THE ENDOCYTIC PATHWAY OF BOVINE PAPILLOMAVIRUS USING EM TOMOGRAPHY

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 病毒通过许多内吞途径进入细胞，包括包被网状蛋白的凹坑、小凹和微吞饮小泡。一旦内吞，无包膜的病毒，即那些没有脂衣的病毒，必须通过进入内体隔室的脂膜来完成感染过程。乳头瘤病毒(PV)是一种重要的人类病原体，通过依赖和独立两种途径进入细胞。由于无法使用天然病毒粒子和适当的宿主细胞，对光伏病毒进入的研究一直处于混乱状态。我们已经从牛疣中提纯了牛乳头瘤病毒，并使用原代牛上皮细胞成功地实现了有效的感染，我们相信这种感染过程非常接近自然感染过程。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。我们用快速冷冻/冷冻替代固定法、塑料切片断层扫描法和冷冻水合全细胞冷冻电子断层扫描法制备了感染细胞。这样做的目的不仅是为了确认细胞生物学检测所暗示的进入途径，也是为了“看到”病毒粒子穿过细胞膜进入胞浆的位置。此外，我们预计我们将观察到病毒在通过内吞途径过程中的构象变化。膜转运点的可视化对于解决许多其他非包膜病毒感染的一般问题具有重要意义。

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.