The globally distributed marine alga Emiliania huxleyi has cooling effect on the Earth's climate. The population density of E. huxleyi is restricted by Nucleocytoviricota viruses, including E. huxleyi virus 201 (EhV-201). Despite the impact of E. huxleyi viruses on the climate, there is limited information about their structure and replication. Here, we show that the dsDNA genome inside the EhV-201 virion is protected by an inner membrane, capsid, and outer membrane. EhV-201 virions infect E. huxleyi by using fivefold vertices to bind to and fuse the virus' inner membrane with the cell plasma membrane. Progeny virions assemble in the cytoplasm at the surface of endoplasmic reticulum-derived membrane segments. Genome packaging initiates synchronously with the capsid assembly and completes through an aperture in the forming capsid. The genome-filled capsids acquire an outer membrane by budding into intracellular vesicles. EhV-201 infection induces a loss of surface protective layers from E. huxleyi cells, which enables the continuous release of virions by exocytosis.

Bijvoet Centre for Biomolecular Research Utrecht University Utrecht Netherlands

Central European Institute of Technology Masaryk University Brno Czech Republic

Department of Botany and Zoology Faculty of Science Masaryk University Brno Czech Republic

Marine Biological Association Plymouth UK

School of Biological and Marine Sciences University of Plymouth Plymouth UK

Veterinary Population Medicine The University of Minnesota St Paul USA

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A. Winter, W. G. Siesser, Coccolithophores (Cambridge Univ. Press, 1994).

Tyrrell T., Holligan P. M., Mobley C. D., Optical impacts of oceanic coccolithophore blooms. J. Geophys. Res. 104, 3223–3241 (1999).

Holligan P. M., Fernandez E., Aiken J., Balch W. M., Boyd P., Burkill P. H., Finch M., Groom S. B., Malin G., Muller K., Purdie D. A., Robinson C., Trees C. C., Turner S. M., van der Wal P., A biogeochemical study of the coccolithophore, Emiliania huxleyi, in the North Atlantic. Glob. Biogeochem. Cycles 7, 879–900 (1993).

Westbroek P., De Jong E. W., van der Wal P., Borman A. H., De Vrind J. P. M., Kok D., De Bruijn W. C., Parker S. B., Mechanism of calcification in the marine alga Emiliania huxleyi. Philos. Trans. R. Soc. B Biol. Sci. 304, 435–444 (1984).

Ziveri P., Broerse A. T. C., van Hinte J. E., Westbroek P., Honjo S., The fate of coccoliths at 48°N 21°W, Northeastern Atlantic. Deep-Sea Res. II Top. Stud. Oceanogr. 47, 1853–1875 (2000).

Malin G., Turner S., Liss P., Holligan P., Harbour D., Dimethylsulphide and dimethylsulphoniopropionate in the Northeast Atlantic during the Summer Coccolithophore Bloom. Deep-Sea Res. I Oceanogr. Res. Pap. 40, 1487–1508 (1993).

Charlson R. J., Lovelock J. E., Andreae M. O., Warren S. G., Oceanic phytoplankton, atmospheric sulphur, cloud albedo and climate. Nature 326, 655–661 (1987).

Westbroek P., Brown C. W., van Bleijswijk J., Brownlee C., Brummer G. J., Conte M., Egge J., Fernandez E., Jordan R., Knappertsbusch M., Stefels J., Veldhuis M., van der Wal P., Young J., A model system approach to biological climate forcing. The example of Emiliania huxleyi. Glob. Planet. Change 8, 27–46 (1993).

Wilson W. H., Tarran G. A., Schroeder D., Cox M., Oke J., Malin G., Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel. J. Mar. Biol. Assoc. U.K. 82, 369–377 (2002).

Bratbak G., Egge J. K., Heldal M., Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms. Mar. Ecol. Prog. Ser. 93, 39–48 (1993).

Castberg T., Larsen A., Sandaa R. A., Brussaard C. P. D., Egge J. K., Heldal M., Thyrhaug R., van Hannen E. J., Bratbak G., Microbial population dynamics and diversity during a bloom of the marine coccolithophorid Emiliania huxleyi (Haptophyta). Mar. Ecol. Prog. Ser. 221, 39–46 (2001).

