Negative-strand RNA viruses, viral transcription
The virus-encoded RNA polymerase of paramyxoviruses is a remarkably versatile enzyme consisting of two subunits, P and L, which is negatively regulated by a third viral protein, the C protein. We have developed mammalian expressions systems utilizing cloned genes to characterize a number of mutations in these genes in Sendai, measles and vesicular stomatitis (vsv) viruses with defective RNA synthesis phenotypes. Further studies of these mutants will allow us to define the molecular mechanisms by which the viral RNA polymerase catalyzes the individual steps in the transcription and replication of the genome RNA.
Some of the viral RNA polymerase activities, like capping, methylation and polyadenylation are analogous to those of cellular enzymes, although the mechanisms are different, whereas others, like RNA editing, are presently considered to be unique to paramyxoviruses and filoviruses. A major focus of our research is to study the functional activities of the L subunit of the Sendai virus RNA polymerase. With the use of site directed mutants we constructed in the six conserved domains of the protein we have characterize mutations in L that are completely defective in all RNA synthesis. Interestingly these inactive L mutants complement one another in transcription en vitro when they are co-expressed.
By reverse genetics we will rescue recombinant viruses containing temperature sensitive and inactive Sendai L mutations, characterize their phenotype during infection in cell culture and in its natural host, the mouse. We will use the mutant defective viruses to isolate and map suppressor mutants. In VSV we have identified amino acids that are important for the methytransferase activities of the L protein by the characterization of the 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, and subsequently extend these observations to Sendai L.
