During the viral replication cycle, entry emerges as a critical juncture, tightly regulated both spatially and temporally, orchestrating the initial steps of infection. Many viral proteins that mediate entry across the host cell membrane are synthesised as inactive precursors, which undergo proteolytic cleavage to become fully active. Processing of viral surface proteins by host proteases, such as furin and trypsin, is a control mechanism that produces mature virions activated for infection across a wide range of viruses.
Rotavirus (RV), the leading cause of severe gastroenteritis with dehydration in children under 5 years of age, serves as a paradigmatic example of a non-enveloped enteric virus whose infectivity depends on trypsin-like digestive enzymes present in the gastrointestinal tract. Despite more than forty years since the discovery of RV virions’ dependency on trypsin [1] and extensive structural characterisation of the RV virion [2-5], the molecular mechanism underlying this proteolytic activation remains poorly understood. Using cryo-electron microscopy (cryo-EM) and advanced image processing techniques, we compared uncleaved and cleaved RV virions and found that the conformation of the non-proteolyzed spike is constrained by the position of loops that surround its structure, linking the lectin domains of the spike head to its body. Proteolysis of these loops removes this structural constraint, enabling the spike to undergo the necessary conformational changes required for cell membrane penetration. Thus, these loops function as regulatory elements that ensure the spike protein is activated precisely when and where it is needed to facilitate a successful infection.