Characterization of vaccinia virus DNA replication mutants with lesions in the D5 gene

Abstract

The vaccinia virus D5 gene encodes a 90 kDa early protein that is essential for viral DNA replication. In this report we map and explore the phenotypes of the temperature sensitive mutants bearing lesions in this gene: ts17, ts24, ts69 (WR strain) and ts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed with ts17, and incorporation of 3H-thymidine ceased rapidly when cultures were shifted to the non-permissive temperature in the midst of replication. The D5 protein may therefore be involved in DNA synthesis at the replication fork. The lesions of the four mutants were localized within the D5 orf by marker rescue, and the single nucleotide changes responsible for the ts phenotype of the three WR mutants were identified. Unexpectedly, the three alleles with N-terminal mutations were impaired in marker rescue when homologous recombination with small (< 2 kb), intragenic DNA fragments at 39.5 degrees C was required. This deficiency was not due to degradation of transfected DNA under non-permissive conditions. Efficient marker rescue could be restored by incubation at the permissive temperature for a brief period after transfection, suggesting a requirement for functional D5 in genome/plasmid recombination. Marker rescue under non-permissive conditions could alternatively be restored by co-transfection of unlinked but contiguous DNA sequences.

Clustered Charge-to-Alanine Mutagenesis of the Vaccinia Virus A20 Gene: Temperature-Sensitive Mutants Have a DNA-Minus Phenotype and Are Defective in the Production of Processive DNA Polymerase Activity

“…6B). This general profile of thymidine incorporation is similar to what has been reported by us and others for wt-infected cells (17,32,35); the decline in thymidine incorporation at a time when DNA replication continues at a robust rate is attributed to feedback inhibition of the viral thymidine kinase as well as changes in the intracellular nucleotide pools (22,40). Both ts mutants showed impaired thymidine incorporation at the nonpermissive temperature.…”

“…Thus, auxiliary functions that might be involved in initiation or template unwinding are not required. The specificity of the defect seen with ts42, tsA20-ER5, and tsA20-6 is underscored by the results obtained with ts mutants carrying lesions in the D5 DNA-independent NTPase (ts17) (15)(16)(17)35) or the B1 protein kinase (ts2) (33,34). Extracts from BSC40 cells infected with ts17 or ts2 at either the permissive or nonpermissive temperature are able to catalyze RFII formation in vitro (Fig.…”

“…The use of conditionally lethal mutants as genetic tools for the study of vaccinia virus has been well established (8). Four complementation groups of ts mutants exhibiting a DNA Ϫ phenotype have been described; these contain lesions in the E9 DNA polymerase, the D5 DNA-independent nucleoside triphosphatase (NTPase), the B1 protein kinase, and the D4 uracil DNA glycosylase (16,17,33,34,36,37,41). Other proteins implicated in genome replication have…”

Poxvirus DNA primase

“…We determined the ability of the D5 constructs to complement VACV Cts24, a conditional lethal mutant defective in DNA replication at 40°C (12,13,26). Following a previously described protocol (13), cells were infected with Cts24 at the permissive temperature, transfected with D5 plasmids at 3 h after infection and shifted to the nonpermissive temperature at 5 h after infection or maintained at the permissive temperature.…”

“…The B1 kinase phosphorylates a cellular DNA-binding protein called BAF and prevents the latter from blocking VACV DNA replication (11). The fast stop DNA replication phenotype of conditional lethal D5 mutants suggests a function at the replication fork (12). D5 also interacts with A20 (8,9) and forms multimers (13).…”