Bromouridine triphosphate inhibits transcription termination and mRNA release by vaccinia virions

Termination of transcription in vitro by purified vaccinia virus RNA polymerase occurs downstream of a cis-acting signal UUUUUNU in the nascent RNA strand and requires a trans-acting termination factor, VTF, that is associated with the viral mRNA capping enzyme. Factor-dependent termination can be inhibited specifically by incorporation of BrUMP (from BrUTP) into nascent RNA in place of UMP. The relevance of VTF action to early vaccinia mRNA biogenesis was demonstrated in the present study of the effects of BrUTP on mRNA synthesis and release by permeabilized vaccinia virions. BrUMP incorporation inhibited the release of newly made transcripts from the virus particle, resulting in the accumulation of transcripts within virus cores. This effect was observed also with IUMP, but not with BrCMP or IMP incorporation. Transcripts synthesized in the presence of BrUTP were heterogeneous in size and severalfold larger than transcripts made in the presence of UTP. The progressive increase in the size of the core-associated, BrUMP-containing transcripts indicated that they were still engaged by elongating RNA polymerase. These results are consistent with a predominant pathway of mRNA 3'-end formation by virions that involves VTF-dependent transcription termination. These data do not support an alternative model of 3'-end formation by endonucleolytic cleavage of larger RNA precursors.