The RNA dependent RNA polymerase of negative strand RNA viruses transcribes and replicates the viral genome. Despite the fact that many members of these viruses cause disease of the central nervous system, little is known about the RNA polymerase and the multiple enzymatic functions it performs. Since many of these funcions are essential to the virus and differ from those of the host cell, they represent potential targets that could specifically be of the host cell, they represent cellular mechanisms. Therefore, the study of these viral specific mechanisms could be exploited in the treatment or prevntion of viral infections in the central nervous system. In order to study the multiple functions of the polymerase complex, we have cDNA cloned, sequenced, assembled and expressd a functional 241 kilodalton polymease protein of Vesicular stomatitis virus (VSV). We have currently established a eukaryotic cell line which constitutively expresses the polymerase protein. Functionality of the polymerase was demonstrated by complementation and rescue of conditional polymerase mutants of VSV. Hence, this unique system will allow for the first time the identification and dissection of the functional domains of the polymerase of negative strand viruses through site specific mutagenesis. The effect of these mutations on the mutation rate of the polymerase, itself, will also be studied with respect to the establishment and maintenance of persistent infections. Towards these ends, we have generated over 20 mutant recombinant L genes and are currently studying their effects on the performance of the multiple functions of the polymerase complex.
