The goal of this project is to understand the molecular mechanisms governing post-initiation events in postreplicative gene transcription during vaccinia virus infection. In recent years it has become increasingly clear that post-initiation events in transcription, including transcription elongation, termination and RNA cleavage, comprise important control points for regulation of gene expression in both prokaryotes and eukaryotes. Our recent work suggests that postreplicative mRNA 3'end formation during vaccinia virus infection is a concerted process that involves both transcription termination and RNA cleavage, that it is coupled to viral DNA replication, and that it is mediated by both viral and host factors in a dynamic complex. The factors include a viral RNA release factor (A18), a viral RNA cleavage/transcription/DNA replication factor (H5), two viral positive transcription elongation factors (G2 and J3), and at least two host factors required for A18 RNA release activity and for RNA cleavage. This project comprises two aims, focusing on understanding the mechanism of transcription termination and endoribonucleolytic RNA cleavage. Studies of termination (Aim 1) involve mechanistic studies on A18 and its required host factor, and studies of mRNA cleavage (Aim 2) involve mechanistic studies of an endoribonuclease comprised of the viral H5 protein and host proteins. The viral gene products under study are highly conserved among vertebrate poxviruses, they are essential for virus replication, they all unquestionably collaborate in mediating postreplicative gene transcription elongation and termination, and yet their precise roles in these processes are only partially understood. The research proposed here will significantly refine our understanding of the roles of these essential genes in poxvirus replication, and it will also impact on our understanding of the interaction between the virus and the host cell. The results will provide critical insight into fundamental mechanisms of vaccinia virus gene expression in particular, and the system may prove to be an important model for study of regulation of transcription elongation in eukaryotes in general.
This project is relevant to public health on three levels. First, it advances our understanding of the basic mechanisms of eukaryotic transcriptional regulation of gene expression, which in turn is a central part of the normal framework on which our understanding of disease is built. Second, it advances our understanding of the molecular mechanisms of virus replication and virus cell interactions, which has implications in general for treatment of virus induced disease. Third, the project provides insight specifically into the replication of poxviruses, which are of particular interest to public health as research tools, as a source of therapeutic proteins, as oncolytic vectors, and as potential bioterrorist weapons.
|D'Costa, Susan M; Bainbridge, Travis W; Kato, Sayuri E et al. (2010) Vaccinia H5 is a multifunctional protein involved in viral DNA replication, postreplicative gene transcription, and virion morphogenesis. Virology 401:49-60|
|D'Costa, Susan M; Bainbridge, Travis W; Condit, Richard C (2008) Purification and properties of the vaccinia virus mRNA processing factor. J Biol Chem 283:5267-75|
|Shatzer, Amber N; Kato, Sayuri E M; Condit, Richard C (2008) Phenotypic analysis of a temperature sensitive mutant in the large subunit of the vaccinia virus mRNA capping enzyme. Virology 375:236-52|
|Cresawn, Steven G; Condit, Richard C (2007) A targeted approach to identification of vaccinia virus postreplicative transcription elongation factors: genetic evidence for a role of the H5R gene in vaccinia transcription. Virology 363:333-41|
|Cresawn, Steven G; Prins, Cindy; Latner, Donald R et al. (2007) Mapping and phenotypic analysis of spontaneous isatin-beta-thiosemicarbazone resistant mutants of vaccinia virus. Virology 363:319-32|
|D'Costa, Susan M; Antczak, James B; Pickup, David J et al. (2004) Post-transcription cleavage generates the 3' end of F17R transcripts in vaccinia virus. Virology 319:1-11|
|Kato, Sayuri E M; Strahl, Audra L; Moussatche, Nissin et al. (2004) Temperature-sensitive mutants in the vaccinia virus 4b virion structural protein assemble malformed, transcriptionally inactive intracellular mature virions. Virology 330:127-46|
|Lackner, Cari A; D'Costa, Susan M; Buck, Charles et al. (2003) Complementation analysis of the dales collection of vaccinia virus temperature-sensitive mutants. Virology 305:240-59|
|Latner, Donald R; Thompson, Joseph M; Gershon, Paul D et al. (2002) The positive transcription elongation factor activity of the vaccinia virus J3 protein is independent from its (nucleoside-2'-O-) methyltransferase and poly(A) polymerase stimulatory functions. Virology 301:64-80|
|Lackner, C A; Condit, R C (2000) Vaccinia virus gene A18R DNA helicase is a transcript release factor. J Biol Chem 275:1485-94|
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