Crystallographic, biophysical,and molecular biological studies were performed by our group and collaborators on insect nodaviruses and picorna-likc viruses. A number of observations were made that apparently relate to specific functions of the particle in the virus life cycle. This renewal application describes experiments in the area of cell biology, as well as crystallography,biophysics and molecular virology,as efforts are made to define viral phenotypes on the basis of chemistry discerned from atomic resolution and in vitro studies. The essence of the proposal is to span the range from atoms to cells for simple, yet elegant, organisms, the non enveloped, ssRNA viruses of insects and plants. The variationsobserved in structure and hypothesized function lead naturally to an investigation of the evolution of these particles. Specifically we seek to: (1) Employ fluoresencc and electron microscopy to determine the mode of entry of flock house virus (FHV) and, to employ biochemistry and biophysics to determine the role of the receptor in RNA release. (2) Employ in vivo expression of GFP fusion proteins in mammalian and drosophila cells to establish the role of a membrane active peptide derived from the capsid protein of FHV by a maturationcleavage. (3) Determine the structure of the RNA directed RNA polymerasc of FHV and establish its novel features that allow it to replicate and CAP RNA molecules in insect, plant, yeast and mammalian cells. (4) Investigate, by crystallography, cryoEM, baculovirus expression and site directed mutagenesis, factors that affect assembly, maturation and genome organization of insect and fish nodaviruses. (5) Determine the sites of FHV RNA replication, capsid protein synthesis and particle assembly by fluorcsence and electron microscopy. (6) Establish evolutionaryrelationships among insect and fish nodaviruses and among insect and plant picorna and picorna-like viruses.
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