Mönttinen H. A. M., Bicep C., Williams T. A., Hirt R. P., The genomes of nucleocytoplasmic large DNA viruses: Viral evolution writ large. Microb. Genom. 7, 000649 (2021). PubMed PMC

Koonin E. V., Dolja V. V., Krupovic M., Varsani A., Wolf Y. I., Yutin N., Zerbini F. M., Kuhn J. H., Global organization and proposed megataxonomy of the virus world. Microbiol. Mol. Biol. Rev. 84, e00061-19 (2020). PubMed PMC

Wilson W. H., Schroeder D. C., Allen M. J., Holden M. T. G., Parkhill J., Barrell B. G., Churcher C., Harnlin N., Mungall K., Norbertczak H., Quail M. A., Price C., Rabbinowitsch E., Walker D., Craigon M., Roy D., Ghazal P., Complete genome sequence and lytic phase transcription profile of a Coccolithovirus. Science 309, 1090–1092 (2005). PubMed

Schroeder D. C., Oke J., Malin G., Wilson W. H., Coccolithovirus (Phycodnaviridae): Characterisation of a new large dsDNA algal virus that infects Emiliana huxleyi. Arch. Virol. 147, 1685–1698 (2002). PubMed

Nissimov J. I., Worthy C. A., Rooks P., Napier J. A., Kimmance S. A., Henn M. R., Ogata H., Allen M. J., Draft genome sequence of four coccolithoviruses: Emiliania huxleyi virus EhV-88, EhV-201, EhV-207, and EhV-208. J. Virol. 86, 2896–2897 (2012). PubMed PMC

Allen M. J., Schroeder D. C., Holden M. T. G., Wilson W. H., Evolutionary history of the coccolithoviridae. Mol. Biol. Evol. 23, 86–92 (2006). PubMed

Mackinder L. C. M., Worthy C. A., Biggi G., Hall M., Ryan K. P., Varsani A., Harper G. M., Wilson W. H., Brownlee C., Schroeder D. C., A unicellular algal virus, Emiliania huxleyi virus 86, exploits an animal-like infection strategy. J. Gen. Virol. 90, 2306–2316 (2009). PubMed

Schatz D., Shemi A., Rosenwasser S., Sabanay H., Wolf S. G., Ben-Dor S., Vardi A., Hijacking of an autophagy-like process is critical for the life cycle of a DNA virus infecting oceanic algal blooms. New Phytol. 204, 854–863 (2014). PubMed PMC

Andres G., Charro D., Matamoros T., Dillard R. S., Abrescia N. G. A., The cryo-EM structure of African swine fever virus unravels a unique architecture comprising two icosahedral protein capsids and two lipoprotein membranes. J. Biol. Chem. 295, 1–12 (2020). PubMed PMC

Liu S., Luo Y., Wang Y., Li S., Zhao Z., Bi Y., Sun J., Peng R., Song H., Zhu D., Sun Y., Li S., Zhang L., Wang W., Sun Y., Qi J., Yan J., Shi Y., Zhang X., Wang P., Qiu H. J., Gao G. F., Cryo-EM structure of the African swine fever virus. Cell Host Microbe 26, 836–843.e3 (2019). PubMed

Vardi A., Van Mooy B. A. S., Fredricks H. F., Popendorf K. J., Ossolinski J. E., Haramaty L., Bidle K. D., Viral glycosphingolipids induce lytic infection and cell death in marine phytoplankton. Science 326, 861–865 (2009). PubMed

Rosenwasser S., Mausz M. A., Schatz D., Sheyn U., Malitsky S., Aharoni A., Weinstock E., Tzfadia O., Ben-Dor S., Feldmesser E., Pohnert G., Vardi A., Rewiring host lipid metabolism by large viruses determines the fate of Emiliania huxleyi, a bloom-forming alga in the ocean. Plant Cell 26, 2689–2707 (2014). PubMed PMC

Zhang X., Xiang Y., Dunigan D. D., Klose T., Chipman P. R., Van Etten J. L., Rossmann M. G., Three-dimensional structure and function of the Paramecium bursaria chlorella virus capsid. Proc. Natl. Acad. Sci. U.S.A. 108, 14837–14842 (2011). PubMed PMC

Agarkova I., Hertel B., Zhang X. Z., Lane L., Tchourbanov A., Dunigan D. D., Thiel G., Rossmann M. G., Van Etten J. L., Dynamic attachment of Chlorovirus PBCV-1 to Chlorella variabilis. Virology 466-467, 95–102 (2014). PubMed PMC

Shao Q., Agarkova I. V., Noel E. A., Dunigan D. D., Liu Y., Wang A., Guo M., Xie L., Zhao X., Rossmann M. G., Van Etten J. L., Klose T., Fang Q., Near-atomic, non-icosahedrally averaged structure of giant virus Paramecium bursaria chlorella virus 1. Nat. Commun. 13, 6476 (2022). PubMed PMC

