Abstract
<jats:p>Orsay virus (OrV), infecting Caenorhabditis elegans, is a valuable model for studying host-virus interactions, yet its bipartite RNA genome packaging and assembly mechanisms remain unclear. To investigate these mechanisms, we combine infection assays, single-molecule imaging, and cryo-electron microscopy. We find that the two RNA segments accumulate asymmetrically, and that productive infection depends on coinfection by multiple particles. We therefore asked whether unequal segment availability generates heterogeneous virions. Cryo-EM analysis of OrV, including symmetric and asymmetric reconstructions to 2.5 Ang resolution, unveils uniform internal density, excluding large populations of empty or semi-empty particles. We show the genome forms a semi-ordered network contacting the electropositive capsid interior through sequence-independent interactions with the N-terminus and penton region. We also resolve the capsid-delta linker, identifying the penton as a likely site coordinating capsid assembly. Together, we propose a model in which stochastic genome assortment emerges during assembly modulated by segment-specific replication dynamics.</jats:p>