THE ENDOCYTIC PATHWAY OF BOVINE PAPILLOMAVIRUS USING EM TOMOGRAPHY

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 病毒通过许多内吞途径进入细胞，包括网格蛋白包被的小凹、小窝和微胞饮小泡。 一旦内吞无包膜病毒，即，那些没有脂质包被的细胞必须穿过进入内体区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非依赖性途径进入细胞。 PV进入的研究由于不能使用天然病毒体和适当的宿主细胞而受到混淆。 我们已经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，我们相信这与自然感染过程非常相似。 我们现在正试图用电镜断层扫描来描述BPV的内吞途径。我们已经准备了感染细胞的方法快速冷冻/冷冻置换固定和塑料切片断层扫描和冷冻水合，全细胞的冷冻电子断层扫描。 其目的不仅是确认细胞生物学测定所提示的进入途径，而且是“看到”病毒体穿过内吞膜进入胞质溶胶的位点。 此外，我们预计，我们将观察到构象变化的病毒，因为它通过内吞途径的进展。膜转运点的可视化将在解决许多其他无包膜病毒感染的一般问题中具有非常重要的意义。

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.