Klose T., Reteno D. G., Benamar S., Hollerbach A., Colson P., La Scola B., Rossmann M. G., Structure of faustovirus, a large dsDNA virus. Proc. Natl. Acad. Sci. U.S.A. 113, 6206–6211 (2016). PubMed PMC

Xian Y., Xiao C., Current capsid assembly models of icosahedral nucleocytoviricota viruses. Adv. Virus Res. 108, 275–313 (2020). PubMed PMC

Yan X., Olson N. H., Van Etten J. L., Bergoin M., Rossmann M. G., Baker T. S., Structure and assembly of large lipid-containing dsDNA viruses. Nat. Struct. Biol. 7, 101–103 (2000). PubMed PMC

Xiao C., Kuznetsov Y. G., Sun S. Y., Hafenstein S. L., Kostyuchenko V. A., Chipman P. R., Suzan-Monti M., Raoult D., McPherson A., Rossmann M. G., Structural studies of the giant mimivirus. PLOS Biol. 7, e92 (2009). PubMed PMC

Yan X., Chipman P. R., Castberg T., Bratbak G., Baker T. S., The marine algal virus PpV01 has an icosahedral capsid with T=219 quasisymmetry. J. Virol. 79, 9236–9243 (2005). PubMed PMC

Yan X., Yu Z., Zhang P., Battisti A. J., Holdaway H. A., Chipman P. R., Bajaj C., Bergoin M., Rossmann M. G., Baker T. S., The capsid proteins of a large, icosahedral dsDNA virus. J. Mol. Biol. 385, 1287–1299 (2009). PubMed PMC

Okamoto K., Miyazaki N., Reddy H. K. N., Hantke M. F., Maia F. R. N. C., Larsson D. S. D., Abergel C., Claverie J.-M., Hajdu J., Murata K., Svenda M., Cryo-EM structure of a Marseilleviridae virus particle reveals a large internal microassembly. Virology 516, 239–245 (2018). PubMed

Chihara A., Burton-Smith R. N., Kajimura N., Mitsuoka K., Okamoto K., Song C., Murata K., A novel capsid protein network allows the characteristic internal membrane structure of Marseilleviridae giant viruses. Sci. Rep. 12, 21428 (2022). PubMed PMC

Xiao C., Rossmann M. G., Structures of giant icosahedral eukaryotic dsDNA viruses. Curr. Opin. Virol. 1, 101–109 (2011). PubMed PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Zidek A., Potapenko A., Bridgland A., Meyer C., Kohl S. A. A., Ballard A. J., Cowie A., Romera-Paredes B., Nikolov S., Jain R., Adler J., Back T., Petersen S., Reiman D., Clancy E., Zielinski M., Steinegger M., Pacholska M., Berghammer T., Bodenstein S., Silver D., Vinyals O., Senior A. W., Kavukcuoglu K., Kohli P., Hassabis D., Highly accurate protein structure prediction with AlphaFold. Nature 596, 583–589 (2021). PubMed PMC

Van Etten J. L., Lane L. C., Meints R. H., Viruses and viruslike particles of eukaryotic algae. Microbiol. Rev. 55, 586–620 (1991). PubMed PMC

Rose S. L., Fulton J. M., Brown C. M., Natale F., Van Mooy B. A. S., Bidle K. D., Isolation and characterization of lipid rafts in Emiliania huxleyi: A role for membrane microdomains in host-virus interactions. Environ. Microbiol. 16, 1150–1166 (2014). PubMed

Abrahao J. S., Dornas F. P., Silva L. C. F., Almeida G. M., Boratto P. V. M., Colson P., La Scola B., Kroon E. G., Acanthamoeba polyphaga mimivirus and other giant viruses: An open field to outstanding discoveries. Virol. J. 11, 120 (2014). PubMed PMC

Schrad J. R., Abrahao J. S., Cortines J. R., Parent K. N., Structural and proteomic characterization of the initiation of giant virus infection. Cell 181, 1046–1061.e6 (2020). PubMed PMC

Gendrault J. L., Steffan A. M., Bingen A., Kirn A., Penetration and uncoating of frog virus 3 (Fv3) in cultured rat Kupffer cells. Virology 112, 375–384 (1981). PubMed

Chinchar V. G., Yu K. H., Jancovich J. K., The molecular biology of frog virus 3 and other iridoviruses infecting cold-blooded vertebrates. Viruses 3, 1959–1985 (2011). PubMed PMC

