The paramyxovirus-encoded RNA polymerase is a remarkably versatile enzyme consisting of two subunits, P and L, which is negatively regulated by a third viral encoded protein, C. We have developed systems utilizing recombinant proteins to identify a number of mutations in these genes with defective RNA synthesis phenotypes, which will collectively allow us to define the molecular mechanisms by which the Sendai virus (SV) RNA polymerase catalyzes the individual steps involved in the transcription and replication of the genome RNA. Some of the viral RNA polymerase activities, like capping, methylation and polyadenylation are analogous superficially to those of cellular enzymes although the mechanisms are different, whereas others, like RNA editing, are presently considered to be novel and unique to paramyxoviruses and filoviruses. We will study a number of aspects of the different multiple activities of the L subunit of SV RNA polymerase with the use of site-directed mutants we have already constructed and characterized in the six conserved domains of the protein. We will rescue recombinant viruses containing temperature sensitive and active SV L mutations, characterize their phenotype during infection and isolate and map suppressor mutants. We will complete the complementation analysis of SV L mutants. We will exploit our collection of mutant L genes both in vitro and within recombinant mutant viruses to study the mechanism of RNA synthesis and the pathogenesis of the virus in the mouse, it's natural host. We will map the P and C binding sites on the L protein. In vesicular stomatitis virus (VSV) we will address the issue of mRNA cap methylation. We will identify amino acids that are important for the methyltransferase activities of the L protein by characterization of the VSV hr1 and hr8 mutations in L protein, which confer the methylation deficient phenotype. We will do site-directed mutagenesis to identify the SAM binding site and the methylation catalytic site in VSV L protein. To study the functional activities of the P subunit of the SV RNA polymerase, we will rescue recombinant viruses containing temperature sensitive SV P mutations, characterize their phenotype during infection and isolate and map suppressors which phenotypically correct P mutations. We will also test for complementation of existing P mutants.
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