Temperature-sensitive mutations in the vaccinia virus H4 gene encoding a component of the virion RNA polymerase

Four previously isolated temperature-sensitive (ts) mutants of vaccinia virus WR (ts1, ts31, ts55, and ts58) comprising a single complementation group (R. C. Condit, A. Motyczka, and G. Spizz, Virology 128:429-443, 1983) have been mapped by marker rescue to the H4L open reading frame located within the genomic HindIII-H DNA fragment. The H4 gene is predicted to encode a 93.6-kDa polypeptide expressed at late times during infection. Nucleotide sequence alterations responsible for thermolabile growth lead to amino acid substitutions in the H4 gene product. All four ts alleles display 'normal' patterns of early and late viral protein synthesis at the nonpermissive temperature (40°C). Mature virion particles, microscopically indistinguishable from wild-type virions, are produced in the cytoplasm of cells infected with ts1 at 40°C. Western immunoblot analysis localizes the H4 protein to the virion core. After solubilization from cores, the H4 protein is associated during purification with transcriptionally active vaccinia virus DNA-dependent RNA polymerase.