Single-stranded DNA bacteriophages of the Microviridae family are major components of the global virosphere. Microviruses are highly abundant in aquatic ecosystems and are prominent members of the mammalian gut microbiome, where their diversity has been linked to various chronic health disorders. Despite the clear importance of microviruses, little is known about the molecular mechanism of host infection. Here, we have characterized an exceptionally large microvirus, Ebor, and provide crucial insights into long-standing mechanistic questions. Cryogenic electron microscopy of Ebor revealed a capsid with trimeric protrusions that recognise lipopolysaccharides on the host surface. Cryogenic electron tomography of the host cell colonized with virus particles demonstrated that the virus initially attaches to the cell via five such protrusions, located at the corners of a single pentamer. This interaction triggers a stargate mechanism of capsid opening along the 5-fold symmetry axis, enabling delivery of the virus genome. Despite variations in specific virus-host interactions among different Microviridae family viruses, structural data indicate that the stargate mechanism of infection is universally employed by all members of the family. Startlingly, our data reveal a mechanistic link for the opening of relatively small capsids made out of a single jelly-roll fold with the structurally unrelated giant viruses.

Department of Biology University of York Wentworth Way York YO10 5DD UK

Department of Experimental Biology Faculty of Science Masaryk University 625 00 Brno Czech Republic

Department of Microbiology and Immunology University of British Columbia Vancouver V6T 1Z3 BC Canada

Department of Microbiology and Molecular Genetics Oklahoma State University US

Materials and Structural Analysis Thermo Fisher Scientific Achtseweg Noord 5 5651 GG Eindhoven The Netherlands

York Biomedical Research Institute University of York York United Kingdom YO10 5NG

York Structural Biology Laboratory Department of Chemistry University of York York YO10 5DD United Kingdom

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Taylor NMI, Prokhorov NS, Guerrero-Ferreira RC, Shneider MM, Browning C, Goldie KN, et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature. 2016. May;533(7603):346–52. PubMed

Sun Y, Roznowski AP, Tokuda JM, Klose T, Mauney A, Pollack L, et al. Structural changes of tailless bacteriophage ΦX174 during penetration of bacterial cell walls. Proc Natl Acad Sci. 2017. Dec 26;114(52):13708–13. PubMed PMC

Bárdy P, MacDonald CIW, Pantůček R, Antson AA, Fogg PCM. Jorvik: A membrane-containing phage that will likely found a new family within Vinavirales. iScience. 2023. Nov 17;26(11):108104. PubMed PMC

Kirchberger PC, Martinez ZA, Ochman H. Organizing the Global Diversity of Microviruses. mBio. 2022. Jun 28;13(3):e0058822. PubMed PMC

Neri U, Wolf YI, Roux S, Camargo AP, Lee B, Kazlauskas D, et al. Expansion of the global RNA virome reveals diverse clades of bacteriophages. Cell. 2022. Oct 13;185(21):4023–4037.e18. PubMed

Brum JR, Schenck RO, Sullivan MB. Global morphological analysis of marine viruses shows minimal regional variation and dominance of non-tailed viruses. ISME J. 2013. Sep;7(9):1738–51. PubMed PMC

Tisza MJ, Pastrana DV, Welch NL, Stewart B, Peretti A, Starrett GJ, et al. Discovery of several thousand highly diverse circular DNA viruses. Kirkegaard K, Pride D, editors. eLife. 2020. Feb 4;9:e51971. PubMed PMC

Yoshida M, Mochizuki T, Urayama SI, Yoshida-Takashima Y, Nishi S, Hirai M, et al. Quantitative Viral Community DNA Analysis Reveals the Dominance of Single-Stranded DNA Viruses in Offshore Upper Bathyal Sediment from Tohoku, Japan. Front Microbiol [Internet]. 2018. [cited 2024 Feb 19];9. Available from: https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2018.00075 PubMed DOI PMC

Zuo T, Sun Y, Wan Y, Yeoh YK, Zhang F, Cheung CP, et al. Human-Gut-DNA Virome Variations across Geography, Ethnicity, and Urbanization. Cell Host Microbe. 2020. Nov 11;28(5):741–751.e4. PubMed

Mayneris-Perxachs J, Castells-Nobau A, Arnoriaga-Rodríguez M, Garre-Olmo J, Puig J, Ramos R, et al. Caudovirales bacteriophages are associated with improved executive function and memory in flies, mice, and humans. Cell Host Microbe. 2022. Mar;30(3):340–356.e8. PubMed

Norman JM, Handley SA, Baldridge MT, Droit L, Liu CY, Keller BC, et al. Disease-specific Alterations in the Enteric Virome in Inflammatory Bowel Disease. Cell. 2015. Jan 29;160(3):447–60. PubMed PMC

