With the discovery that cloned cDNA copies of poliovirus RNA can yield infectious virus after transfection into mammalian cells, poliovirus became amenable to genetic manipulation. Using well-defined poliovirus mutants as tools, many new functions of the polioviral genome have been discovered, and similar functions have been discovered in other positive-strand RNA viruses and in cellular RNA molecules as well. The goal of this proposal is to study functions located in the 5' noncoding region (5' NCR) of poliovirus, and to explore the virus as a tool to design ribozyme-based vaccines. First, host cell gene products will be identified that mediate the unusual internal ribosome binding to the 5' NCR of poliovirus. We have found that dicistronic mRNAs containing the poliovirus 5'NCR between two cistrons can be expressed in the yeast Saccharomyces cerevisiae and translated to produce the gene products of both the first and second cistrons. Yeast strains which express these dicistronic mRNAs will be mutagenized, and strains will be selected that fail under certain conditions to accumulate the product of the second cistron, which is required for yeast viability. Subsequent cloning of the defective yeast genes will show whether cellular proteins, RNA molecules or both mediate internal ribosome binding. Second, we will test whether internal ribosome binding requires a free 5' end of the mRNA. Specifically, 5' ends of mRNA will be selectively blocked with biotinylated nucleotides, adsorbed to a streptavidin-containing solid support, and the translation of free and blocked mRNAs will be examined in vitro. Third, a strategy has been developed to identify sequences in the viral negative-strand RNA that direct the synthesis of positive-strand RNA molecules. Plasmids containing the promoter for T7 RNA polymerase, reading the luciferase gene and the 5' NCR of poliovirus in antisense orientation, will be transfected into cell line KJT7 that constitutively expresses T7 RNA polymerase. T7 RNA polymerase will produce mRNA that contains the authentic 3' end of the viral negative-strand. Replication of these RNAs by poliovirus polymerase, provided in am by infection, should produce RNAs that can be translated to yield luciferase. This system will be used to study the effects of defined mutations in the viral 5' NCR on initiation of viral positive strands. Lastly, poliovirus will be used as a tool to design ribozyme-based vaccines. Viral mutants bearing specific nucleotide insertions in the viral 5' NCR will be tested for hybridization with a neuron-specific mRNA. Hybridization will result in the formation of a hammerhead-ribozyme structure, and in the specific cleavage of the viral 5' NCR. Results from this study should provide valuable insight into the use of ribozymes as antiviral agents.
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|Thompson, S R; Sarnow, P (2000) Regulation of host cell translation by viruses and effects on cell function. Curr Opin Microbiol 3:366-70|