GENETICS OF FIVE NONCODING REGIONS OF PICORNAVIRUS RNAS

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The proposed project will utilize picornavirus molecular recombinants and mutants to assay the functional domains of the 5'-non-coding region of poliovirus RNA. We will determine the effects of genetic alteration of the 5'-untranslated region of the polio genome on RNA replication and ribosome binding/viral- specific protein synthesis. We will carry out in vitro manipulation of cloned cDNAs to construct a number of different site-specific recombinant and mutant genomes within the 5'-noncoding regions of polio-virus and coxsackievirus RNAs. The 5'-noncoding cDNA chimeras and mutants will then be ligated to the coding region from an infectious cDNA clone of poliovirus RNA. Plasmids containing the complete recombinant/mutant genomes will be assayed for infectivity by transfection of cultured primate cells. Infectious recombinant and mutant viruses will be recovered and analyzed for altered or temperature-sensitive expression of (i) RNA replication functions,(ii) viral protein synthesis functions, and (iii) host cell shut off functions. Finally, any of the recombinant plasmids that do not produce infectious virus in the transfection assay will be analyzed for in vitro translation efficiency using in vitro transcripts derived from a bacteriophage T7 promoter and RNA polymerase system. Our overall goal in this project is to understand the role of specific blocks of noncoding nucleotides within a viral genome in the regulation of expression of the single translational unit of polio gene products. The health-relatedness of the proposed project is based upon previous studies that implicate the 5'-noncoding region of poliovirus RNA in both the replicative fitness of the virus and the full expression of viral neurovirulence determinants. The information gained from the proposed project should provide a rational scheme for the genetic modification of vaccine strains of picornaviruses by recombinant DNA methodology. Such noncoding region modifications have the potential advantage of reducing vaccine strain cytopathogenicity and instability without perturbing the virion structure that is critical for eliciting a fully- protective immune response.