Biophysical characterization of the assembly and disassembly of the ssDNA viruses

Viruses are the causative agents of many diseases that affect plants and animals and the mechanisms by which they assemble and disassemble represent critical steps in their replicative life cycle. The focus of this study was to elucidate the steps involved in the formation and disruption of the viral capsid of the Geminiviridae and Pavoviridae family of viruses.Maize Streak Virus (MSV-N) belongs to the Geminiviridae family of virus. The Geminiviridae have caused significant economic loses and hardship due to their infection of a number of horticultural crops worldwide. MSV-N is a severe pathogen of maize and is used in this study as a model for understanding assembly and disassembly of this unique virus family. The goal of the study was to decipher the steps involved in the formation and disruption of the MSV capsid. This was accomplished by isolating and characterizing the capsid components and intermediates from maize agro-inoculated with MSV-N. These components and intermediates include the characteristic geminate capsid, a T=1 icosahedral (single) capsid, pentamers, and dimers of pentamers. This study also included a pH assembly/diasassembly assay that has been generated for MSV and could be developed as a method to test peptide inhibitors of these processes.Adeno-Associated Virus serotype 2 (AAV2) belongs to the Parvoviridae family and it is an important therapeutic agent for clinical gene therapy. AAV2 has been extensively studied and it has been used in the treatment of several genetic diseases. However, there is still a large deficit in the amount of information available on the mechanism by which it assembles and disassembles. We have isolated and characterized subassemblies of recombinant AAV2. These include what appear to be pentamers, dimers of pentamers (DOPs) and pentamers of dimers (PODs). In the final aspect of this work, we used the crystal structure of AAV2, other AAV serotypes, other parvoviruses and site directed mutagenesis to identify residues and regions of the capsid that are important to the assembly and stability of the virus capsid.