Simian virus 40 (SV40) infects cells by binding to plasma membrane MHC class I molecules and then entering via a unique mechanism mediated by caveolae, rather than clathrin-coated pits. A notable feature of the unusual SV40 entry pathway is that it transports the virus to the endoplasmic reticulum (ER), rather than to endosomes. Importantly, the ER is the site in which the SV40 internal capsid proteins VP2 and VP3 separate from virions. VP2/3 then enter the cytosol, whereas virions and the major capsid protein VP1 remain in the ER. The goal of this project is to investigate the molecular features of SV40 that underlie cell recognition, entry and intracellular trafficking, and to clarify the process of release of the SV40 genome (the minichromosome) and its transport to the nucleus. Specific objectives are as follows. First, to determine the roles of the SV40 capsid proteins in the binding and intracellular trafficking of SV40, and to identify the molecular features of those proteins that underlie the unusual entry and trafficking of this virus. Second, to identify the.cellular compartment.into which wild type SV40 virions release their minichromosomes and the route of the minichromosomes to the nucleus. Third, to characterize the function of VP2 and VP3 in the processes of uncoating the minichromosome and transporting it out of the ER. A particular aim here is to determine whether myristylated VP2 has a role in these events. Fourth, to determine whether the cellular cytoskeleton has a role in transporting the minichromosome to the nucleus. If it does, then experiments will be carried out to identify the relevant motor proteins and to evaluate the possible role of VP2/3 in the cytoskeleton-mediated transport process. Experimental procedures will make use of confocal microscopy, in which virions, virus proteins, and cellular organelles will be visualized by immunofluorescent staining. In some experiments, fluorescent in situ hybrization (FISH) will be combined with immunocytochemistry, to visualize minichromosomes with respect to cellular organelles, the cytoskeleton, and virus proteins. Experimental samples will include wild type virus, constructed virus mutants, and in vitro assembled virus particles that contain specific combinations of virus proteins. Recent studies report evidence that SV40 infection may lead to neoplastic disease in humans, including mesotheliornas, osteosarcomas, childhood brain tumors, and non-Hodgkin's Iymphoma.
