Single-Particle Cryo-EM of Naturally Coexisting Mycoviruses Enables Structural Characterization of Conserved Capsid Folds, Divergent Architectures, and dsRNA Genome Organization
Mycoviruses frequently coexist within fungal hosts, yet high-resolution structural studies have traditionally relied on the purification of individual viral species, limiting the structural analysis of naturally occurring mixed infections. Here, we show that single-particle cryo-electron microscopy (cryo-EM) can simultaneously resolve multiple coexisting mycoviruses directly from naturally virus-infected fungal hosts. Using Saccharomyces cerevisiae strain TF229, which naturally harbours the closely related totiviruses Saccharomyces cerevisiae virus L-A (ScV-L-A) and L-BC (ScV-L-BC), we separated both viral populations in silico and reconstructed their capsids at near-atomic resolution under identical experimental conditions. Despite sharing only 10% sequence identity, their capsid proteins retain a highly conserved structural fold, whereas a [~]20{degrees} difference in asymmetric dimer orientation remodels capsid curvature and generates distinct virion architectures. Structural comparison further highlighted the C-terminal extension of ScV-L-BC, which mediates molecular swapping between neighbouring subunits and contributes to capsid stabilization. Symmetry relaxation revealed that, in both viruses, the encapsidated genome adopts a conserved spool-like organization, with ordered dsRNA filaments arranged into concentric layers. The outermost genome layer remains separated by [~]10-15 [A] from the inner capsid surface, consistent with its predominantly electronegative character, while specific capsid-genome contacts are maintained mainly through the C-terminal regions of Gag. These findings reveal how conserved capsid folds can generate structurally distinct viral particles and establish mixed-sample cryo-EM as a scalable strategy for the high-resolution characterization of complex mycovirus communities. IMPORTANCEFungal viruses are widabstacttespread and frequently occur as mixed infections, yet most structural studies rely on the purification of individual viruses, limiting our understanding of their diversity. This study demonstrates that cryo-electron microscopy can resolve closely related mycoviruses directly from naturally mixed samples, enabling their structural characterization under identical conditions. Application of this strategy to ScV-L-A and ScV-L-BC reveals how conserved capsid protein architectures can generate distinct viral particles and uncovers principles governing dsRNA genome organization. This work establishes a strategy to connect the rapidly expanding discovery of fungal viruses with structural and functional understanding.