Emiliania huxleyi is a worldwide distributed unicellular marine alga whose cells are covered by calcite disks called coccoliths. By reflecting light, the coccoliths influence retention of heat in oceans, which impacts planetary climate . Emiliania huxleyi virus 201 (EhV-201) and related nucleocytoplasmic large DNA viruses limit the population growth of E. huxleyi .
Virion of EhV-201 is pleiomorphic in shape, therefore we used localised reconstruction of small fractions of the virion edges to elucidate its complex ultrastructure, comprising an inner membrane, capsid, outer membrane, and surface protein envelope. Furthermore, we used focused ion beam milling and cryo-electron tomography to characterize the formation of EhV-201 virions in E. huxleyi cells. The particle assembly is initiated on membrane fragments, which separate from the endoplasmic reticulum. Assembly of the capsid proteins at the outer surface of the membrane fragment induces its bending and gradual formation of capsids containing a membrane sack. Virus DNA is packaged into the pre-formed particles through an opening in the capsid and inner membrane. The genome-filled intermediates bud into intracellular vesicles, and in this process, acquire the outer membrane and protein envelope. Virions are released from the cell by exocytosis or lysis of the infected alga. Our results give structural insight into the formation of EhV-201 – a pathogen that influences the Earth’s climate.