Research Article: New Insights into Rotavirus Entry Machinery: Stabilization of Rotavirus Spike Conformation Is Independent of Trypsin Cleavage

Date Published: May 29, 2014

Publisher: Public Library of Science

Author(s): Javier M. Rodríguez, Francisco J. Chichón, Esther Martín-Forero, Fernando González-Camacho, José L. Carrascosa, José R. Castón, Daniel Luque, Félix A. Rey.


The infectivity of rotavirus, the main causative agent of childhood diarrhea, is dependent on activation of the extracellular viral particles by trypsin-like proteases in the host intestinal lumen. This step entails proteolytic cleavage of the VP4 spike protein into its mature products, VP8* and VP5*. Previous cryo-electron microscopy (cryo-EM) analysis of trypsin-activated particles showed well-resolved spikes, although no density was identified for the spikes in uncleaved particles; these data suggested that trypsin activation triggers important conformational changes that give rise to the rigid, entry-competent spike. The nature of these structural changes is not well understood, due to lack of data relative to the uncleaved spike structure. Here we used cryo-EM and cryo-electron tomography (cryo-ET) to characterize the structure of the uncleaved virion in two model rotavirus strains. Cryo-EM three-dimensional reconstruction of uncleaved virions showed spikes with a structure compatible with the atomic model of the cleaved spike, and indistinguishable from that of digested particles. Cryo-ET and subvolume average, combined with classification methods, resolved the presence of non-icosahedral structures, providing a model for the complete structure of the uncleaved spike. Despite the similar rigid structure observed for uncleaved and cleaved particles, trypsin activation is necessary for successful infection. These observations suggest that the spike precursor protein must be proteolytically processed, not to achieve a rigid conformation, but to allow the conformational changes that drive virus entry.

Partial Text

To initiate infection, viruses must overcome the complex membranous system that surrounds and resides within the cell. The ability of the virus to penetrate this barrier is one of the elements that define virulence and host range. Entry into the host cell is thus a key factor in viral infectivity, and a natural target for the design of efficient strategies against virus infections [1].

Recent reports suggest that the conformational changes undergone by the rotavirus spike protein VP4 are the main driving force behind membrane disruption and virus entry into the host cell [11], [17], [20]. For this process to be efficient, the rotavirus particle must be primed by proteolytic cleavage of VP4 into its mature products, VP8* and VP5*. Despite the availability of an atomic structure for the trypsinized rotavirus particle, our understanding of the molecular mechanisms that underlie the proteolytic enhancement of rotavirus infectivity has been hindered by the lack of a structure of the undigested spike. The aim of this study was to determine, using cryo-EM and cryo-ET, the structure of the uncleaved rotavirus to improve comprehension of the structural mechanisms underlying the proteolytic enhancement of rotavirus infectivity.




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