Electron microscopy studies of novel poliovirus genome-releasing intermediates

说明(申请人提供)：微小核糖核酸病毒是无包膜的病毒，不能依靠病毒膜与宿主细胞膜的融合来进入细胞质。它们依赖于衣壳蛋白经历迄今尚未确定的构象变化的能力，使病毒RNA突破宿主膜屏障。这些不可逆转的变化包括衣壳内部的多肽外化。脊髓灰质炎病毒是典型的小核糖核酸病毒，是一个很好的模式系统。我们的主要目标是通过电子显微镜和三维数据分析(3DEM)获得与宿主膜附着和基因组释放相关的病毒结构动力学知识。小核糖核酸病毒彼此之间具有高度的结构同源性，因此，在本研究期间开发的模型和方法将与其他小核糖核酸病毒相关。这项研究的两个具体目标是1)确定脊髓灰质炎病毒中间体在基因组释放过程中的3DEM结构，以及2)确定这些中间体附着在脂质体上的3DEM结构。将进行的实验步骤如下：a.在RNA释放的特定阶段(早期或晚期)，优化制备富含脊髓灰质炎病毒颗粒的玻璃化EM网格的条件。B.使用Polara和Tecnai F30显微镜收集EM数据(包括未倾斜和断层扫描)。C.使用用于未倾斜数据的PFT和用于断层图像的IMOD程序，从2D EM图像确定RNA释放中间体的二十面体和不对称3D重建。D.建立伪原子模型以适应更高分辨率的EM图，以帮助解释密度变化。公共卫生相关性：皮科纳病毒家族包括心脏病毒、口蹄疫病毒、鼻病毒、甲型肝炎病毒和肠道病毒。其中，有导致人类和兽医疾病的重要原因。详细了解这些病毒用于膜附着和基因组释放的分子机制将有助于开发抗病毒策略，以阻止感染的早期阶段，并降低致病性。

DESCRIPTION (provided by applicant): Picornaviruses are non-enveloped viruses and cannot rely on fusion of a viral membrane with a host cell membrane to gain access to the cytoplasm. They rely on the ability of capsid proteins to undergo as-yet-uncharacterized conformational changes for the viral RNA to breach the host-membrane barrier. These irreversible changes include externalization of peptides from the interior of the capsid shell. Poliovirus is the prototypical picornavirus and serves as an excellent model system. Our broad aim is to gain knowledge of viral structural dynamics associated with host-membrane attachment and genome release by using electron microscopy and three-dimensional data analysis (3DEM). The picornaviruses share a high degree of structural homology with one another and, therefore, the models and methodologies developed during this study will be relevant to other picornaviruses. The two specific aims of this proposed study are 1) to determine the 3DEM structures of poliovirus intermediates during genome-release, and 2) to determine 3DEM structures of such intermediates attached to liposomes. The experimental steps to be carried out are listed below: a. Optimize conditions for preparing vitrified EM grids enriched with poliovirus particles at a particular stage (early or late) of RNA-release. b. Collect EM data (both un-tilted and tomographic) using the Polara and Tecnai F30 microscopes. c. Determine both icosahedral and asymmetric 3D reconstructions of RNA-release intermediates from the 2D EM images using the program PFT for un-tilted data and IMOD for tomograms. d. Build pseudo-atomic models to fit the higher-resolution EM maps, to help account for density changes. PUBLIC HEALTH RELEVANCE: The Picornavirus family includes cardioviruses, foot-and-mouth disease virus, rhinoviruses, hepatitis A virus and enteroviruses. Among these, there are significant causes of human and veterinary diseases. Detailed knowledge of the molecular mechanisms that these viruses use for membrane attachment and genome release would help in the development of antiviral strategies to block the earliest stages of infection and reduce pathogenicity.