CHARACTERIZATION OF THE INTERACTIONS AMONG VACCINIA VIRUS TRANSCRIPTION FACTORS G2R, A18R, AND H5R

Prior genetic analysis suggests that there may exist an interaction be tween the products of the vaccinia virus genes A18R, a putative negati ve transcription elongation factor, and G2R, a putative positive trans cription elongation factor In addition, affinity purification of polyh istidine-tagged G2R protein overexpressed in vaccinia virus-infected c ells, reported here, results in copurification of the vaccinia H5R pro tein, previously characterized as a late viral transcription factor. W e have therefore used several methods to screen further for interactio ns among the G2R, A18R, and H5R proteins. Methods include copurificati on or co-immunoprecipitation of proteins overexpressed during vaccinia virus infection, activation of the gal 4 promoter by gal 4 fusions in the yeast two-hybrid system, and co-immunoprecipitation of proteins s ynthesized in vitro in a rabbit reticulocyte lysate. The results revea l interactions which include all possible pairwise combinations of the three proteins G2R, A18R, and H5R; however, not all possible permutat ions of the interactions are observed and the interactions are not obs erved in all environments tested. The results suggest that the vaccini a Virus proteins G2R, A18R, and H5R interact as part of a higher order transcription complex. (C) 1998 Academic Press.