The viability of all viruses is dependent upon faithful replication of their entire genome, including terminal sequences. Many viruses use protein primers and produce full-length genomic DNA/RNA molecules with the 5% end covalently linked to a polypeptide, often called VPg in RNA viruses or terminal protein (TP) in DNA viruses. Among those protein-priming viruses are picornaviruses, caliciviruses, adenoviruses and hepadnaviruses that cause serious human diseases. As viral polymerases are often targeted for antiviral therapy, mechanistic studies of protein priming should provide promising leads in the development of innovative antiviral compounds. Here we propose to use birnaviruses as a paradigm to elucidate the structural basis of viral protein-primed RNA synthesis. Birnaviruses form a unique family of dsRNA viruses with a VPg-linked genome. With protein priming and polymerase catalytic functions provided by the same polypeptide, birnavirus VP1 serves as an excellent model for structural studies of the protein-priming mechanism. Infectious bursal disease virus (IBDV), the best studied birnavirus, causes Gumboro disease in avian species by destroying immature B-lymphocytes in the Bursa of Fabricius. In addition to a VPg-linked genome, mature virion contains the major capsid proteins VP2 and VP3, the viral polymerase VP1, and possibly the viral protease VP4. To this end, we have obtained a crystal structure of VP1 and have shown that VP1 exhibits both self-guanylylation and polymerase activities in vitro. The structure of VP1 shows an unprecedented active site formed by five essential RNA polymerase motifs arranged in the permuted order of C-A-B-D-E. Through site-directed mutagenesis, we showed that a fatal polymerase mutant remains active in self-guanylylation, suggesting that VP1 gunaylylation and nucleotide polymerization are catalyzed by two separate active sites. These intriguing discoveries, and our other preliminary results, indicate that structure-function studies of VP1 will not only unveil the atomic details of viral protein priming, but also enhance our understanding of polymerase catalysis as well as the evolutionary history of the birnavirus family. This proposal will: (1) elucidate the mechanism of guanylylation in protein-primed replication;(2) elucidate the mechanism of terminal priming;and (3) characterize the functional roles of viral proteins VP2, VP3 and VP4 in RNA transcription. A diverse array of methods, encompassing biochemical assays, X-ray crystallography, electron microscopy and reverse genetics, will be utilized in addressing these questions.
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