Olo Ndela E, Roux S, Henke C, Sczyrba A, Sime Ngando T, Varsani A, et al. Reekeekee- and roodoodooviruses, two different Microviridae clades constituted by the smallest DNA phages. Virus Evol. 2023. Jan 1;9(1):veac123. PubMed PMC

Zhang L, Li Z, Bao M, Li T, Fang F, Zheng Y, et al. A Novel Microviridae Phage (CLasMV1) From “Candidatus Liberibacter asiaticus”. Front Microbiol [Internet]. 2021. [cited 2024 Jan 4];12. Available from: https://www.frontiersin.org/articles/10.3389/fmicb.2021.754245 PubMed DOI PMC

Kirchberger PC, Ochman H. Microviruses: A World Beyond phiX174. Annu Rev Virol. 2023. Sep 29;10(1):99–118. PubMed

Walker PJ, Siddell SG, Lefkowitz EJ, Mushegian AR, Adriaenssens EM, Alfenas-Zerbini P, et al. Changes to virus taxonomy and to the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses (2021). Arch Virol. 2021. Sep 1;166(9):2633–48. PubMed

Chipman PR, Agbandje-McKenna M, Renaudin J, Baker TS, McKenna R. Structural analysis of the spiroplasma virus, SpV4: implications for evolutionary variation to obtain host diversity among the Microviridae. Struct Lond Engl 1993. 1998. Feb 15;6(2):135–45. PubMed PMC

Kirchberger PC, Ochman H. Resurrection of a global, metagenomically defined gokushovirus. Zambrano MM, Kirkegaard K, Breitbart M, editors. eLife. 2020. Feb 26;9:e51599. PubMed PMC

Lee H, Baxter AJ, Bator CM, Fane BA, Hafenstein SL. Cryo-EM Structure of Gokushovirus ΦEC6098 Reveals a Novel Capsid Architecture for a Single-Scaffolding Protein, Microvirus Assembly System. J Virol. 96(21):e00990–22. PubMed PMC

Sun L, Young LN, Zhang X, Boudko SP, Fokine A, Zbornik E, et al. Icosahedral bacteriophage ΦX174 forms a tail for DNA transport during infection. Nature. 2014. Jan 16;505(7483):432–5. PubMed

Zucker F, Bischoff V, Olo Ndela E, Heyerhoff B, Poehlein A, Freese HM, et al. New Microviridae isolated from Sulfitobacter reveals two cosmopolitan subfamilies of single-stranded DNA phages infecting marine and terrestrial Alphaproteobacteria. Virus Evol. 2022. Jul 1;8(2):veac070. PubMed PMC

Schrad JR, Abrahão JS, Cortines JR, Parent KN. Structural and Proteomic Characterization of the Initiation of Giant Virus Infection. Cell. 2020. May 28;181(5):1046–1061.e6. PubMed PMC

Zauberman N, Mutsafi Y, Halevy DB, Shimoni E, Klein E, Xiao C, et al. Distinct DNA Exit and Packaging Portals in the Virus Acanthamoeba polyphaga mimivirus. PLoS Biol. 2008. May;6(5):e114. PubMed PMC

Bárdy P, Füzik T, Hrebík D, Pantůček R, Thomas Beatty J, Plevka P. Structure and mechanism of DNA delivery of a gene transfer agent. Nat Commun. 2020. Jun 15;11(1):3034. PubMed PMC

Strnad H, Lapidus A, Paces J, Ulbrich P, Vlcek C, Paces V, et al. Complete Genome Sequence of the Photosynthetic Purple Nonsulfur Bacterium Rhodobacter capsulatus SB 1003. J Bacteriol. 2010. Jul;192(13):3545–6. PubMed PMC

Das B, Martínez E, Midonet C, Barre FX. Integrative mobile elements exploiting Xer recombination. Trends Microbiol. 2013. Jan;21(1):23–30. PubMed

Ding H, Moksa MM, Hirst M, Beatty JT. Draft Genome Sequences of Six Rhodobacter capsulatus Strains, YW1, YW2, B6, Y262, R121, and DE442. Genome Announc. 2014. Feb 13;2(1):e00050–14. PubMed PMC

Solioz M, Marrs B. The gene transfer agent of Rhodopseudomonas capsulata,. Arch Biochem Biophys. 1977. May 1;181(1):300–7. PubMed

Yen HC, Hu NT, Marrs BL. Characterization of the gene transfer agent made by an overproducer mutant of Rhodopseudomonas capsulata. J Mol Biol. 1979. Jun 25;131(2):157–68. PubMed

