Crown protein is dispensable for OmRV entry but required for efficient transmission.

Non-enveloped icosahedral dsRNA viruses, such as taxons of the Totiviridae family, have evolved surface features that enable infection of multicellular hosts through receptor binding and membrane penetration via the canonical endosomal pathway. Unlike totiviruses that infect unicellular hosts, toti-like viruses, including artiviruses, infect invertebrate multicellular hosts and possess an additional surface crown protein (CrP) on their conserved T = 1 icosahedral capsid. The mosquito-specific artivirus Omono River virus (OmRV) also expresses this CrP, which has been suggested to facilitate viral infection and propagation, although it may not be essential. To clarify its role in viral infection, we generated a CrP deletion mutant of OmRV to directly assess its functional significance. The absence of CrP does not affect infectivity in mosquito cells or alter the capsid structure. However, cytopathic effects (CPEs), vesicular virus factories, and viral efficient transmission, mediated by viral release and cell-to-cell spread, are still affected. These findings indicate that CrP is dispensable for OmRV infection but may play a crucial role in viral release, providing important insight into its previously unclear function in the viral life cycle.