Vaccinia, the prototype of the poxvirus family, is a large and complex virus. The virion encapsidates the 185 kb genome and sufficient enzymes to initiate the transcription of early viral genes immediately upon infection. Infection is accompanied by the rapid shut-off of host transcription and replication; early transcription is followed by DNA replication, which is an obligate step for the subsequent switch to the transcription of late viral genes. The virus replicates entirely within the cytoplasm of infected cells; this physical autonomy from host nuclear functions is mirrored in the apparent genetic autonomy of the virus. Vaccinia appears to encode all of the functions required for nucleic acid metabolism. The genome is a linear duplex with covalently-closed hairpin termini reminiscent of yeast telomeres. The autonomy of the virus from host functions, the ease of preparation of biochemically pure material, and the recent advances in the molecular biological analysis of the genome make it an excellent model system for the study of DNA replication. We propose a broad analysis of viral replication which combines genetic, molecular and biochemical analyses.
Our aims i nclude: (1) The identification and characterization of viral genes encoding known enzymes with a role in DNA metabolism. (2) The molecular genetic analysis of genes affected in known vaccinia replication mutants. (3) The isolation of additional mutants with replication defects, using classical genetic techniques as well as the targeting of mutations to defined DNA fragments. (4) The refinement of a system for the in vitro mutagenesis and expression of the vaccinia DNA polymerase gene. These experiments will facilitate structure-function analysis and ready the technique for application to genes whose function has eluded classical genetic study. (5) Refinement of an in vitro replication system to aid in the study of replication mutants and the identification of replication functions. These studies should provide significant insight into the regulation and mechanism of faithful DNA replication. Vaccinia's role as the agent used so successfully in the worldwide campaign to eradicate smallpox by vaccination, and its current potential as a recombinant vaccine engineered to carry foreign antigens, augments the importance of understanding the intricacies of viral replication.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI021758-08
Application #
3132071
Study Section
Virology Study Section (VR)
Project Start
1984-12-01
Project End
1993-06-30
Budget Start
1991-12-01
Budget End
1993-06-30
Support Year
8
Fiscal Year
1992
Total Cost
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Type
Schools of Medicine
DUNS #
201373169
City
New York
State
NY
Country
United States
Zip Code
10065
Greseth, Matthew D; Czarnecki, Maciej W; Bluma, Matthew S et al. (2018) Isolation and Characterization of v?I3 Confirm that Vaccinia Virus SSB Plays an Essential Role in Viral Replication. J Virol 92:
Czarnecki, Maciej W; Traktman, Paula (2017) The vaccinia virus DNA polymerase and its processivity factor. Virus Res 234:193-206
Boyle, Kathleen A; Greseth, Matthew D; Traktman, Paula (2015) Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication. J Virol 89:6312-27
Greseth, Matthew D; Boyle, Kathleen A; Bluma, Matthew S et al. (2012) Molecular genetic and biochemical characterization of the vaccinia virus I3 protein, the replicative single-stranded DNA binding protein. J Virol 86:6197-209
Boyle, Kathleen A; Stanitsa, Eleni S; Greseth, Matthew D et al. (2011) Evaluation of the role of the vaccinia virus uracil DNA glycosylase and A20 proteins as intrinsic components of the DNA polymerase holoenzyme. J Biol Chem 286:24702-13
Wiebe, Matthew S; Traktman, Paula (2007) Poxviral B1 kinase overcomes barrier to autointegration factor, a host defense against virus replication. Cell Host Microbe 1:187-97
Boyle, Kathleen A; Arps, Lisa; Traktman, Paula (2007) Biochemical and genetic analysis of the vaccinia virus d5 protein: Multimerization-dependent ATPase activity is required to support viral DNA replication. J Virol 81:844-59
Stanitsa, Eleni S; Arps, Lisa; Traktman, Paula (2006) Vaccinia virus uracil DNA glycosylase interacts with the A20 protein to form a heterodimeric processivity factor for the viral DNA polymerase. J Biol Chem 281:3439-51
Boyle, Kathleen A; Traktman, Paula (2004) Members of a novel family of mammalian protein kinases complement the DNA-negative phenotype of a vaccinia virus ts mutant defective in the B1 kinase. J Virol 78:1992-2005
Traktman, Paula; Boyle, Kathleen (2004) Methods for analysis of poxvirus DNA replication. Methods Mol Biol 269:169-86

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