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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 病毒通过多种内吞途径进入细胞，包括网格蛋白包被的小凹、小凹和微胞饮小泡。 一旦内吞​​无包膜病毒，即那些没有脂质外壳的病毒，必须穿过进入内体隔室的脂质膜以完成感染过程。 乳头瘤病毒（PV）是重要的人类病原体，通过网格蛋白依赖性和独立途径进入细胞。 由于无法使用天然病毒体和适当的宿主细胞，PV 进入的研究受到了困扰。 我们从牛疣中纯化了牛乳头瘤病毒病毒粒子，并使用原代牛上皮细胞成功地实现了有效的感染，我们认为这与自然感染过程非常相似。 我们现在正尝试使用这些细胞的 EM 断层扫描来描述 BPV 的内吞途径。我们通过快速冷冻/冷冻替代固定和塑料切片断层扫描方法以及冷冻水合全细胞的冷冻电子断层扫描方法制备了感染细胞。 目标不仅是确认细胞生物学测定所建议的进入途径，而且是“看到”病毒粒子穿过内吞膜进入胞质溶胶的位点。 此外，我们预计我们将观察病毒在通过内吞途径进展时的构象变化。膜转运点的可视化对于解决许多其他无包膜病毒感染的一般问题非常重要。