CCA initiation boxes without unique promoter elements support in vitro transcription by three viral RNA-dependent RNA polymerases

It has previously been observed that the only specific requirement for tran scriptional initiation on viral RNA in vitro by the RNA-dependent RNA polym erase (RdRp) of turnip yellow mosaic virus is the CCA at the 3' end of the genome. We now compare the abilities of this RdRp, turnip crinkle virus RdR p, and Q beta replicase, an enzyme capable of supporting the complete viral replication cycle in vitro, to transcribe RNA templates containing multipl e CCA boxes but lacking specific viral sequences. Each enzyme is able to in itiate transcription from several CCA boxes within these RNAs, and no speci al reaction conditions are required for these activities. The transcription al yields produced from templates comprised of multiple CCA or CCCA repeats relative to templates derived from native viral RNA sequences vary between 2:1 and 0.1:1 for the different RdRps. Control of initiation by such redun dant sequences presents a challenge to the specificity of viral transcripti on and replication. We identify 3'-preferential initiation and sensitivity to structural presentation as two specificity mechanisms that can limit ini tiation among potential CCA initiation sites. These two specificity mechani sms are used to different degrees by the three RdRps. The finding that thre e viral RdRps representing two of the three supergroups within the positive -strand RNA viral RdRp phylogeny support substantial transcription in the a bsence of unique promoters suggests that this phenomenon may be common amon g positive-strand viruses. A framework is presented arguing that replicatio n of viral RNA in the absence of unique promoter elements is feasible.