Martinez-Romero, J. M. | bioRxiv Science

bioRxiv2026

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.

molecular biology↗

bioRxiv2025

Calicivirus assembly and stability are mediated by the N-terminal domain of the capsid protein with the involvement of the viral genome

Caliciviruses are important human and animal pathogens that cause varying clinical signs including gastroenteritis, respiratory illness, and hepatitis. Despite the availability of numerous calicivirus structures, relatively little is known about the mechanisms of capsid assembly and stability, or about genome packaging. Here we present the atomic structure of the RHDV virion and several related non-infectious virus-like particles, determined using cryo-EM at 2.5-3.3 [A] resolution. The inherent molecular switch, responsible for the conformational flexibility of the capsid protein VP1, is located in its N-terminal arm (NTA). The NTA establishes an extensive network of interactions on the inner capsid surface that stabilizes the hexamers and pentamers. For this structural polymorphism, we show that the NTA must interact with the RNA viral genome, that is, the genomic RNA acts concomitantly with the NTA as a molecular switch. The NTA-RNA interaction leads to specific conformational states that result in two types of VP1 dimers (the basic building blocks) necessary for T=3 capsid assembly. In addition, we used atomic force microscopy (AFM) to assess whether differences in genomic RNA content influence viral properties such as capsid stiffness in physiological conditions. These analyses highlight the mechanical role of packed RNA genome in RHDV virions, as the virion capsid pentamers are strengthened by interactions of the NTA star-like structure promoted by the viral genome. These results indicate that the interactions between the NTA and the viral genome guide the conformational states of VP1 dimers, directing capsid assembly and modulating its mechanical properties. Through interference with intermediate assemblies, the NTA network promoted by the genome could be an attractive target in future antiviral strategies. Author SummaryRabbit hemorrhagic disease virus (RHDV) is a highly contagious, often fatal calicivirus that poses a significant threat to rabbit populations worldwide. Although considerable progress has been made in elucidating the structural organization of RHDV capsids at near-atomic resolution, information regarding the nucleic acid organization within these virions and its interactions with the viral capsid remains limited. Understanding these interactions is essential for identifying mechanisms of structural polymorphism of the capsid protein, as well as virion assembly and stability. We report the cryo-electron microscopy structure, at near-atomic resolution, of RHDV virions and distinct virus-like particles, together with characterization of their biophysical properties using atomic force microscopy. The N-terminal region of the capsid protein, essential for its structural polymorphism, requires its interaction with genomic RNA. The interaction between N-terminal arms and the viral genome governs the conformational states of capsid protein dimers, thereby orchestrating capsid assembly and influencing its biophysical properties. By interfering with intermediate assemblies, the NTA network promoted by the genome could constitute an attractive target in antiviral strategies for several major human calicivirus pathogens that cause acute gastroenteritis and respiratory illness, including norovirus and sapovirus.

biophysics↗