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

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.

轮状病毒（rotavirus）是引发儿童腹泻的主要病原体，其感染性依赖于宿主肠道腔内胰蛋白酶样蛋白酶（trypsin-like proteases）对细胞外病毒粒子的激活。该激活过程需将VP4刺突蛋白酶解切割为成熟产物VP8*与VP5*。此前针对经胰蛋白酶激活的病毒粒子所开展的冷冻电子显微镜（cryo-electron microscopy, cryo-EM）分析显示，刺突结构分辨率良好，但未在未切割的病毒粒子中观察到刺突的密度信号；上述数据提示，胰蛋白酶激活可引发关键构象变化，进而形成具有侵染能力的刚性刺突。由于缺乏未切割刺突的结构数据，此类构象变化的本质尚未得到充分阐明。本研究借助冷冻电子显微镜（cryo-EM）与冷冻电子断层扫描（cryo-electron tomography, cryo-ET），对两种模式轮状病毒毒株的未切割病毒体结构进行了表征。对未切割病毒体的冷冻电镜三维重构结果显示，其刺突结构与切割后刺突的原子模型兼容，且与经酶解处理的粒子刺突结构无明显差异。结合分类方法的冷冻电镜断层扫描与亚体积平均分析，明确了非二十面体结构的存在，进而构建了未切割刺突的完整结构模型。尽管未切割与切割后的病毒粒子刺突均呈现相似的刚性结构，但胰蛋白酶激活仍是成功侵染的必要条件。上述观测结果表明，刺突前体蛋白需经酶解加工，其目的并非形成刚性构象，而是为驱动病毒侵染的构象变化提供基础。