Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways

Single-stranded RNA and DNA bacteriophages constitute a significant portion of the global virome, but the host genetic networks facilitating their infection cycles are not well understood. In this study, we conducted a genome-wide analysis of the genetic factors orchestrating the infection processes of F pilus-dependent RNA and DNA phages within Escherichia coli.

By screening genetic variations across RNA phages representing all four groups of Leviviricetes, we identified a highly conserved network of host dependencies, with the exception of the F plasmid gene traD. The primary structural components of the receptor and dsbA-mediated disulfide bond formation are essential across all viral lineages for F pilus integrity.

However, traD shows variable essentiality across different viral groups despite sharing the same primary receptor. Our gene dosage screens revealed that elevated copy numbers of the hslU protease or the RNA chaperone stpA hinder infection, thereby establishing genetic barriers that can impede the viral life cycle. Similar assays with filamentous DNA phages produced host factor profiles consistent with existing literature, while also revealing additional variations in host dependency.

These screens confirmed that DNA phages rely exclusively on the host TolQRA complex for entry following pilus engagement. The experiments tracked significant negative fitness signatures across homeostatic clusters, highlighting the strain on the host envelope due to the continuous non-lytic production of virions. Our comparative approach also traced the host selection of our isolation strain (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella.

This surface shield prevents the binding of several double-stranded DNA phages isolated on various E. coli strains, while still allowing the engagement of ssDNA and ssRNA phages through the extended F pilus and bypassing the barrier through native pilus retraction. Overall, this comprehensive study offers a systematic, class-wide map of single-stranded phage-host interactions, bridging classical genetics with modern viral discovery.

It establishes a robust host platform for investigating uncultured viral diversity and provides a functional blueprint for the development of next-generation diagnostics, protein antibiotics, and biocontrol tools to mitigate horizontal gene transfer.