Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways
- 1Lawrence Berkeley National Laboratory
Abstract
Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.
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BibTeX
@misc{murray2026genomewide,
title = {{Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways}},
author = {I. Murray and R. T. Magar and D. Piya and H. Selvakumar and L. Moriniere and S. Koderivalappil and M. Svab and J. Thongchol and A. Kazakov and N. H. Elisabeth and M. Alayouni and P. P. Singh and A. Muszynski and P. Azadi and S. V. Owen and J. Zhang and S. D. Friedman and A. P. Arkin and A. Deutschbauer and S. Roux and V. K. Mutalik},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.17.752499},
url = {https://www.biorxiv.org/content/10.64898/2026.09.17.752499v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Genome-wide characterization of host factors involved in single-stranded RNA and DNA phage infection pathways
AU - I. Murray
AU - R. T. Magar
AU - D. Piya
AU - H. Selvakumar
AU - L. Moriniere
AU - S. Koderivalappil
AU - M. Svab
AU - J. Thongchol
AU - A. Kazakov
AU - N. H. Elisabeth
AU - M. Alayouni
AU - P. P. Singh
AU - A. Muszynski
AU - P. Azadi
AU - S. V. Owen
AU - J. Zhang
AU - S. D. Friedman
AU - A. P. Arkin
AU - A. Deutschbauer
AU - S. Roux
AU - V. K. Mutalik
PY - 2026
DA - 2026/09/17
PB - bioRxiv
DO - 10.64898/2026.09.17.752499
UR - https://www.biorxiv.org/content/10.64898/2026.09.17.752499v1
AB - Single stranded RNA (ssRNA) and single stranded DNA (ssDNA) bacteriophages represent a key component of the global virome, yet the host genetic networks supporting their infection cycles remain poorly understood. Here, we present a comprehensive, genome-wide mapping of the genetic landscape regulating infection cycles for F pilus-dependent ssRNA and ssDNA phages in Escherichia coli. Genetic screens across ssRNA phages spanning all four genogroups of the Leviviricetes revealed a highly conserved network of host dependencies, with the notable exception of the F plasmid gene traD. While primary structural receptor components and dsbA mediated disulfide bond formation are universally required across all lineages to ensure F pilus integrity, traD exhibits a strict genogroup-specific requirement during entry, showing variable essentiality across different viral groups despite sharing an identical primary receptor. Our gene dosage screens revealed that an elevated copy number of the hslU protease or the RNA chaperone stpA restricts infection, identifying clear genetic barriers that can perturb the viral life cycle. Parallel assays with filamentous ssDNA phages produced host factor profiles consistent with published literature, while revealing additional variations in host dependency. These screens confirmed that ssDNA phages strictly rely on the host TolQRA complex for entry downstream of pilus engagement. The assays tracked prominent negative fitness signatures across homeostatic clusters, highlighting how the physiological burden of continuous, non-lytic virion extrusion strains the host envelope. Finally, this comparative approach traced the selectivity of our isolation host (E. coli HSF) to a horizontally acquired capsule architecture from Klebsiella. This surface shield excludes a large panel of double stranded DNA phages isolated on diverse E. coli strains, while allowing virions from ssDNA and ssRNA phages to engage the extended F pilus and bypass the barrier via native pilus retraction. Together, this work provides a systematic, class-wide map of single stranded phage-host interactions, bridging classical genetics with modern viral discovery while establishing a robust host platform to access uncultured viral diversity and a functional blueprint to design next generation diagnostics, protein antibiotics, and biocontrol tools to halt horizontal gene transfer.
ER -