Poxvirus transcriptome analysis.

Expression of approximately two-thirds of the vaccinia virus (VACV) genome has been previously analyzed gene by gene, and the analysis has led to the classification of early-, intermediate-, and late-stage genes with distinct promoters and associated transcription factors. Assarsson et al. (1) reported the complete VACV transcriptome by using a tiling array. There are problems in application of this technology to poxviruses: intergenic regions are typically short or nonexistent (2); early gene termination can occur in downstream ORFs; and extensive run-on transcription occurs with intermediate and late genes producing large amounts of double-stranded RNA (3). Supporting information figure 6A in ref. 1 shows decreased signal between ORFs 125 and 123 to support resolution of individual ORFs. However, intervening ORF 124 is transcribed from the opposite strand under an early/late promoter (4), and that transcript could compete with 25-mer probes for hybridization of mRNA. Similarly, genes transcribed oppositely are on either side of ORFs 124 and 127 (figure 1A in ref. 1). Showing more typical regions of the genome in which adjacent ORFs are transcribed in the same direction would have helped assess the run-on problem. Because most VACV genes are known to occur in clusters transcribed at the same stage, the failure to resolve adjacent transcripts might not be perceived. However, run-on transcription can explain discrepancies between early and late gene assignments in supporting information table 2 in ref. 1 and the previous literature as shown in Fig. 1. We suggest that alternative methods that specifically analyze 5 RNA start sites are preferable to tiling arrays for analysis of the poxvirus transcriptome. ACKNOWLEDGMENTS. This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.