| Positive strand RNA viruses represent one third of all virus genera and include significant human pathogens. Alfalfa Mosaic Virus (AMV), a member of the Bromoviridae, consists of a tripartite, capped, non-adenylated genome. Two distinctive features make AMV a valuable model system for studying RNA-protein interactions in the replication cycle of plus stranded viruses. First, the AMV genomic RNAs are insufficient for replication; that is coat protein (CP) binding to the 3' end of the genomic RNAs is necessary for replication. Second, the highly conserved 3' UTRs of AMV lack the canonical features of the tRNA-like structure (TLS) that is common to most other bromoviruses. The CP-3'UTR ribonucleoprotein complex provides a simple system for understanding the functional significance of structural determinants of the RNA-coat protein interactions in AMV replication. The work presented in this dissertation demonstrates limitations of an existing model for coat protein's role in replication and describes an alternative model of AMV replication. This 3' organization model proposes that a defining feature of bromovirus replication strategies is the requirement for a structurally organized 3' terminus. For AMV, this organization is achieved by the formation of the coat protein-3 'UTR complex. The conformation of this ribonucleoprotein complex is established by determinants present in both the RNA and coat protein, some of which are not essential for high affinity interactions. This dissertation also describes the development of an improved experimental approach to study the mechanisms of coat protein mediated activation of replication. The broad goal of this work is to understand how viral RNAs are recognized as templates for replication and how the viral RNA-dependent RNA polymerise is positioned for accurate initiation of minus strand synthesis. |
