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

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.