The long-term objectives of our studies are to understand the molecular mechanisms governing genome expression and replication of unsegmented negative-strand RNA viruses and their defective interfering particles. Vesicular stomatitis virus (VSV) is the best characterized member of this group because it grows easily and is readily amenable to biochemical and genetic analysis. Its replication strategy is virtually identical to the less tractable paramyxoviruses which are responsible for many important human diseases. Moreover, recent progress in the study of VSV transcription reveals parallels with that of host cells such as acidic protein activation domains and phosphorylation control. This proposal is a continuation of our investigations on factors regulating VSV polymerase activity. The unique polR mutants which bear a single missense mutation in their nucleocapsid protein gene have played a prominent role in uncovering an ATP-linked antitermination function and will be used to further our understanding of transcription and replication control. We propose to test three basic hypotheses: 1) second-site mutations which suppress transcriptional readthrough in polR VSV map to an important regulatory domain of the large multifunctional polymerase protein; 2) phosphorylation of the VSV NS transcription factor regulates antitermination and replication perhaps via a protein kinase activity intrinsic to the polymerase protein; and 3) synthesis of readthrough products by polR VSV in vivo activates the dsRNA-dependent cellular protein kinase responsible for phosphorylating eIF2a and leads to growth restriction in mouse L cells. The MBRS student involved in these studies will be working under the direct supervision of the principal investigator and will be interacting at both the technical and academic levels with all other members of the laboratory.
