Illuminating the newly produced viruses within the virosphere with bioorthogonal noncanonical amino acid tagging and single-virus genomic sequencing technologies
Creators
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1.
University of Alicante
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2.
California Institute of Technology
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3.
Centre for Genomic Regulation
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4.
Pompeu Fabra University
Abstract
Marine viruses impact biogeochemical cycles through cell lysis, releasing organic matter and nutrients that fuel ocean productivity. Identifying and quantifying the specific viruses active in these processes remain a priority in the field. Here, we introduce a click-chemistry method to fluorescently label, sort, and sequence the genomes of newly produced viral particles (viral progeny) released from transcriptionally active host microbial cells, alongside the analysis of co-occurring inactive cells and pre-existing viruses in environmental samples. This approach, called viral bioorthogonal noncanonical amino acid tagging (BONCAT)-fluorescence-activated cell sorting (FACS), combines BONCAT with environmental sample incubation, followed by single-virus and single-cell sorting by flow cytometry (FACS). Genomic analysis of translationally active cells and new viral progeny in coastal seawater incubations confirmed BONCAT labeling and successful sorting of diverse marine bacteria, microeukaryotic cells, and virioplankton, with stark differences in the predicted turnover of specific groups of infecting viruses, including pelagiphages, methylophages, a Flavobacteriales-associated novel “Far-T4” clade, noncanonical DNA viruses of Naomiviridae using dU instead of dT, algae-infecting giant NCLDV viruses, and parasitic virophages. Sequenced BONCAT-active cells showed a strong enrichment in viral contigs relative to the inactive cell fraction, suggestive of a large proportion of translationally active virocells. This study illustrates the effectiveness of viral BONCAT-FACS for uncovering genome-resolved virus–host dynamics. By providing a direct approach for tracking active viral infections in natural environments, this method enhances our ability to investigate behavior and interactions of these nanoscale predators, expanding our understanding of their role in ecosystem dynamics.
Copyright and License
Acknowledgement
We thank the manager S. Ranson at Caltech’s Kerckhoff Marine Laboratory for facilitating water collections. We are also appreciative of the reviewer’s comments and suggestions, which improved this work.
Funding
This work was funded by the US Department of Energy, Office of Science, Office of Biological and Environmental Research under the award number DE-SC0022991 and subaward no. S591062, with additional support from the Spanish Ministry of Universities (MARSALAS21-15), Agencia Estatal de Investigación (PID2021-125175OB-I00), Generalitat Valenciana (APOSTD/2020/237), the Simons Collaboration on Principles of Microbial Ecosystems (PriME) under award number SFI-LS-PRIME-00011855, and an investigator grant from the NOMIS foundation (to V.J.O.).
Data Availability
The raw reads from the active and non-active cell and viral progeny and pre-existing viruses are available on the SRA data bank under accession numbers SAMN53290052, SAMN53290053, SAMN53290054, SAMN53290055, SAMN53290056, SAMN53290057, SAMN53290058, SAMN53290059, and SAMN53290060 from the BioProject PRJNA1365689.
Ethics
This report was prepared as an account of work sponsored by an agency of the US Government. Neither the US Government nor any agency thereof, nor any of their employees, makes any warranty, express or implied; assumes any legal liability or responsibility for the accuracy, completeness, or usefulness of any information, apparatus, product, or process disclosed; or represents that its use would not infringe privately owned rights. Reference herein to any specific commercial product, process, or service by trade name, trademark, manufacturer, or otherwise does not necessarily constitute or imply its endorsement, recommendation, or favoring by the US Government or any agency thereof. The views and opinions of authors expressed herein do not necessarily state or reflect those of the US Government or any agency thereof.
Supplemental Material
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Additional details
Additional titles
- Alternative title
- Illuminating the active virosphere with BONCAT and single virus genomic sequencing technologies
Identifiers
Related works
- Is new version of
- Discussion Paper: 10.1101/2025.08.05.666878 (DOI)
Funding
- United States Department of Energy
- DE-SC0022991
- United States Department of Energy
- S591062
- Ministerio de Ciencia, Innovación y Universidades
- MARSALAS21-15
- Agencia Estatal de Investigación
- PID2021-125175OB-I00
- Generalitat Valenciana
- APOSTD/2020/237
- Simons Foundation
- SFI-LS-PRIME-00011855
- Nomis Foundation
Dates
- Submitted
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2025-09-03
- Accepted
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2026-02-27
- Available
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2026-03-06Published
- Available
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2026-03-31Corrected and typeset
Caltech Custom Metadata
- Caltech groups
- Division of Geological and Planetary Sciences (GPS) , Division of Biology and Biological Engineering (BBE)
- Publication Status
- Published