Wahlund T. M., Hadaegh A. R., Clark R., Nguyen B., Fanelli M., Read B. A., Analysis of expressed sequence tags from calcifying cells of marine coccolithophorid (Emiliania huxleyi). Mar. Biotechnol. (N.Y.) 6, 278–290 (2004). PubMed

Hernaez B., Guerra M., Salas M. L., Andres G., African swine fever virus undergoes outer envelope disruption, capsid disassembly and inner envelope fusion before core release from multivesicular endosomes. PLOS Pathog. 12, e1005595 (2016). PubMed PMC

Haunost M., Riebesell U., Bach L. T., The calcium carbonate shell of Emiliania huxleyi provides limited protection against viral infection. Front. Mar. Sci. 7, 735 (2020).

Walker C. E., Heath S., Salmon D. L., Smirnoff N., Langer G., Taylor A. R., Brownlee C., Wheeler G. L., An extracellular polysaccharide-rich organic layer contributes to organization of the coccosphere in coccolithophores. Front. Mar. Sci. 5, 306 (2018).

Rybicka K., Glycosomes (protein-glycogen complex) in the canine heart - Ultrastructure, histochemistry and changes induced by acidic treatment. Virchows Arch. B Cell Pathol. Incl. Mol. Pathol. 30, 335–347 (1979). PubMed

Rybicka K., Simultaneous demonstration of glycogen and protein in glycosomes of cardiac tissue. J. Histochem. Cytochem. 29, 4–8 (1981). PubMed

Sturgess J. M., Mitranic M. M., Moscarello M. A., Extraction of glycoproteins during tissue preparation for electron-microscopy. J. Microsc. 114, 101–105 (1978). PubMed

Vardi A., Haramaty L., Van Mooy B. A. S., Fredricks H. F., Kimmance S. A., Larsen A., Bidle K. D., Host-virus dynamics and subcellular controls of cell fate in a natural coccolithophore population. Proc. Natl. Acad. Sci. U.S.A. 109, 19327–19332 (2012). PubMed PMC

Brussaard C. P. D., Kempers R. S., Kop A. J., Riegman R., Heldal M., Virus-like particles in a summer bloom of Emiliania huxleyi in the North Sea. Aquat. Microb. Ecol. 10, 105–113 (1996).

Vincent F., Sheyn U., Porat Z., Schatz D., Vardi A., Visualizing active viral infection reveals diverse cell fates in synchronized algal bloom demise. Proc. Natl. Acad. Sci. U.S.A. 118, e2021586118 (2021). PubMed PMC

Mutsafi Y., Zauberman N., Sabanay I., Minsky A., Vaccinia-like cytoplasmic replication of the giant Mimivirus. Proc. Natl. Acad. Sci. U.S.A. 107, 5978–5982 (2010). PubMed PMC

Schramm B., Locker J. K., Cytoplasmic organization of POXvirus DNA replication. Traffic 6, 839–846 (2005). PubMed

Schulz F., Abergel C., Woyke T., Giant virus biology and diversity in the era of genome-resolved metagenomics. Nat. Rev. Microbiol. 20, 721–736 (2022). PubMed

Yakubovskaya E., Zaliznyak T., Martinez J. M., Taylor G. T., Raman microspectroscopy goes viral: Infection dynamics in the cosmopolitan microalga, Emiliania huxleyi. Front. Microbiol. 12, 686287 (2021). PubMed PMC

Han G., Gable K., Yan L., Allen M. J., Wilson W. H., Moitra P., Harmon J. M., Dunn T. M., Expression of a novel marine viral single-chain serine palmitoyltransferase and construction of yeast and mammalian single-chain chimera. J. Biol. Chem. 281, 39935–39942 (2006). PubMed

Allen M. J., Howard J. A., Lilley K. S., Wilson W. H., Proteomic analysis of the EhV-86 virion. Proteome Sci. 6, 11 (2008). PubMed PMC

Malitsky S., Ziv C., Rosenwasser S., Zheng S. N., Schatz D., Porat Z., Ben-Dor S., Aharoni A., Vardi A., Viral infection of the marine alga Emiliania huxleyi triggers lipidome remodeling and induces the production of highly saturated triacylglycerol. New Phytol. 210, 88–96 (2016). PubMed

Sun S., Rao V. B., Rossmann M. G., Genome packaging in viruses. Curr. Opin. Struc. Biol. 20, 114–120 (2010). PubMed PMC

Chaudhari H. V., Inamdar M. M., Kondabagil K., Scaling relation between genome length and particle size of viruses provides insights into viral life history. iScience 24, 102452 (2021). PubMed PMC

