Biochemical and Genetic Analysis of the Vaccinia Virus D5 Protein: Multimerization-Dependent ATPase Activity Is Required To Support Viral DNA Replication

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Abstract
The vaccinia virus-encoded D5 protein is an essential ATPase involved in viral DNA replication. We have expanded the genotypic and phenotypic analysis of six temperature-sensitive ( ts ) D5 mutants (C ts 17, C ts 24, E ts 69, D ts 6389 [also referred to as D ts 38], D ts 12, and D ts 56) and shown that at nonpermissive temperature all of the ts D5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For C ts 17 and C ts 24, this restriction reflects the thermolability of the D5 proteins. The D ts 6389, D ts 12, and D ts 56 D5 proteins become insoluble at 39.7°C, while the E ts 69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7°C. To investigate which structural features of D5 are important for its biological and biochemical activities, we generated targeted mutations in invariant residues positioned within conserved domains found within D5. Using a transient complementation assay that assessed the ability of D5 variants to sustain ongoing DNA synthesis during nonpermissive C ts 24 infections, only a wt D5 allele supported DNA synthesis. Alleles of D5 containing targeted mutations within the Walker A or B domains, the superfamily III helicase motif C, or the AAA+ motif lacked biological competency. Furthermore, purified preparations of these variant proteins revealed that they all were defective in ATP hydrolysis. Multimerization of D5 appeared to be a prerequisite for enzymatic activity and required the Walker B domain, the AAA+ motif, and a region located upstream of the catalytic core. Finally, although multimerization and enzymatic activity are necessary for the biological competence of D5, they are not sufficient.

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