GENETICS OF 5 NONCODING REGIONS OF PICORNAVIRUS RNAS

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DESCRIPTION: The applicant will continue his studies aimed at understanding the roles of RNA-protein interactions in functions directed by the 5'-NCR of picornavirus RNAs. Although other activities (eg. virion assembly) may be associated with the 5'-NCR of picornavirus genomic RNAs, he will focus his efforts on functions (ie. viral translation and RNA replication) known to be directed by this region of viral RNAs. For translation functions he will test the hypothesis that specific ribonucleoprotein complexes between cellular proteins and uncapped viral RNAs are required prior to binding ribosomes for initiation of cap-independent protein synthesis. For the replication functions, the PI will test the hypothesis that interaction of a cellular RNA binding protein (PCBP2) with a specific sequence in the 5'-NCR of poliovirus RNA and the viral protein 3CD is necessary for initiation of viral RNA synthesis. He will take advantage of his collection of viable mutants and pseudorevertants containing site-directed lesions in the 5'-NCR as well as a number of biochemical assays for functional associations between the 5'-NCR and cellular or viral proteins. Specifically, the PI will: (1) Elucidate the role of neuronal-cell specific factors in poliovirus translation initiation. (2) Determine the cellular expression pattern and biochemical properties of PCBP2. (3) Define the function of PCBP2 in cap-independent translation via its interaction with stem-loop IV in the IRES of poliovirus RNAs. (4) Determine the relationship between PCBP2 ribonucleoprotein complex formation with stem-loop I and viral RNA replication. The applicant states that these approaches, coupled with the analysis of mutant viral growth properties in HeLa cells and neuronal cells, should provide new insights into the mechanisms employed by the picornaviruses to insure the faithful translation and replication of viral RNAs during an infection of mammalian cells. He also indicated that, more broadly, his studies will lead to a high resolution "map" of specific macromolecular interactions between RNAs and protein in the orchestration of higher order structures required for effecting functions in the cytoplasm of eukaryotic cells.