Rokitta S. D., de Nooijer L. J., Trimborn S., de Vargas C., Rost B., John U., Transcriptome analyses reveal differential gene expression patterns between the life-cycle stages of Emiliania huxleyi (Haptophyta) and reflect specialization to different ecological NICHES1. J. Phycol. 47, 829–838 (2011). PubMed

Guillard R. R., Ryther J. H., Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (cleve) Gran. Can. J. Microbiol. 8, 229–239 (1962). PubMed

Klaveness D., Guillard R. R. L., The requirement for silicon in Synura petersenii (Chrysophyceae)1,2. J. Phycol. 11, 349–355 (1975).

Derelle E., Ferraz C., Escande M. L., Eychenie S., Cooke R., Piganeau G., Desdevises Y., Bellec L., Moreau H., Grimsley N., Life-cycle and genome of OtV5, a large DNA virus of the pelagic marine unicellular green alga Ostreococcus tauri. PLOS ONE 3, e2250 (2008). PubMed PMC

Zheng S. Q., Palovcak E., Armache J.-P., Verba K. A., Cheng Y., Agard D. A., MotionCor2: Anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). PubMed PMC

Zhang K., Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016). PubMed PMC

Wagner T., Merino F., Stabrin M., Moriya T., Antoni C., Apelbaum A., Hagel P., Sitsel O., Raisch T., Prumbaum D., Quentin D., Roderer D., Tacke S., Siebolds B., Schubert E., Shaikh T. R., Lill P., Gatsogiannis C., Raunser S., SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019). PubMed PMC

Zivanov J., Nakane T., Forsberg B. O., Kimanius D., Hagen W. J. H., Lindahl E., Scheres S. H. W., New tools for automated high-resolution cryo-EM structure determination in RELION-3. eLife 7, e42166 (2018). PubMed PMC

Schneider C. A., Rasband W. S., Eliceiri K. W., NIH image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675 (2012). PubMed PMC

Mastronarde D. N., Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005). PubMed

Turonova B., Hagen W. J. H., Obr M., Mosalaganti S., Beugelink J. W., Zimmerli C. E., Krausslich H. G., Beck M., Benchmarking tomographic acquisition schemes for high-resolution structural biology. Nat. Commun. 11, 876 (2020). PubMed PMC

Tegunov D., Xue L., Dienemann C., Cramer P., Mahamid J., Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 Å in cells. Nat. Methods 18, 186–193 (2021). PubMed PMC

Mastronarde D. N., Held S. R., Automated tilt series alignment and tomographic reconstruction in IMOD. J. Struct. Biol. 197, 102–113 (2017). PubMed PMC

Himes B. A., Zhang P., emClarity: Software for high-resolution cryo-electron tomography and subtomogram averaging. Nat. Methods 15, 955–961 (2018). PubMed PMC

Kimanius D., Dong L., Sharov G., Nakane T., Scheres S. H. W., New tools for automated cryo-EM single-particle analysis in RELION-4.0. Biochem. J. 478, 4169–4185 (2021). PubMed PMC

Goddard T. D., Huang C. C., Meng E. C., Pettersen E. F., Couch G. S., Morris J. H., Ferrin T. E., UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018). PubMed PMC

Searle S. R., Speed F. M., Milliken G. A., Population marginal means in the linear model: An alternative to least squares means. Am. Stat. 34, 216–221 (1980).

J. Fox, S. Weisberg, An R Companion to Applied Regression (SAGE, ed. 3, 2019).

Bates D., Machler M., Bolker B. M., Walker S. C., Fitting Linear mixed-effects models using lme4. J. Stat. Softw. 67, 1–48 (2015).

H. Wickham, ggplot 2: Elegant Graphics for Data Analysis, Use R! (Springer, 2009).

Camacho C., Coulouris G., Avagyan V., Ma N., Papadopoulos J., Bealer K., Madden T. L., BLAST+: Architecture and applications. BMC Bioinformatics 10, 421 (2009). PubMed PMC

Katoh K., Standley D. M., MAFFT multiple sequence alignment software version 7: Improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013). PubMed PMC

Waterhouse A. M., Procter J. B., Martin D. M. A., Clamp M., Barton G. J., Jalview Version 2—a multiple sequence alignment editor and analysis workbench. Bioinformatics 25, 1189–1191 (2009). PubMed PMC

Capella-Gutierrez S., Silla-Martinez J. M., Gabaldon T., trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009). PubMed PMC