TEMPERATURE-SENSITIVE MUTATIONS IN THE GENE ENCODING THE SMALL-SUBUNIT OF THE VACCINIA VIRUS EARLY TRANSCRIPTION FACTOR IMPAIR PROMOTER-BINDING, TRANSCRIPTION ACTIVATION, AND PACKAGING OF MULTIPLE VIRION COMPONENTS

The vaccinia virus D6R open reading frame encodes the small subunit of the heterodimeric vaccinia virus early transcription factor (VETF) th at activates transcription of early genes in vitro. VETF binds early g ene promoters and has a DNA-dependent ATPase activity that is essentia l for activation of transcription. To examine the relationship between the structure and function of VETF, we have localized the mutations i n two temperature-sensitive viruses whose lesions previously were mapp ed to the D6R gene. For both mutants, a single G-to-A nucleotide chang e that would alter protein coding potential was identified. In mutant E93, the codon for alanine 25 was changed to that of threonine, and in mutant S4 the codon for valine 278 was replaced with that for methion ine. The molecular phenotype of each mutant was assessed by expressing mutant transcription factors in HeLa cells by using a vaccinia virus- T7 system and characterizing the proteins' activities in vitro. The A2 5T mutant activated transcription to a lesser extent than wild-type VE TF, and the V278M mutant had no demonstrable transcription factor acti vity. Both mutant proteins were shown to be defective for promoter bin ding, accounting for their impairment in transcription activation. The functional defects for both mutants were observed at permissive as we ll as nonpermissive temperatures. The mutant proteins retained ATPase activity but required higher DNA concentrations to activate the ATPase . These results indicate that the small subunit of VETF is essential f or its promoter binding activity and likely contacts the promoter DNA. Immunoblotting experiments showed that the virion particles from the two mutant viruses contained about half the VETF of wild-type virus, s uggesting that promoter binding may contribute to packaging of VETF in to the virion particle. RNA polymerase, mRNA capping enzyme, and nucle oside triphosphate phosphohydrolase I were found at similarly reduced levels in the virion, indicating that packaging of some virion core en zymes may be interdependent.