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

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.The ability of poxviruses to replicate in the cytoplasm depends on the presence within the infectious particle of a system for the synthesis of translatable early mRNAs (29). The protein components of this system have been isolated from vaccinia virus (VACV) and comprise a multisubunit DNA-dependent RNA polymerase (RPO), RPO-associated protein of 94 kDa (RAP94), the VACV early transcription factor (VETF), capping and methylating enzymes, poly(A) polymerase, two nucleic acid-dependent ATPases, and topoisomerase. The products of the early mRNAs include proteins needed for the replication of the viral genome and intermediate gene transcription factors, thereby allowing the next stage of gene expression. Newly synthesized viral DNA serves as a template for the successive transcription of intermediate genes encoding late transcription factors and late genes encoding early transcription factors. In this way, the orderly expression of early, intermediate, and late genes is tightly programmed.Previous studies suggested that the components of the early transcription system are associated in a labile complex. The complex, which could initiate and terminate transcription from an exogenous DNA template containing an early promoter, was released by the disruption of purified virions and isolated by glycerol gradient sedimentation (8). After further treatments and...

Cholesterol Flux Is Required for Endosomal Progression of African Swine Fever Virions during the Initial Establishment of Infection

“…This mode of entry, related to endocytosis, could be somewhat inhibited by blocking cholesterol efflux. Next, early gene transcription and mRNA export would be initiated via the action of viral RNA polymerases and transcription factors inside the core (62). The activation of the transcription machinery is considered a priming step, as it is a prerequisite for the next phase of uncoating.…”

Interactions of the Vaccinia Virus A19 Protein

“…Repression of RAP94 results in the failure to package RNA polymerase, capping enzyme, and NPH I, but not VETF, leading to a model in which VETF binds to early promoters and attracts the transcription enzyme complex through RAP94 (22,25). However, repression of A19 did not impair the incorporation of the transcription complex or the A3 core protein (2), into virus particles.…”

“…RAP94 was also detected by Western blotting, but not the transcription factor subunits possibly because of low sensitivity of Western blotting with the available antibodies and an indirect association with A19. Since the components of the early transcription complex, including the RNA polymerase, RAP94, capping enzyme, NPH I, and early transcription factors, are associated through multiple interactions (22,25,(40)(41)(42), it is difficult to determine which polypeptide interacts directly with A19. However, the available evidence points to the RNA polymerase for the following reasons.…”

“…Therefore, A19 most likely interacts directly with the RNA polymerase, though interaction with the capping enzyme subunits, which also have early promoters, could not be excluded. In our current model, RAP94 is pulled down with A19, because the latter interacts with RNA polymerase (30,38); NPH I (42) and A7 and D6 (25) are pulled down because of their association with RAP94. At this time, we are unable to predict the interacting subunit(s) of the RNA polymerase.…”

“…The recombinant viruses vT7LacOI (21), vFS-A19 (2), vFS-A19i (2), vRAP94i (22), vE10i (23), vL3i (24), v3XFlag-H4 (25), vA7-3XFlag (25), and vD6-3XFlag (25) have been described previously ( Table 1).…”