A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell¹. For rotaviruses, membrane perforation is a principal function of the viral outer-layer protein, VP4^2,3. Here we describe the use of electron cryomicroscopy to determine how VP4 performs this function and show that when activated by cleavage to VP8* and VP5*, VP4 can rearrange on the virion surface from an ‘upright’ to a ‘reversed’ conformation. The reversed structure projects a previously buried ‘foot’ domain outwards into the membrane of the host cell to which the virion has attached. Electron cryotomograms of virus particles entering cells are consistent with this picture. Using a disulfide mutant of VP4, we have also stabilized a probable intermediate in the transition between the two conformations. Our results define molecular mechanisms for the first steps of the penetration of rotaviruses into the membranes of target cells and suggest similarities with mechanisms postulated for other viruses.

非包膜病毒需要膜病灶才能将其基因组传递到靶细胞^1中。对于轮状病毒，膜穿孔是病毒外层蛋白VP4 ^2,3的主要功能。在这里，我们描述了使用电子冷冻显微镜来确定VP4如何执行此功能，并显示当通过裂解为VP8 *和VP5 *激活时，VP4可以在病毒体表面上从“直立”重新排列为“反向”构象。反向结构将先前埋藏的“脚”结构域向外投射到病毒体已附着的宿主细胞的膜中。病毒颗粒进入细胞的电子冰冻图与该图一致。使用VP4的二硫键突变体，我们还在两个构象之间的过渡中稳定了可能的中间体。我们的结果确定了轮状病毒渗透到靶细胞膜的第一步的分子机制，并暗示了与其他病毒所假定的机制的相似性。