Roux S, Krupovic M, Poulet A, Debroas D, Enault F. Evolution and Diversity of the Microviridae Viral Family through a Collection of 81 New Complete Genomes Assembled from Virome Reads. PLOS ONE. 2012. Jul 11;7(7):e40418. PubMed PMC

Gabler F, Nam SZ, Till S, Mirdita M, Steinegger M, Söding J, et al. Protein Sequence Analysis Using the MPI Bioinformatics Toolkit. Curr Protoc Bioinforma. 2020;72(1):e108. PubMed

McKenna R, Xia D, Willingmann P, Ilag LL, Krishnaswamy S, Rossmann MG, et al. Atomic structure of single-stranded DNA bacteriophage phi X174 and its functional implications. Nature. 1992. Jan 1;355(6356):137–43. PubMed PMC

Ilag LL, McKenna R, Yadav MP, BeMiller JN, Incardona NL, Rossmann MG. Calcium Ion-induced Structural Changes in Bacteriophage φ X174. J Mol Biol. 1994. Dec 1;244(3):291–300. PubMed

Leung MM, Brimacombe CA, Spiegelman GB, Beatty JT. The GtaR protein negatively regulates transcription of the gtaRI operon and modulates gene transfer agent (RcGTA) expression in Rhodobacter capsulatus. Mol Microbiol. 2012. Feb;83(4):759–74. PubMed PMC

Adams PG, Lamoureux L, Swingle KL, Mukundan H, Montaño GA. Lipopolysaccharide-Induced Dynamic Lipid Membrane Reorganization: Tubules, Perforations, and Stacks. Biophys J. 2014. Jun 3;106(11):2395–407. PubMed PMC

Stacey JCV, Tan A, Lu JM, James LC, Dick RA, Briggs JAG. Two structural switches in HIV-1 capsid regulate capsid curvature and host factor binding. Proc Natl Acad Sci. 2023. Apr 18;120(16):e2220557120. PubMed PMC

Nielsen TK, Forero-Junco LM, Kot W, Moineau S, Hansen LH, Riber L. Detection of nucleotide modifications in bacteria and bacteriophages: Strengths and limitations of current technologies and software. Mol Ecol. 2023;32(6):1236–47. PubMed

Bernal RA, Hafenstein S, Esmeralda R, Fane BA, Rossmann MG. The phiX174 protein J mediates DNA packaging and viral attachment to host cells. J Mol Biol. 2004. Apr 9;337(5):1109–22. PubMed

Nasir A, Caetano-Anollés G. Identification of Capsid/Coat Related Protein Folds and Their Utility for Virus Classification. Front Microbiol. 2017. Mar 10;8:380. PubMed PMC

Buchta D, Füzik T, Hrebík D, Levdansky Y, Sukeník L, Mukhamedova L, et al. Enterovirus particles expel capsid pentamers to enable genome release. Nat Commun. 2019. Mar 8;10(1):1138. PubMed PMC

Ormerod JG, Ormerod KS, Gest H. Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch Biochem Biophys. 1961. Sep 1;94(3):449–63. PubMed

Wall JD, Weaver PF, Gest H. Gene transfer agents, bacteriophages, and bacteriocins of Rhodopseudomonas capsulata. Arch Microbiol. 1975. Nov 7;105(3):217–24. PubMed

Beatty JT, Gest H. Generation of succinyl-coenzyme A in photosynthetic bacteria. Arch Microbiol. 1981. Jul 1;129(5):335–40.

Lin Y, Yuan J, Kolmogorov M, Shen MW, Chaisson M, Pevzner PA. Assembly of long error-prone reads using de Bruijn graphs. Proc Natl Acad Sci. 2016. Dec 27;113(52):E8396–405. PubMed PMC

Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinforma Oxf Engl. 2014. Jul 15;30(14):2068–9. PubMed

Besemer J, Lomsadze A, Borodovsky M. GeneMarkS: a self-training method for prediction of gene starts in microbial genomes. Implications for finding sequence motifs in regulatory regions. Nucleic Acids Res. 2001. Jun 15;29(12):2607–18. PubMed PMC

Carver T, Harris SR, Berriman M, Parkhill J, McQuillan JA. Artemis: an integrated platform for visualization and analysis of high-throughput sequence-based experimental data. Bioinformatics. 2012. Feb 15;28(4):464–9. PubMed PMC

Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997. Sep 1;25(17):3389–402. PubMed PMC

Jumper J, Evans R, Pritzel A, Green T, Figurnov M, Ronneberger O, et al. Highly accurate protein structure prediction with AlphaFold. Nature. 2021. Aug;596(7873):583–9. PubMed PMC

