The assembly of proteins and nucleic acids into viruses involves numerous molecular interactions. Often assembly is dependent on scaffolding proteins. Analogous to scaffoldings used in building construction, these proteins assist assembly, but are not found in the final product. After the virus is constructed, it can be regarded as a small machine. Its purpose is to deliver genetic material into the target cell, which is achieved by a series of programmed structural changes in viral proteins. The broad objective of this research is to elucidate the molecular mechanisms of scaffolding-mediated viral morphogenesis and DNA delivery, using the Microviridae, a family of small single stranded DNA viruses. Alterations (mutations) have been designed into the external scaffolding protein that confer dominant lethal effects. When the mutant (antiviral) protein is present in infected cells, it is able to interact with other viral proteins and unaltered scaffolding proteins (wild-type) to block viral replication in a manner similar to antiviral chemicals. If wildtype virus is continuously propagated in the presence of increasing concentrations of the antiviral protein, it selects for a multiple mutant virus that is not only highly resistant to the antiviral protein but is actually stimulated by it. Thus, the virus is able to evolve a mechanism to convert an antiviral agent into an assembly stimulator. Biochemical and genetic experiments will be conducted to test a model that seeks to explain this phenomenon. The results of this study should provide insights into the mechanism of viral assembly, protein engineering and viral evolution. After the virus recognizes its target cell, the DNA pilot protein delivers the viral genome into the cell. In a microvirus infection the DNA pilot protein and genome are first deposited in the outer membrane. The protein then directs the transport of the genome to the inner membrane, which is the site of DNA replication. The mechanism by which the protein accomplishes this function is largely unknown. During particle assembly, the internal scaffolding protein incorporates 12 individual copies of the DNA pilot protein into the forming virus. However, the results of biochemical and genetic experiments indicate that the individual DNA pilot proteins must associate with each other to deliver the viral DNA at the onset of infection. The domain within the DNA pilot protein that most likely mediates this self-association has been identified as well as the domain that initially interacts with the host cell membrane. Biochemical, genetic and structural analyses will be conducted to elucidate the mechanism of viral DNA delivery. Deciphering this mechanism should have direct applications to the fields of nanotechnology and protein engineering. Broader Impacts: The project's broader impact goes beyond the standard education of graduate and undergraduate students within the investigator's program. The impact reaches the students enrolled in the courses taught by the investigator. Incorporating research into curricula is essential at larger universities, where undergraduates who desire research opportunities far outnumber the available positions. Part of the proposed research will be conducted in a virology laboratory course in which students will conduct hypothesis-driven experiments that may uncover novel mutations and assembly mechanisms. The educational paradigm for this course has already proven successful, yielding a class-generated scientific manuscript and elevating scientific literacy.
