High-resolution transcriptome analysis reveals a multilayered and dynamic transcriptional architecture in the archaeal virus SSV1

Abstract

Fuselloviridae infecting thermoacidophilic Sulfolobales are among the best-studied archaeal viruses, with the Saccharolobus Spindle Shaped Virus 1 (SSV1)-Saccharolobus solfataricus interaction serving as a model system. After infection, SSV1 establishes a chronic infection with new virions continuously budding from the infected cell. UV irradiation triggers virus replication, increased virion production and a tightly orchestrated temporal transcription cycle. However, gene regulatory mechanisms post-UV-induction remain elusive. In this study, we investigated the SSV1 transcriptomic landscape post-UV-induction using high-resolution, long-read ONT-cappable-RNA-sequencing. Through end-to-end sequencing of primary transcripts, we generated a comprehensive transcript map of SSV1, identifying 21 transcription start sites (TSSs), including 12 newly described sites, and 19 termination sites (TTSs), of which 9 are novel. This enabled refinement of transcription units and operon structures across the genome. Five new antisense RNAs were discovered, including one encoded antisense to the open reading frame of a putative viral toxin gene a291, which we hypothesize to be a cis-encoded regulatory RNA. In addition, spontaneous recombination between two direct repeats in the SSV1 structural genes vp1 and vp3 was confirmed. To our knowledge, this is the first implementation of cappable-seq for an archaeal virus, and the first base-resolution analysis of the SSV1 transcriptome. It reveals a previously unappreciated level of transcriptional complexity, illustrating how high-resolution transcriptomics can deepen understanding of viral transcriptome architecture and regulatory networks. ImportanceArchaeal viruses remain among the least understood viruses in the virosphere, despite their ecological and evolutionary significance. Saccharolobus Spindle Shaped Virus 1 (SSV1) is a key model for the study of virus-host interactions in archaea. Yet, its gene regulatory mechanisms have remained poorly resolved. Here, we provide the first base-pair resolution transcriptome of an archaeal virus, revealing a complex and multi-layered transcriptional architecture. We uncover alternative transcription start and termination sites, as well as antisense transcription, pointing to regulatory mechanisms beyond a simple temporal gene expression program. These findings suggest that archaeal viruses use sophisticated transcriptional and post-transcriptional mechanisms similar to those in bacterial and eukaryotic viruses. By establishing a high-resolution map of the SSV1 transcriptome, this work advances our understanding of archaeal virus biology.