Underwood AP, Green J. MOP-UP: an online tool for finding strain-specific primers or motifs in DNA or protein alignments. Clin Microbiol Infect Off Publ Eur Soc Clin Microbiol Infect Dis. 2004. Oct;10(10):948–50. PubMed

Sievers F, Higgins DG. Clustal Omega for making accurate alignments of many protein sequences. Protein Sci. 2018;27(1):135–45. PubMed PMC

Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinforma Oxf Engl. 2014. May 1;30(9):1312–3. PubMed PMC

Lemoine F, Domelevo Entfellner JB, Wilkinson E, Correia D, Dávila Felipe M, De Oliveira T, et al. Renewing Felsenstein’s phylogenetic bootstrap in the era of big data. Nature. 2018. Apr;556(7702):452–6. PubMed PMC

Fogg MJ, Wilkinson AJ. Higher-throughput approaches to crystallization and crystal structure determination. Biochem Soc Trans. 2008. Jul 22;36(4):771–5. PubMed

Benešík M, Nováček J, Janda L, Dopitová R, Pernisová M, Melková K, et al. Role of SH3b binding domain in a natural deletion mutant of Kayvirus endolysin LysF1 with a broad range of lytic activity. Virus Genes. 2018. Feb;54(1):130–9. PubMed

Zheng SQ, Palovcak E, Armache JP, Verba KA, Cheng Y, Agard DA. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat Methods. 2017. Apr;14(4):331–2. PubMed PMC

Zivanov J, Nakane T, Forsberg BO, Kimanius D, Hagen WJ, Lindahl E, et al. New tools for automated high-resolution cryo-EM structure determination in RELION-3. Egelman EH, Kuriyan J, editors. eLife. 2018. Nov 9;7:e42166. PubMed PMC

Kremer JR, Mastronarde DN, McIntosh JR. Computer visualization of three-dimensional image data using IMOD. J Struct Biol. 1996;116(1):71–6. PubMed

Rohou A, Grigorieff N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J Struct Biol. 2015. Nov;192(2):216–21. PubMed PMC

Pettersen EF, Goddard TD, Huang CC, Meng EC, Couch GS, Croll TI, et al. UCSF ChimeraX: Structure visualization for researchers, educators, and developers. Protein Sci Publ Protein Soc. 2021. Jan;30(1):70–82. PubMed PMC

Sanchez-Garcia R, Gomez-Blanco J, Cuervo A, Carazo JM, Sorzano COS, Vargas J. DeepEMhancer: a deep learning solution for cryo-EM volume post-processing. Commun Biol. 2021. Jul 15;4(1):1–8. PubMed PMC

Zivanov J, Nakane T, Scheres SHW. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ. 2019. Jan 1;6(Pt 1):5–17. PubMed PMC

Emsley P, Lohkamp B, Scott WG, Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010. Apr;66(Pt 4):486–501. PubMed PMC

Liebschner D, Afonine PV, Baker ML, Bunkóczi G, Chen VB, Croll TI, et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr Sect Struct Biol. 2019. Oct 1;75(Pt 10):861–77. PubMed PMC

Chen VB, Arendall WB, Headd JJ, Keedy DA, Immormino RM, Kapral GJ, et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010. Jan 1;66(Pt 1):12–21. PubMed PMC

Chen M, Bell JM, Shi X, Sun SY, Wang Z, Ludtke SJ. A complete data processing workflow for cryo-ET and subtomogram averaging. Nat Methods. 2019. Nov;16(11):1161–8. PubMed PMC

Bepler T, Morin A, Rapp M, Brasch J, Shapiro L, Noble AJ, et al. Positive-unlabeled convolutional neural networks for particle picking in cryo-electron micrographs. Nat Methods. 2019. Nov;16(11):1153–60. PubMed PMC

Schwab J, Kimanius D, Burt A, Dendooven T, Scheres SHW. DynaMight: estimating molecular motions with improved reconstruction from cryo-EM images [Internet]. bioRxiv; 2023. [cited 2024 Apr 22]. p. 2023.10.18.562877. Available from: https://www.biorxiv.org/content/10.1101/2023.10.18.562877v1 PubMed DOI PMC

Jurrus E, Engel D, Star K, Monson K, Brandi J, Felberg LE, et al. Improvements to the APBS biomolecular solvation software suite. Protein Sci Publ Protein Soc. 2018. Jan;27(1):112–28. PubMed PMC

Chen M, Dai W, Sun SY, Jonasch D, He CY, Schmid MF, et al. Convolutional neural networks for automated annotation of cellular cryo-electron tomograms. Nat Methods. 2017. Oct;14(10):983–5. PubMed PMC

Carnoy C, Roten CA. The dif/Xer Recombination Systems in Proteobacteria. PLoS ONE. 2009. Sep 3;4(9):e6531. PubMed PMC