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The vaccinia virus DNA polymerase and its processivity factor
Maciej W Czarnecki1, Paula Traktman2
1Departments of Biochemistry & Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, United States; Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226, United States.
Virus Research
|February 5, 2017
Summary
Vaccinia virus DNA polymerase (E9L) gains processivity through interaction with A20 and D4 proteins. This complex, including a DNA repair enzyme, is crucial for viral replication and offers targets for antiviral drug discovery.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Vaccinia virus, a prototypic poxvirus, possesses a large DNA genome encoding its replication machinery.
- The vaccinia virus DNA polymerase, encoded by E9L, is a B family replicative polymerase with essential polymerase and exonuclease activities.
- Understanding the vaccinia virus DNA polymerase complex is key to elucidating viral replication mechanisms.
Purpose of the Study:
- To investigate the molecular interactions governing vaccinia virus DNA polymerase processivity.
- To characterize the roles of A20 and D4 proteins in the vaccinia virus DNA holoenzyme.
- To explore the potential coupling of DNA replication and repair in vaccinia virus.
Main Methods:
- Purification and characterization of the A20/D4 heterodimer and its interaction with E9.
- Genetic analysis of E9 to identify key residues and motifs.
- Structural studies including X-ray crystallography of D4 and its complexes.
- Biochemical assays to assess DNA synthesis and enzyme activities.
- Screens for small molecules inhibiting the A20/D4 interaction.
Main Results:
- Vaccinia virus DNA polymerase (E9L) requires the A20/D4 heterodimer for processivity, forming a 1:1 holoenzyme.
- A20 acts as a bridge between E9 and D4; D4 is a uracil DNA glycosylase involved in DNA scanning.
- Crystal structures reveal the complex architecture of D4 with A20 and DNA.
- Small molecule screens identified compounds disrupting the A20/D4 interface and inhibiting viral DNA synthesis and plaque formation.
- The D4 protein's DNA repair function suggests a link between replication and repair.
Conclusions:
- The vaccinia virus DNA holoenzyme comprises E9L, A20, and D4, with D4 contributing DNA scanning and repair capabilities.
- The A20/D4 heterodimer is essential for conferring processivity to the viral DNA polymerase.
- Targeting the A20/D4 interface represents a potential strategy for developing novel antiviral therapeutics.
- The integration of a DNA repair enzyme into the replication machinery suggests coupled DNA replication and repair pathways in vaccinia virus.
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