Molecular Characterization of a Novel Class of DNA Binding Proteins

Ascovirus infection of lepidopteran larvae leads to an accumulation of virion- containing vesicles in the hemolymph turning it opaque white. The unusual feature of the formation of virion-filled vesicles by these large, complex, double-stranded DNA viruses results from a process similar to apoptosis, but one modified to rescue the developing apoptotic bodies for viral reproduction and virion dissemination. Another unusual feature of these viruses is their dependence on transmission by endoparasitic wasps that deposit their eggs in a lepidopteran host. Four species of ascoviruses have been sequenced and reveal a range of genes involved in apoptosis, RNA metabolism, and lipid metabolism. In the present dissertation, the research focuses on characterization of a novel type of DNA binding protein, P64 along with its homologs, responsible for condensing viral genomic DNA as part of the process of virion assembly. Proteomic, electron microscopic, and biochemical studies demonstrated that P64 is a major virion protein which bound to and condensed DNA prior to virion core formation and envelopment. Mobility shift assays, bioinformatics, and additional electron microscopy data provided strong evidence that P64 and its homologs constitute a novel family of DNA condensing proteins. The combination of P64's highly basic nature, its large size relative to known viral/eukaryotic genome condensing proteins, its non-specific DNA binding activity, and the presence of two distinct and unique domains suggest that these proteins evolved in certain complex DNA viruses to condense and assist the packaging of newly synthesized viral genomes as part of the assembly of progeny virions. Preliminary data suggest that P64 might associate with other virion structural proteins once it has bound to condensed DNA. Finally, phylogenetic analyses and results of binding studies showed that P64 is related to DNA binding proteins from viruses of the closely related Iridoviridae family, which provided additional evidence that the ascoviruses evolved from iridoviruses