Gene therapy holds promise as a new treatment approach for a variety of diseases, though optimal vehicles for gene transfer remain elusive. A novel alternative to currently favored gene delivery strategies is in vitro packaging of nucleic acid into virus-like particles using purified recombinant viral capsids, which might circumvent problems with conventional gene transfer. This proposal seeks to develop a such a model system using the well studied papovavirus capsid proteins. Purified recombinant VP1, the major coat protein of murine polyomavirus and simian virus 40 (SV40), and purified recombinant L1, the major coat protein of human papillomaviruses (HPV), self-assemble in vitro into virus-like particles. As yet, however, packaging of viral genomes or foreign DNA in vitro using VP1 or L1 has been unsuccessful. DNA encapsidation may require viral or cellular chaperone proteins to stabilize sub-capsomer intermediates during assembly. Therefore, the interaction of viral coat proteins with chaperone proteins in both virus-infected cells and in vitro capsid assembly reactions will be investigated. The initial aim of this project is to biochemically and genetically characterize the in vitro interactions of papovavirus coat proteins with the cellular chaperone hsc70 and viral chaperone agnoprotein. The project will also determine the in vivo role of hsc7O in capsid protein nuclear transport and the effect of the agnoprotein and hsc70 on in vitro viral assembly. Finally, if infectious virus-like particles are successfully reconstructed in vitro, their capacity for gene transfer will be assessed. In addition to elucidating mechanisms of viral assembly, this project may lead to a new system of gene delivery potentially useful in future gene therapy protocols.
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