Molecular mechanisms of rhesus rotavirus entry

Abstract

For non-enveloped viruses, genome delivery into the cytosol must include a cellular membrane disruption step. Some non-enveloped viruses appear to create protein channels just large enough to secrete viral nucleic acids into the cytoplasm. Reovirus family members, however, transfer sub-viral particles 70 nm in diameter from endocytic compartments into the cytosol, indicating that substantially sized lesions in the membrane must be formed to allow entry. Rotaviruses, members of a genus within the reovirus family, rely on the two components of their outer protein layer, VP4 and VP7, to gain entry into a cell. The mechanism requires proteolytic cleavage of VP4 by trypsin into VP5* and VP8*. VP8* promotes attachment of virus particles to cell membranes by binding headgroups of glycosylated lipids. Membrane contacts appear to drive engulfment into endocytic vesicles, with loss of Ca2+ from the particle leading to further membrane disruption and virus escape into the cytosol. Ca2+ leakage and membrane disruption are thought to be promoted by VP5*. Previous structural and biochemical studies have shown that VP5* can undergo a large-scale conformational refolding and that a set of hydrophobic loops in some way links this conformational change with membrane disruption. In this study, I have used cryo-electron microscopy to show that the VP5* conformational change can occur spontaneously on the surface of the virion, transforming it from an upright, asymmetric trimer to a reversed symmetric trimer. The conformational change projects the so-called "foot domain" of VP5*, which in the upright conformation is anchored by VP7 onto the particle surface, outwards and potentially into the membrane to which the virion is bound. I have trapped a likely conformational intermediate, by creating a mutant VP4 (A567C-S590C VP4). The potential sequence of states (upright-intermediate-reversed) can occur without dissociation of VP4 from the virus particle. Using a combination of cryo-EM structure analysis, liposome disruption assays, and Ca2+ leakage assays, I show that the foot domain can integrate into lipid bilayers in the first step of the mechanism by which VP5* promotes membrane disruption.