Functional refolding of the penetration protein on a non-enveloped virus

Abstract

Non-enveloped viruses must create a transient membrane lesion to initiate infection by transferring their genomes into a target cell 1 . Rotaviruses offer a particularly favorable opportunity to visualize the mechanism for subviral particle delivery, the principal function of their outer-layer protein, VP4 2 – 4 . We show here by electron cryomicroscopy (cryo-EM) that VP4, activated by cleavage to VP8* and VP5*, rearranges on the virion surface from an “upright” to a “reversed” conformation. The reversed structure projects an initially buried, “foot” domain outward into the host cell membrane to which the virion has attached. Analysis of cryo-tomograms of virus particles entering cells is consistent with this picture. We have stabilized with a disulfide mutant a likely intermediate in this transition. The results define molecular mechanisms for the first steps in viral membrane penetration and suggest similarities with mechanisms postulated for other viruses.

Rotavirus Interactions With Host Intestinal Epithelial Cells

“…As illustrated in Figure 1 , during RV release, RV particles can be assembled in five steps., (i) RV particles relocate from the endoplasmic reticulum (ER) to the apical plasma membrane bypassing the Golgi complex; (ii) RV NSP4 protein halts the early secretory pathway and arrests normal ER-to-Golgi membrane trafficking; (iii) VP4 and VP7 are detected in a filamentous array; (iv) VP4 present at the plasma membrane associated with microtubules; and (v) interaction of NSP4 and lipid membranes mimicking endocytosis. Most recently, Herrmann and colleagues using electron cryomicroscopy demonstrated the involvement of VP4 gene in the initial RV penetration into the membranes of target cells ( 41 ). They showed that when VP4 is cleaved into VP5* and VP8*, it transitions from an upright to a reversed conformation leading to membrane perforation and virion attachment.…”

Engineering a Vaccine Platform using Rotavirus A to Express SARS-CoV-2 Spike Epitopes

“…It is possible that reduced infectivity of VP4 mutants could be due to inefficient conformational transition of VP4 following proteolytic cleavage [1,23,87]. Following cell attachment, VP8* lectin domains at the tip of VP4 dissociate and expose the hydrophobic loops of the VP5* β-barrel domains [21,67,68]. This enables interaction of the VP5* hydrophobic loops with the lipid bilayer and perforation of the target membrane by the VP5* foot domain, leading to RV entry, analogous to refolding of influenza virus haemagglutinin during membrane fusion [67,68,[88][89][90][91][92][93].…”

Mechanisms of Cell Entry by dsRNA Viruses: Insights for Efficient Delivery of dsRNA and Tools for Improved RNAi-Based Pest Control

“…Rice of BTV of dipteran cells and plant reoviruses of hemipteran cells (see sections 7 and 8). Regardless of whether core particles are double-or singlelayered, the outer capsid layers of orthoreoviruses, rotaviruses and BTV contain the host receptor-binding proteins and the machinery for the penetration of the cellular membrane for their efficient delivery in the cytoplasm during their infection of mammalian cells (Herrmann et al, 2021;Roth et al, 2021). During this process, the outer layer proteins are discarded in the endosomes or at the cell surface while the core particles enter the cytoplasm as intact entities that initiate transcription without further disassembly (Lourenco and Roy, 2011;Patel and Roy, 2014).…”

“…In the processed spike protein VP4, the main function of VP8 ∗ consists of the attachment to the cell surface, while VP5 ∗ provides the means to perforate the cellular or vesicular membrane ( Herrmann et al, 2021 ). VP4 is a trimer but structurally rearranges on the surface of the virions in an asymmetric configuration that consists of foot, stalk, body and head regions ( Rodríguez et al, 2014 ).…”

“…During “loose” contacts with the cell membrane, the spike protein displays an extended asymmetric configuration in which the head and body are formed by VP8 ∗ and VP5 ∗ domains, respectively, of two VP4 spike proteins; the VP5 ∗ domain of the third VP4 constitutes the stalk through unique interactions with VP7; and the third VP8 ∗ domain is either lost or sequestered at the foot domain ( Rodríguez et al, 2014 ). After initial interactions of the glycan-interacting lectin domains of VP8 ∗ with the cell surface, VP4 undergoes a transition from an upright conformation to a so-called “reverse” formation: the third VP5 ∗ of the stalk flips outward and forms a trimer of β-barrel domains with the two VP5 ∗ domains of the body, and zippering of a three-strand α-helical coiled-coil at the center causes the unfolding and outward projection of the foot domain ( Herrmann et al, 2021 ). “Tight” contacts with the cell membrane may be triggered by lateral movements of the lectin domains that will expose the hydrophobic loops of the two β-barrel domains of VP5 ∗ in the body of the spike; after adopting the reverse conformation, hydrophobic regions of the protruded foot domain can insert in the membrane and likely cause larger scale disruptions (perforations) for delivery of the DLPs into the cytoplasm ( Herrmann et al, 2021 ).…”