THE ENDOCYTIC PATHWAY OF BOVINE PAPILLOMAVIRUS USING EM TOMOGRAPHY

University Of Colorado, Boulder CO

Investigators

Linked publications & trials

Abstract

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.