Interaction of the Vaccinia Virus RNA Polymerase-Associated 94-Kilodalton Protein with the Early Transcription Factor

Abstract

ABSTRACT A multisubunit RNA polymerase (RPO) encoded by vaccinia virus (VACV), in conjunction with specific factors, transcribes early, intermediate, and late viral genes. However, an additional virus-encoded polypeptide referred to as the RPO-associated protein of 94 kDa (RAP94) is tightly bound to the RPO for the transcription of early genes. Unlike the eight RPO core subunits, RAP94 is synthesized exclusively at late times after infection. Furthermore, RAP94 is necessary for the packaging of RPO and other components needed for early transcription in assembling virus particles. The direct association of RAP94 with NPH I, a DNA-dependent ATPase required for transcription termination, and the multifunctional poly(A) polymerase small subunit/2′- O -methyltransferase/elongation factor was previously demonstrated. That RAP94 provides a structural and functional link between the core RPO and the VACV early transcription factor (VETF) has been suspected but not previously demonstrated. Using VACV recombinants that constitutively or inducibly express VETF subunits and RAP94 with affinity tags, we showed that (i) VETF associates only with RPO containing RAP94 in vivo and in vitro, (ii) the association of RAP94 with VETF requires both subunits of the latter, (iii) neither viral DNA nor other virus-encoded late proteins are required for the interaction of RAP94 with VETF and core RPO subunits, (iv) different domains of RAP94 bind VETF and core subunits of RPO, and (v) NPH I and VETF bind independently and possibly simultaneously to the N-terminal region of RAP94. Thus, RAP94 provides the bridge between the RPO and proteins needed for transcription initiation, elongation, and termination.