The long term objectives of this research program are to define the mechanisms of fundamental replication processes, virus-host interactions, and cytopathologies common to many important (+) strand RNA viruses, and to use these results to improve virus control and beneficial uses of viruses and their components in biotechnology and medicine. New directions on the functions and interactions of viral proteins, RNAs and host factors in RNA replication and gene expression will be pursued using brome mosaic virus (BMV), a representative member of the alphavirus-like superfamily and a productive, advanced model system. Valuable new opportunities in these areas follow from our recent findings that BMV RNA replication occurs in virus-induced mini-organelles (spherules) whose structure, assembly and function show parallels with replicative cores of retroviruses and dsRNA viruses; from our advancing definition of the functions and interactions of BMV replication factors 1a, 2apol and cis-acting RNA signals; and from our use of the rare ability of BMV to replicate in yeast to conduct the first genome-wide analysis of host gene functions in virus replication. Building on these, we will use BMV replication in natural plant host cells and yeast with synergistic genetic, biochemical and cell biology approaches, to analyze virus replication and virus-host interactions. Among other steps, we will use electron microscope tomography and related approaches to define the three dimensional in vivo ultrastructure of RNA replication complexes arrested at consecutive stages, to resolve crucial issues about their organization, morphogenesis and function. The mechanisms by which BMV 1a protein alone induces these membrane-enveloped spherule replication compartments will be determined, including the roles of multiple 1a-1a interactions and 1a interactions with membranes and lipid rafts. We will determine how 1a interaction with specific cis-acting viral RNA signals inhibits translation and recruits RNAs to replication, and if, as our results suggest, the 1a NTPase/helicase domain parallels dsRNA phage NTPases in translocating viral RNA replication templates into pre-formed replication compartments. We will analyze the contributions of selected host genes implicated in RNA replication, and use novel genetic approaches to perform genome-wide cataloging of the contributions of essential host genes to RNA replication. The results will advance understanding and control of widespread viral diseases, and understanding of normal cell functions relevant to other disease processes. ? ?
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