Identification of a transcription factor, encoded by two vaccinia virus early genes, that regulates the intermediate stage of viral gene expression

Vaccinia virus early, intermediate, and late stage genes are sequentially t ranscribed by the viral RNA polymerase within the cytoplasm of infected cel ls. We found that the 34- and 45-kDa polypeptides encoded by vaccinia virus ORFs A8R and A23R, respectively, were necessary to reconstitute transcript ion of a template with an intermediate stage promoter. Coexpression of the A8R and A23R genes in Escherichia coil was required for in vitro activity. In addition, the two polypeptides copurified, indicating their association as protein subunits of a vaccinia virus intermediate transcription factor, This factor, which we named VITF-3, complemented three viral proteins-namel y, the RNA polymerase. capping enzyme, and a 30-kDa protein called VITF-1 t hat is also a subunit of the RNA polymerase-and an unidentified cell factor called VITF-2, Expression of the A8R and A23R genes occurred between I and 5 h after vaccinia virus infection and was not prevented by an inhibitor o f DNA replication, consistent with a role for VITF-3 in specifically regula ting intermediate transcription in vivo. The vaccinia virus A8R and A23R ge nes are highly conserved among vertebrate poxviruses, but no other viral or cellular homologs were identified.