Fine structure mapping and phenotypic analysis of five temperature-sensitive mutations in the second largest subunit of vaccinia virus DNA-dependent RNA polymerase

Abstract

We have used plasmid clones spanning the region encoding the 132-kDa subunit of the cowpox virus RNA polymerase (CPV rpo132) to marker rescue each of five vaccinia virus (VV) temperature sensitive ( ts) mutants, ts 27, ts 29, ts 32, ts 47, and ts 62, which together constitute a single complementation group. The experiments fine-map the vaccinia mutations to a 1.3-kb region containing the 3′ end of the CPV rpol 32 gene. Phenotypic characterization shows that all five mutants are affected to varying extents in their ability to synthesize late viral proteins at the nonpermissive temperature, similar to other is mutants with lesions in the 22- and the 147-kDa subunits of the VV RNA polymerase. Two mutants, ts 27 and ts 32, exhibit a delay in the synthesis of late viral proteins at both the permissive and the nonpermissive temperatures. We conclude that the five W mutants affect the 132-kDa subunit of the VV RNA polymerase. Additional genetic experiments demonstrate intragenic complementation between ts 62 and three other members of this complementation group, ts 27, ts 29, and ts 32.