Abstract
Poxviruses are large DNA viruses that replicate within the cytoplasm and encode a complete transcription system, including a multisubunit RNA polymerase, stage-specific transcription factors, capping and methylating enzymes, and a poly(A) polymerase. Expression of the more than 200 open reading frames by vaccinia virus, the prototype poxvirus, is temporally regulated: early mRNAs are synthesized immediately after infection, whereas intermediate and late mRNAs are synthesized following genome replication. The postreplicative transcripts are heterogeneous in length and overlap the entire genome, which pose obstacles for high resolution mapping. We used tag-based methods in conjunction with high throughput cDNA sequencing to determine the precise 5'-capped and 3'-polyadenylated ends of postreplicative RNAs. Polymerase slippage during initiation of intermediate and late RNA synthesis results in a 5'-poly(A) leader that allowed the unambiguous identification of true transcription start sites. Ninety RNA start sites were located just upstream of intermediate and late open reading frames, but many more appeared anomalous, occurring within coding and non-coding regions, indicating pervasive transcription initiation. We confirmed the presence of functional promoter sequences upstream of representative anomalous start sites and demonstrated that alternative start sites within open reading frames could generate truncated isoforms of proteins. In an analogous manner, poly(A) sequences allowed accurate mapping of the numerous 3'-ends of postreplicative RNAs, which were preceded by a pyrimidine-rich sequence in the DNA coding strand. The distribution of postreplicative promoter sequences throughout the genome provides enormous transcriptional complexity, and the large number of previously unmapped RNAs may have novel functions.