THE ENDOCYTIC PATHWAY OF BOVINE PAPILLOMAVIRUS USING EM TOMOGRAPHY

使用电子断层扫描技术研究牛乳头状病毒的内吞途径

• 批准号: 7955053
• 负责人: Robert L Garcea
• 金额: $ 2.14万
• 依托单位: UNIVERSITY OF COLORADO
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2010-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955053
• 关键词: Biological Assay; Bovine Papillomavirus; Cattle; Caveolae; Cells; Cellular Structures; Cellular biology; Clathrin; Computer Retrieval of Information on Scientific Projects Database; Cytosol; Endocytosis; Epithelial Cells; Freeze Substitution; Freezing; Funding; Goals; Grant; Human; Imagery; Infection; Institution; Lipids; Membrane; Membrane Lipids; Papillomavirus; Pathway interactions; Pinocytotic Vesicle; Plastics; Process; Research; Research Personnel; Resources; Site; Source; United States National Institutes of Health; Virion; Virus; coated pit; electron tomography; pathogen; rapid technique; sample fixation; tomography

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Viruses enter cells via numerous endocytic pathways, including clathrin-coated pits, caveolae, and micro-pinocytotic vesicles. Once endocytosed non-enveloped viruses, i.e., those without a lipid coat, have to transit the lipid membrane of the entry endosomal compartment to complete the infection process. Papillomaviruses (PV) are important human pathogens that enter cells by both clathrin-dependent and independent pathways. Studies on PV entry have been confounded by the inability to use native virions and appropriate host cells. We have purified bovine papillomavirus virions from cow warts, and using primary bovine epithelial cells have succeeded in achieving efficient infection that we believe closely mimicks the natural infection process. We are now attempting to describe the endocytic pathway of BPV using EM tomography of these cells. We have prepared infected cells both by methods for rapid freezing/freeze-substitution fixation and plastic section tomography and for cryo-electron tomography of frozen-hydrated, whole cells. The goal is not only to confirm the pathway of entry suggested by cell biology assays, but also to "see" the site where the virion transits the endocytic membrane to enter the cytosol. In addition we anticipate that we will observe conformational changes in the virus as it progresses through the endocytic pathway. Visualization of the membrane transit point would be highly significant in solving a general question in infection by numerous other non-enveloped viruses.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 病毒通过众多内吞途径进入细胞，包括涂有网格蛋白的凹坑，小窝和微型细胞囊泡。 一旦内吞​​非发育的病毒，即没有脂质涂层的病毒，就必须将入口内体隔室的脂质膜转移才能完成感染过程。 乳头瘤病毒（PV）是重要的人类病原体，通过依赖性途径和独立途径进入细胞。 无法使用天然病毒体和适当的宿主细胞的研究使有关PV进入的研究困惑。 我们已经从牛疣中纯化了牛乳头瘤病毒病毒体，并且使用原发性牛上皮细胞已经成功地实现了有效的感染，我们认为我们密切模仿了自然感染过程。 我们现在正在尝试使用这些细胞的EM层析成像来描述BPV的内吞途径。我们已经通过快速冷冻/冻结固定固定和塑性片层造影以及冷冻保湿全细胞的冷冻电子层析成像的方法制备了感染的细胞。 该目标不仅是要确认细胞生物学测定法建议的进入途径，而且还要“参见”病毒体将内吞膜转移以进入细胞质的位点。 此外，我们预计我们将在病毒通过内吞途径发展时观察到构象变化。膜过境点的可视化对于解决许多其他非发达病毒在感染中的一般问题方面将非常重要。