Long-read sequencing (LRS) techniques enable the identification of full-length RNA molecules in a single run eliminating the need for additional assembly steps. LRS research has exposed unanticipated transcriptomic complexity in various organisms, including viruses. Herpesviruses are known to produce a range of transcripts, either close to or overlapping replication origins (Oris) and neighboring genes related to transcription or replication, which possess confirmed or potential regulatory roles. In our research, we employed both new and previously published LRS and short-read sequencing datasets to uncover additional Ori-proximal transcripts in nine herpesviruses from all three subfamilies (alpha, beta and gamma). We discovered novel long non-coding RNAs, as well as splice and length isoforms of mRNAs. Moreover, our analysis uncovered an intricate network of transcriptional overlaps within the examined genomic regions. We demonstrated that herpesviruses display distinct patterns of transcriptional overlaps in the vicinity of or at the Oris. Our findings suggest the existence of a 'super regulatory center' in the genome of alphaherpesviruses that governs the initiation of both DNA replication and global transcription through multilayered interactions among the molecular machineries.
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