Nanopore direct RNA sequencing (DRS) of MS2 bacteriophages in E. coli throughout its life cycles reveals a complex transcriptional activity to control and maintain its growth.

backgroundThe RNA bacteriophage MS2 is an RNA phage that infects the bacterium E. coli and is one of the most studied and prototypical model phages in molecular biology and microbiology. Previous research revealed complex translational control and fine-tuning for MS2 replication. However, the dynamics of its transcriptional activity and replication during the life cycles within the bacteria remain elusive.

methodsHere, we employed Nanopore Direct RNA sequencing (DRS) to investigate the transcriptome and epitranscriptome landscape of the MS2 in infected E. coli throughout multiple life cycles.

resultsWe discovered that MS2 phages sustain a high level of transcriptional activity required for replication. We found large amounts of subgenomic small transcripts from RNA degradation, Nanopore DRS bias, and transcripts containing the coat-encoding region, required for virion assembly. We found the error-prone activity of the MS2 replicase produced hybrid reads from the template-switching mechanism. We finally evidenced that RNA modification is conserved throughout the entire life cycle in full-length transcripts without the acquisition of new modifications, whereas small transcripts did acquire newly modified sites. The conserved sequence and secondary structure (U-rich hairpin) of Ψ installation sites were the most amenable to RNA modification, from potentially the host RluA-mediated installation.

conclusionsOverall, our investigation revealed a more complex transcriptional dynamics of MS2 phages than anticipated within E. coli to maintain its growth and replication under host pressure.