Vaccinia Virus Gene A18R DNA Helicase Is a Transcript Release Factor

Abstract

Prior phenotypic analysis of a vaccinia virus gene A18R mutant, Cts23, showed the synthesis of longer than wild type (Wt) length viral transcripts during the intermediate stage of infection, indicating that the A18R protein may act as a negative transcription elongation factor. The purpose of the work described here was to determine a biochemical activity for the A18R protein.Pulse-labeled transcription complexes established from intermediate virus promoters on bead-bound DNA templates were assayed for transcript release during an elongation step that contained nucleotides and various proteins. Pulse-labeled transcription complexes elongated in the presence of only nucleotides were unable to release nascent RNA. The addition of Wt extract during the elongation phase resulted in release of the nascent transcript, indicating that additional factors present in the Wt extract are capable of inducing transcript release. Extract from Cts23 or mock-infected cells was unable to induce release. The lack of release upon addition of Cts23 extract suggests that A18R is involved in release of nascent RNA. By itself, purified polyhistidine-tagged A18R protein (His-A18R) was unable to induce release; however, release did occur in the presence of purified His-A18R protein plus extract from either Cts23 or mock-infected cells. These data taken together indicate that A18R is necessary but not sufficient for release of nascent transcripts. We have also demonstrated that the combination of A18R protein and mock extract induces transcript release in an ATP-dependent manner, consistent with the fact that the A18R protein is an ATP-dependent helicase. Further analysis revealed that the release activity is not restricted to a vaccinia intermediate promoter but is observed using pulse-labeled transcription complexes initiated from all three viral gene class promoters. Therefore, we conclude that A18R and an as yet unidentified cellular factor(s) are required for the in vitro release of nascent RNA from a vaccinia virus transcription elongation complex.Elongation and termination are key control points in both prokaryotic and eukaryotic transcription (1). Transcriptional events such as pausing, arrest, and termination are regulated by cis-and trans-acting factors that decide the fate of a given transcript, either elongation or termination. A paused complex, in which the 3Ј end of the nascent RNA is retained in the polymerase catalytic site, can be induced by a DNA sequencespecific element, such as a T-rich sequence, or blockage of the RNA polymerase by so-called negative elongation factors. Elongation of a paused complex may resume either spontaneously or in response to positive elongation factors. An arrested complex, in which the catalytic site of the polymerase has slipped backwards and out of context with the 3Ј end of the nascent transcript, must cleave the nascent RNA to return the 3Ј end to the catalytic site in order to relieve arrest. Cleavage is an endogenous activity of the RNA polymerase but is activated in response to trans-a...

Vaccinia virus transcription

“…The A18R polypeptide has classical DNA-dependent nucleic acid helicase motifs and DNA helicase activity specific for DNA:DNA hybrids less than 20 nt in length (Simpson & Condit, 1995). Extracts from infected cells depleted of A18R by growth of the mutant under non-permissive conditions did not support transcript release in an in vitro assay (Lackner & Condit, 2000). Interestingly, purified A18R was incapable of supporting transcript release unless extract from uninfected cells was also provided.…”

Identification of Vaccinia Virus Replisome and Transcriptome Proteins by Isolation of Proteins on Nascent DNA Coupled with Mass Spectrometry

“…The genes encoding these proteins all have early promoters, except for the three late transcription factors, which have intermediate promoters. The A18 protein, a putative helicase with a role in the release of RNA from transcription complexes (79,80), was detected, but the peptide abundance was below the threshold that we chose for significance. A transient association with the transcription complex, as well as other technical factors, could explain this result.…”

Duplication of the A17L Locus of Vaccinia Virus Provides an Alternate Route to Rifampin Resistance

“…Although any part of the duplicated region in RifR9 could be responsible for resistance to rifampin, we focused on A17 because previous studies had shown an interaction between D13 and the N terminus of A17 (5,14), whereas A18 is a DNA-dependent helicase involved in transcription (35,36) and A19 is involved in a late stage of morphogenesis (37,38). A17 is a 23-kDa protein that undergoes several modifications, including C-and N-terminal cleavages and phosphorylation.…”