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New insights into rotavirus entry machinery: stabilization of rotavirus spike conformation is independent of trypsin cleavage.

ABSTRACT: 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.

SUBMITTER: Rodriguez JM

PROVIDER: S-EPMC4038622 | biostudies-literature | 2014 May

REPOSITORIES: biostudies-literature

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New insights into rotavirus entry machinery: stabilization of rotavirus spike conformation is independent of trypsin cleavage.

Rodríguez Javier M JM Chichón Francisco J FJ Martín-Forero Esther E González-Camacho Fernando F Carrascosa José L JL Castón José R JR Luque Daniel D

PLoS pathogens 20140529 5

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 ...[more]

PMID: 24873828

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Project description:Because the rotavirus spike protein VP4 contains conserved Cys residues at positions 216, 318, 380, and 774 and, for many animal rotaviruses, also at position 203, we sought to determine whether disulfide bonds were structural elements of VP4. Electrophoretic analysis of untreated and trypsin-treated rhesus rotavirus (RRV) and simain rotavirus SA11 in the presence and absence of the reducing agent dithioerythritol revealed that VP4 and its cleavage fragments VP5* and VP8* possessed intrachain disulfide bonds. Given that the VP8* fragments of RRV and SA11 contain only two Cys residues, those at positions 203 and 216, these data indicated that these two residues were covalently linked. Electrophoretic examination of truncated species of VP4 and VP4 containing Cys-->Ser mutations synthesized in reticulocyte lysates provided additional evidence that Cys-203 and Cys-216 in VP8* of RRV were linked by a disulfide bridge. VP5* expressed in vitro was able to form a disulfide bond analogous to that in the VP5* fragment of trypsin-treated RRV. Analysis of a Cys-774-->Ser mutant of VP5* showed that, while it was able to form a disulfide bond, a Cys-318-->Ser mutant of VP5* was not. These results indicated that the VP4 component of all rotaviruses, except B223, contains a disulfide bond that links Cys-318 and Cys-380 in the VP5* region of the protein. This bond is located between the trypsin cleavage site and the putative fusion domain of VP4. Because human rotaviruses lack Cys-203 and, hence, unlike many animal rotaviruses cannot possess a disulfide bond in VP8*, it is apparent that VP4 is structurally variable in nature, with human rotaviruses generally containing one disulfide linkage and animal rotaviruses generally containing two such linkages. Considered with the results of anti-VP4 antibody mapping studies, the data suggest that the disulfide bond in VP5* exists within the 2G4 epitope and may be located at the distal end of the VP4 spike on rotavirus particles.

Dataset Information

New insights into rotavirus entry machinery: stabilization of rotavirus spike conformation is independent of trypsin cleavage.

Publications

New insights into rotavirus entry machinery: stabilization of rotavirus spike conformation is independent of trypsin cleavage.

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