Our laboratory will study the mechanism(s) involved in the regulation of the functions of the virus-specific, virion-associated RNA polymerases of the two minus-stranded RNA animal viruses, vesicular stomatitis virus (VSV) and influenza A. Specifically we have designed experiments to examine the modification(s) of these two enzymes which allows them to switch from transcription of their genomes into virus-specific mRNAs which are capped, methylated and polyadenylated and are incomplete copies of the genomic sequence to a replicative mode which leads to synthesis of full length (+) and (-) copies of the genome that are assembled during synthesis with nucleocapsid proteins to form RNP complexes. We are presently cloning the small subunit of the VSV polymerase (NS) for expression in E. coli and in animal cells to obtain sufficient amounts of NS with which to carry out analyses of the phosphorylation sites on NS. We will also sequence the cDNA to determine the amino acid sequence of this protein. If we can obtain reasonable levels of expression for NS we hope to use the NS protein for in vitro reconstitution studies with purified RNPs and L protein. Using monospecific antisera versus both VSV enzyme subunits (L1, NS1) we are studying the interaction of this enzyme with its RNP template by immunoelectron microscopic techniques. Since we can inhibit influenza transcription (due to its reliance on host cell mRNA for caps and primers) with actino D, we will establish an in vitro replication system from cell nuclear extracts for flu by coupling transcription and translation to drive flu replication in a manner similar to our in vitro VSV replication system. Using specific antibodies to the flu RNA polymerase (PB1, PB2 PA) we will establish whether all three subunits are involved in flu replication as well as transcription. In a similar manner we will study the role, if any, of flu nonstructural protein (NS1) in vitro replication system in which we can selectively inhibit transcription (actino D) or replication (cycloheximide) to study the enzyme modification(s) involved and the role of cellular and/or virus factors in it.
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