Poxviruses encode enzymes and factors needed for transcription and replication of their genomes within the cytoplasm of infected cells. Vaccinia virus, the prototypic member of the poxvirus family, provides a unique system for combining biochemical and genetic approaches for investigating mechanisms of gene regulation, mRNA biosynthesis, and DNA replication. Studies with vaccinia virus indicated that the genes are divided into three temporal classes - early, intermediate and late. Each gene class has a consensus DNA promoter sequence and corresponding transcription factors that interact with the virus-encoded multisubunit RNA polymerase. The transcription system for early genes is packaged within the infectious virus particle during its assembly, whereas the factors for intermediate and late gene transcription are synthesized successively after infection.The DNA is synthesized as concatemers that are resolved into unit length genomes and packaged during virus assembly. All poxviruses have a highly conserved DNA topoisomerase that is a potential target of antiviral drugs. Although the structure and catalytic activity of the enzyme were well studied, little was known about its biological function. The viral topoisomerase was thought to be essential; and roles in DNA replication, recombination, concatemer resolution and transcription were suggested. We have now demonstrated that the topoisomerase is not essential for replication of vaccinia virus in cultured cells, although deletion mutants formed fewer and smaller plaques on cell monolayers than wild type virus. Purified mutant virus particles were able to bind and enter cells, but exhibited reduced viral early transcription and a delay in DNA replication. The data suggest that the primary, perhaps only, role of the poxvirus topoisomerase is to increase early transcription, which takes place within virus cores in the cytoplasm of infected cells. Because the topoisomerase functions early in infection, drugs capable of penetrating the virus core and irreversibly damaging DNA by trapping nicked DNA-topoisomerase intermediates could make potent antiviral agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Intramural Research (Z01)
Project #
1Z01AI000307-23
Application #
6985033
Study Section
(LVD)
Project Start
Project End
Budget Start
Budget End
Support Year
23
Fiscal Year
2004
Total Cost
Indirect Cost
Name
Niaid Extramural Activities
Department
Type
DUNS #
City
State
Country
United States
Zip Code
De Silva, Frank S; Paran, Nir; Moss, Bernard (2009) Products and substrate/template usage of vaccinia virus DNA primase. Virology 383:136-41
Katsafanas, George C; Moss, Bernard (2007) Colocalization of transcription and translation within cytoplasmic poxvirus factories coordinates viral expression and subjugates host functions. Cell Host Microbe 2:221-8
Parrish, Susan; Moss, Bernard (2007) Characterization of a second vaccinia virus mRNA-decapping enzyme conserved in poxviruses. J Virol 81:12973-8
Hebben, Matthias; Brants, Jan; Birck, Catherine et al. (2007) High level protein expression in mammalian cells using a safe viral vector: modified vaccinia virus Ankara. Protein Expr Purif 56:269-78
Parrish, Susan; Resch, Wolfgang; Moss, Bernard (2007) Vaccinia virus D10 protein has mRNA decapping activity, providing a mechanism for control of host and viral gene expression. Proc Natl Acad Sci U S A 104:2139-44
Garcia, Alonzo D; Otero, Joel; Lebowitz, Jacob et al. (2006) Quaternary structure and cleavage specificity of a poxvirus holliday junction resolvase. J Biol Chem 281:11618-26
Parrish, Susan; Moss, Bernard (2006) Characterization of a vaccinia virus mutant with a deletion of the D10R gene encoding a putative negative regulator of gene expression. J Virol 80:553-61
Domi, Arban; Moss, Bernard (2005) Engineering of a vaccinia virus bacterial artificial chromosome in Escherichia coli by bacteriophage lambda-based recombination. Nat Methods 2:95-7
De Silva, Frank S; Moss, Bernard (2005) Origin-independent plasmid replication occurs in vaccinia virus cytoplasmic factories and requires all five known poxvirus replication factors. Virol J 2:23
Katsafanas, George C; Moss, Bernard (2004) Vaccinia virus intermediate stage transcription is complemented by Ras-GTPase-activating protein SH3 domain-binding protein (G3BP) and cytoplasmic activation/proliferation-associated protein (p137) individually or as a heterodimer. J Biol Chem 279:52210-7

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