The long-term objectives of this application are to understand the mechanisms of cell entry by the mouse polyoma virus. This virus has a broad host range. It replicates and induces tumors in a wide variety of cell types in its natural host. The Specific Aims are first to determine whether certain glycolipids constitute the only class of cell receptors for the virus or whether certain glycoproteins may also serve as functional receptors. This will be done using a mouse deficient in glycolipid biosynthesis but unaffected in glycoprotein pathways. Investigations will be carried out to identify the steps in virus disassembly in the endoplasmic reticulum and the cellular factors the virus utilizes to undergo the necessary steps of disassembly and to enter the nucleus. Factors under investigation in this regard are the oxidoreductase ERp29, protein disulfide isomerase, the derlin chaperones, and Ran GTPase. A specific goal is to generate in vitro and characterize the altered particle that is primed for membrane insertion and escape from the ER. Finally, in collaboration with Dr. Xiaowei Zhuang's lab at Harvard, attempts will be made to follow single virus particles in live cells as they enter and establish infection. We will attempt to produce polyoma particles labeled both on the minichromosome and the outer capsid protein with fluorescent probes to study decapsidation and separation of these components. The processes of cell entry by many viruses are not fully understood. The polyoma group includes several viruses that are pathogenic in humans, including JC, BK and possibly SV40. Understanding the pathways of cell entry by these viruses may suggest opportunities for anti-viral therapy or prevention.