Molecular Genetic and Biochemical Characterization of the Vaccinia Virus I3 Protein, the Replicative Single-Stranded DNA Binding Protein

Authors

Matthew D. Greseth, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
Kathleen A. Boyle, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
Matthew S. Bluma, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
Bethany Unger, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
Matthew S. Wiebe, Department of Microbiology and Molecular Genetics, Medical College of WisconsinFollow
Jamaria A. Soares-Martins, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
Nadi T. Wickramasekera, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
James Wahlberg, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin
Paula Traktman, Department of Microbiology and Molecular Genetics, Medical College of Wisconsin

Document Type

Article

Date of this Version

2012

Citation

June 2012 Volume 86 Number 11 Journal of Virology p. 6197–6209 jvi.asm.org

Comments

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

Abstract

Vaccinia virus, the prototypic poxvirus, efficiently and faithfully replicates its approximately 200-kb DNA genome within the cytoplasm of infected cells. This intracellular localization dictates that vaccinia virus encodes most, if not all, of its own DNA replication machinery. Included in the repertoire of viral replication proteins is the I3 protein, which binds to single-stranded DNA (ssDNA) with great specificity and stability and has been presumed to be the replicative ssDNA binding protein (SSB). We substantiate here that I3 colocalizes with bromodeoxyuridine (BrdU)-labeled nascent viral genomes and that these genomes accumulate in cytoplasmic factories that are delimited by membranes derived from the endoplasmic reticulum. Moreover, we report on a structure/ function analysis of I3 involving the isolation and characterization of 10 clustered charge-to-alanine mutants. These mutants were analyzed for their biochemical properties (self-interaction and DNA binding) and biological competence. Three of the mutant proteins, encoded by the I3 alleles I3-4, -5, and -7, were deficient in self-interaction and unable to support virus viability, strongly suggesting that the multimerization of I3 is biologically significant. Mutant I3-5 was also deficient in DNA binding. Additionally, we demonstrate that small interfering RNA (siRNA)-mediated depletion of I3 causes a significant decrease in the accumulation of progeny genomes and that this reduction diminishes the yield of infectious virus.