The structures of several plant and animal viruses are now known to near atomic resolution, and represent some of the most complex biological assemblies understood. These structures provide a rational basis for studying virus-cell interactions, as well as designing anti-viral therapeutic agents. Although the final virion structures are defined, the mechanisms involved in assembling these structures are still largely unknown. This knowledge is significant not only to the problems of protein folding, regulation of other protein assemblies, and the interaction of structural proteins with eukaryotic chromosomes, but also provides another target (i.e. the assembly process rather than the final structure) for possible therapeutic intervention. In addition to other DNA viruses, the papovaviruses themselves are a relevant target for anti-viral agents. Papillomaviruses are already of recognized medical importance, and in an era of immunosuppression secondary to transplantation regimens, cancer chemotherapy, and HIV infection, human polyomaviruses also may be more frequently encountered as pathogenic agents. The purpose of this proposal is to use papovaviruses as a model system to understand how DNA virus assembly is regulated, and to define the relative importance of different protein bonding interactions in forming the final virus structure. Three experimental approaches utilize purified recombinant polyomavirus capsid proteins to study protein-protein and protein-chromatin interactions in vitro. Site-directed mutagenesis of recombinant polyoma VP1 oat proteins expressed in bacteria will be used to assess the relative contribution of specific intercapsomeric bonds to capsid stability and assembly in vitro. Recombinant VP1 proteins will also be used to identify assembly intermediates in forming capsids in vitro, and determine whether bonding interactions between coat proteins are functionally conserved between the murine polyoma and SV40 viruses. Recombinant VP2 and VP3 coat proteins will be used to study their possible interaction with VP1 and the viral minichromosome. Capsid proteins expressed in insect cells using baculovirus vectors will e used to study the regulation of capsid assembly in vivo. Combinatorial infections with vectors expressing these proteins will determine the contribution of the VP2 and VP3 proteins to the nuclear transport of VP1 and to the ultrastructure of the viral capsid. Finally, approaches used to study polyomavirus assembly will be extended to investigate bovine papillomavirus (BPV) coat proteins, and BPV virus purified from bovine warts will be used to crystallize this prototype papillomavirus for high resolution structural analysis. The ultimate goal of these studies will be to compare the strategies of assembly between polyoma and papillomaviruses, and identify general principles in constructing a T=7 icosahedral DNA virus.
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