Transcription regulatory program of SSV1 infecting Saccharolobus solfataricus

Fuselloviridae infecting thermoacidophilic Sulfolobales are undoubtedly the best-studied archaeoviruses, with the SSV1-Saccharolobus solfataricus interaction as a model. Around one-third of fusellovirus core genes encode putative DNA-binding proteins (DBPs), which, although little is known about their functions and mechanisms of action, suggests that DNA-binding processes such as transcription regulation are a critical aspect of the infection cycle.
In this work, we aim to identify and characterize SSV1-encoded DBPs to elucidate transcription regulatory mechanisms in the virus-host interaction.
Host and virus transcriptome dynamics post-infection and post-induction are being studied by performing high-resolution, long-read ONT-cappable RNA-sequencing. This end-to-end sequencing of primary transcripts will provide a full transcriptional blueprint of SSV1, including transcription start and stop sites, operon structures and UTRs. This transcriptomic study will be complemented with genome-wide binding profiles of all SSV1 putative DBPs by performing ChIP-seq experiments using engineered virus variants in which each DBP is epitope-tagged.
Molecular mechanisms of individual SSV1-encoded DBPs will be characterized through protein-DNA binding assays and in vitro transcription. To date, we have already cloned, heterologously overexpressed and purified 6 SSV1 putative transcription regulators: F93, F112, B115, A100, C80 and E51. Through EMSAs, we have confirmed their DNA-binding properties, exhibiting different DNA-binding behavior on native PAGE gels.
This work is expected to greatly enhance our understanding of the SSV virus-host interaction by applying state-of-the-art techniques to study SSV1. By bridging critical knowledge gaps, we might contribute to the development of virus-based biological parts for gene expression engineering in Sulfolobales, an archaeal order with great biotechnological potential.