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) andts6389 (IHD strain). Viral DNA synthesis was virtually undetectable during non-permissive infections performed withts17, and incorporation of3H-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 D5orf by marker rescue, and the single nucleotide changes responsible for thets 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°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. |