罗河病毒[2 3 4 5 6]和登革热病毒[7 8 2 9 10 11 12 13 14] 对西尼罗河病毒晶体的分析工作正在进行中，这种晶体的分辨率约为16埃。这些晶体中的病毒粒子似乎与晶格中的相邻粒子发生了融合。黄病毒的囊膜蛋白（E）、前成熟膜蛋白（prM）和PrM-E复合物等组分的结构正在研究中。 还在研究各种Fab片段与E. 此外，我们正在通过冷冻电镜研究与整个病毒复合的各种Fab片段。结合这些晶体学和EM研究，使我们深入了解抗体中和的机制以及病毒进入细胞期间发生的融合过程。 2.甲病毒[15 16 17 18 7 19 13 14]： α病毒（辛德毕斯病毒罗斯河病毒基孔肯雅病毒）的电子显微镜研究与E1和E2表面糖蛋白的晶体研究相结合。E1-E2融合蛋白的晶体结构（省略了跨膜区域）显示了三聚体刺突，其结构与全病毒的EM研究中发现的结构相同。该结构仍然需要相位改进（使用SeMet SAD数据），以便完全建立E2的先前未知的结构。 我们最近获得了基孔肯雅病毒（CHIKV）样颗粒（VLP）的优异cryoEM结构。CHIKV是在东南亚具有致命影响的新兴病原体。我们已经开始研究中和抗体的一些Fab片段的晶体结构。将这些结构与它们与病毒的复合物的cryoEM结构相结合将提供关于中和病毒上的抗原位点的模式的信息以及用于设计VLP疫苗的其它基本信息。 我们还致力于风疹病毒（德国麻疹的病原体）的冷冻电子显微镜与其E1和E2糖蛋白组分及其衣壳蛋白的结构测定相结合。 我们正在使用与我们用于研究登革病毒13和辛德毕斯病毒17相同的克隆表达和结晶技术。 3.细小病毒科： 已经确定了几种细小病毒的衣壳结构[20 21]，包括人细小病毒（B19）[22 23]。一种虾和一种蚕的细小病毒的晶体结构正在测定中。正在尝试使B19衣壳蛋白的PLA 2独特区域结晶。 4. T4噬菌体的结构组分： 到目前为止，我们已经解决了大约40种蛋白质中的15种[24 25 26 27 28 31]。我们最近已经确定了T4的结构和晶体的T4的临时和临时的两个次要的头部辅助蛋白。 5.其他建筑物的结构组成： 我们有phiX 174 29的H pilot蛋白片段的晶体，并有phi 29的phiKZ 30 gp 13 gp 9细胞裂解酶的各种蛋白的晶体[32 33]。 6.拟病毒结构蛋白 我们正在用冷冻电镜和冷冻断层扫描研究巨型拟态病毒。我们目前正在鉴定用于重组蛋白结晶的各种结构蛋白，包括主要衣壳蛋白。 由于篇幅所限，参考文献作为单独文件附于附件。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We study the structure of viruses and their components. We are interested in viral assembly pathways viral interaction with the host and entry mechanisms. We make extensive use of electron microscopy to study viruses or fragments of viruses that cannot be crystallized. These relatively low resolution maps can be augmented by crystallographic studies of the protein components. 1. Flaviviruses [1]: (West Nile virus [2 3 4 5 6] and Dengue virus [7 8 2 9 10 11 12 13 14] The analysis of West Nile virus crystals that diffract to about 16Angstrom resolution is in progress. The virions in these crystals appear to have undergone fusion with neighboring particles in the crystal lattice. Structure of components of flaviviruses such as envelope protein (E) the pre maturation membrane protein (prM) and PrM-E complex are being studies. Also being studied are crystal structures of various Fab fragments in complex with E. In addition we are studying various Fab fragments complexed with the whole virus by cryoEM. Combining these crystallographic and EM studies has given us insight into the mechanisms of neutralization by the antibodies as well as the fusion process that occurs during virus entry into cells. 2. Alphaviruses [15 16 17 18 7 19 13 14]: Electron microscopy studies of alpha viruses (Sindbis virus Ross River virus Chikungunya virus) are being combined with crystal studies of the E1 and E2 surface glycoprotein. The crystal structure of an E1-E2 fusion protein (with the trans-membrane regions omitted) shows a trimeric spike which has a structure the same as found in the EM studies of the whole virus. This structure still requires phase improvement (using SeMet SAD data) in order to completely establish the the previousily unknown structure of E2. We have recently obtained an excellent cryoEM structure of Chikungunya virus(CHIKV) like particles (VLPs). CHIKV is an emerging pathogen in SE Asia with lethal implications. We have initiated started to work on the crystal structures of a number of Fab fragments of neutralizing antibodies. Combining these structures with the cryoEM structures of their complexes with virus will be informative as to the mode of neutralization the antigenic sites on the virus and other essential information for the design of VLP vaccines. We are also working on Rubella virus (the causative agent of German Measels) by combining cryo electron microscopy with the structure determination of its E1 and E2 glycoprotein components and its capsid protein. We are using the same cloning expression and crystalization techniques as we used for the study of dengue virus 13 and Sindbis virus17. 3. Parvoviridae: Capsid structures of several parvoviruses [20 21] including a human parvovirus (B19) [22 23] have been determined. The crystal structures of a shrimp pavovirus and a silk worm pavovirus are being determined. Attempts are being made to crystallize the PLA2 unique region of the capsid protein of B19. 4. Structural components of T4 phage: We have solved about 15 out of about 40 proteins so far [24 25 26 27 28 31]. We have recently determined the structure of T4 soc and crystals of T4 hoc and soc the two minor head accessory proteins. 5. Structural components of other phages: We are have crystals of fragments of the H pilot protein of phiX174 29and have crystalized various proteins of phiKZ 30 gp13 gp9 cell lysing enzymes of phi29 [32 33]. 6. Mimivirus structural proteins We are studying the giant Mimivirus using cryoEM and cryo tomography. We are currently identifying various structural proteins for crystallization of recombinant proteins including the major capsid protein. References are attached as a separate file due to space limitation.