VACCINIA VIRUS BIOCHEMICAL GENETICS

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In recent years it has become increasingly clear that transcription elongation is an important control point for regulation of eucaryotic gene expression. Our recent work demonstrates that postreplicative gene transcription elongation is regulated during vaccinia virus infection. A study of this regulation is important for understanding regulation of vaccina virus gene expression in particular, and the system may prove to be an important model for study of regulation of transcription elongation in eucaryotes in general. The goal of this project is to understand the regulation of vaccinia virus postreplicative (intermediate and late) gene transcription elongation. The project centers on two vaccinia genes, G2R and A18R, which seem to regulate postreplicative gene transcription elongation with complementing activities. Genetic experiments indicate that the A18R gene product, a DNA helicase, restricts the processivity of the viral RNA polymerase and therefore acts as negative transcription elongation factor, while the G2R gene product enhances the processivity of the viral RNA polymerase and therefore acts as a positive transcription elongation factor. Biochemical and genetic experiments implicate several other viral gene products in the regulation of postreplicative transcription elongation, including a viral late transcription initiation factor (H5R), a viral poly A polymerase subunit (J3R), and two viral RNA polymerase subunits (J4R and A24R). Our working hypothesis is that these gene products work together, perhaps as a transcription elongation complex, to regulate formation of 3' ends of intermediate and late vaccinia viral mRNAs. We propose to test, refine, and extend this hypothesis through 1) an in vitro transcription elongation assay, 2) phenotypic analysis of existing virus mutants defective in transcription elongation, and 3) isolation and characterization of additional elongation defective mutants.