The Nodaviridae are a family of small icosahedral viruses with bipartite, single-stranded, positive-sense RNA genomes. Containing only 4.5 kb, their genomes are among the smallest and simplest of all known animal viruses, yet they replicate up to 100-fold more abundantly than most other viral RNAs, and can do so in cells from vertebrates, insects, and even plants. These and other features of the nodaviruses allow steps in their replication cycles to be examined at levels of resolution that cannot yet be achieved with other animal viruses. The advances in our basic understanding of the dynamic RNA-protein interactions involved in nodavirus replication can be confidently expected to illuminate the study of the many positive-strand RNA viruses with medical and economic importance. Moreover, because of the simplicity, robustness, and extraordinary amplifying power of the nodavirus RNA replicases, they are promising candidates for development as components of expression vectors for a wide range of applications. The work described in this proposal focuses on three aspects of the molecular biology of the nodaviruses. The first is a detailed study of the mechanism of RNA replication that combines genetic and biochemical approaches to examine the interactions of the RNA replicase with its templates; the possibility that components of the cellular splicing machinery are involved in RNA replication; and the properties of the RNA replicase and other enzymes that mediate replication. The second addresses how virus assembly and disassembly occur, focusing on the functions of the RNA encapsidation signals and exploiting the ability to carry out both the disassembly and assembly of infectious virus in a cell-free system. The third explores how RNA replication can be harnessed most effectively to drive the amplification and expression of heterologous RNAs.
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