Isolation and properties of the vaccinia virus DNA-dependent RNA polymerase.

A vaccinia virus-specified DNA-dependent RNA polymerase was isolated from the cytoplasm of infected HeLa cells. The enzyme appeared in cells only after infection; it differed in its chromatographic properties from all cellular RNA polymerases; and its subunit composition (seven subunits ranging in size from 13,500 to 135,000 daltons) was completely different from that of HeLa cell RNA polymerases I and II. The latter two enzymes were also isolated and their subunit compositions were found to resemble closely those of mouse plasmacytoma MOPC 315 RNA polymerases I and II. The three largest subunits of the isolated vaccinia virus-specified RNA polymerase migrated in sodium dodecyl sulfate-polyacrylamide gels at the same rate as virion core polypeptides VP1c, VP1d, and VP2c; the enzyme may therefore be the same as the vaccinia virus core-associated RNA polymerase. The purified enzyme was free of detectable exonuclease and poly(A) polymerase activity. It was maximally stimulated by 0.08 M KCl and 0.001 M MN/sup 2 +/; Mg/sup 2 +/ could not substitute for MN/sup 2 +/ to a significant degree. The enzyme was very resistant to ..cap alpha..-amanitin. It was 10 times more active with denatured calf thymus or vaccinia virus DNA as template than with the corresponding nativemore » DNAs, but transcribed the synthetic double-stranded poly(d(A-T)) almost as rapidly as single-stranded natural DNA. It transcribed all sequences of native vaccinia virus DNA.« less