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

Single-stranded RNA (ssRNA) and single-stranded DNA (ssDNA) bacteriophages form a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here we present a genome-wide map of the host factors involved in infection by F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Screens across ssRNA phages spanning all four genogroups of the Leviviricetes reveal a conserved network of host dependencies, with the notable exception of the F plasmid gene traD. Primary structural receptor components and DsbA-mediated disulfide bond formation are universally required to ensure F pilus integrity, whereas traD shows a strict genogroup-specific requirement during entry despite all lineages sharing the same receptor. Gene dosage screens identify elevated copy number of the genomic fragments encoding the HslU protease or the RNA chaperone StpA as barriers that restrict infection. Parallel assays with filamentous ssDNA phages confirm reliance on the host TolQRA complex for entry downstream of pilus engagement and track negative fitness signatures across envelope homeostasis clusters, reflecting the physiological burden of non-lytic virion extrusion. Comparative genomics and high-throughput genetics trace the selectivity of the isolation host, E. coli HSF, to a horizontally acquired Klebsiella capsule that excludes a large panel of double-stranded DNA phages isolated on other E. coli strains while allowing ssRNA and ssDNA virions to engage the extended F pilus and bypass the barrier through native pilus retraction. The work delivers a systematic, class-wide map of single-stranded phage-host interactions, a host platform for accessing uncultured viral diversity, and a functional blueprint for diagnostics, protein antibiotics and biocontrol tools that halt horizontal gene transfer.