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° 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 Å 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.