10779/crick.11558223.v1 Charlotte E Melia Charlotte E Melia Christopher J Peddie Christopher J Peddie Anja WM de Jong Anja WM de Jong Eric J Snijder Eric J Snijder Lucy M Collinson Lucy M Collinson Abraham J Koster Abraham J Koster Hilde M van der Schaar Hilde M van der Schaar Frank JM van Kuppeveld Frank JM van Kuppeveld Montserrat Bárcena Montserrat Bárcena Origins of enterovirus replication organelles established by whole-cell electron microscopy. The Francis Crick Institute 2020 CLEM SBF-SEM correlative light and electron microscopy coxsackievirus membrane structure picornavirus replication organelle biogenesis serial block-face scanning electron microscopy volume electron microscopy EM 0605 Microbiology 2020-01-09 11:43:41 Journal contribution https://crick.figshare.com/articles/journal_contribution/Origins_of_enterovirus_replication_organelles_established_by_whole-cell_electron_microscopy_/11558223 Enterovirus genome replication occurs at virus-induced structures derived from cellular membranes and lipids. However, the origin of these replication organelles (ROs) remains uncertain. Ultrastructural evidence of the membrane donor is lacking, suggesting that the sites of its transition into ROs are rare or fleeting. To overcome this challenge, we combined live-cell imaging and serial block-face scanning electron microscopy of whole cells to capture emerging enterovirus ROs. The first foci of fluorescently labeled viral protein correlated with ROs connected to the endoplasmic reticulum (ER) and preceded the appearance of ROs stemming from the trans-Golgi network. Whole-cell data sets further revealed striking contact regions between ROs and lipid droplets that may represent a route for lipid shuttling to facilitate RO proliferation and genome replication. Our data provide direct evidence that enteroviruses use ER and then Golgi membranes to initiate RO formation, demonstrating the remarkable flexibility with which enteroviruses usurp cellular organelles.IMPORTANCE Enteroviruses are causative agents of a range of human diseases. The replication of these viruses within cells relies on specialized membranous structures termed replication organelles (ROs) that form during infection but whose origin remains elusive. To capture the emergence of enterovirus ROs, we use correlative light and serial block-face scanning electron microscopy, a powerful method to pinpoint rare events in their whole-cell ultrastructural context. RO biogenesis was found to occur first at ER and then at Golgi membranes. Extensive contacts were found between early ROs and lipid droplets (LDs), which likely serve to provide LD-derived lipids required for replication. Together, these data establish the dual origin of enterovirus ROs and the chronology of their biogenesis at different supporting cellular membranes.