Faculty Collaboration Database

Biochemical and genetic analysis of the vaccinia virus d5 protein: Multimerization-dependent ATPase activity is required to support viral DNA replication. J Virol 2007 Jan;81(2):844-59

Date

11/10/2006

Pubmed ID

17093187

Pubmed Central ID

PMC1797480

DOI

10.1128/JVI.02217-06

Scopus ID

2-s2.0-33846086914 (requires institutional sign-in at Scopus site)   50 Citations

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 (Cts17, Cts24, Ets69, Dts6389 [also referred to as Dts38], Dts12, and Dts56) and shown that at nonpermissive temperature all of the tsD5 viruses exhibit a dramatic reduction in DNA synthesis and virus production. For Cts17 and Cts24, this restriction reflects the thermolability of the D5 proteins. The Dts6389, Dts12, and Dts56 D5 proteins become insoluble at 39.7 degrees C, while the Ets69 D5 protein remains stable and soluble and retains the ability to oligomerize and hydrolyze ATP when synthesized at 39.7 degrees 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 Cts24 infections, only a wtD5 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.

Author List

Boyle KA, Arps L, Traktman P

MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphatases
Amino Acid Sequence
Animals
Cell Line
DNA Replication
Dimerization
Gene Expression Regulation, Viral
Humans
Mice
Molecular Sequence Data
Mutation
Structure-Activity Relationship
Temperature
Vaccinia virus
Viral Proteins
Virus Replication