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Rotavirus live-attenuated vaccines, both mono- and pentavalent, generate broadly heterotypic protection. B-cells isolated from adults encode neutralizing antibodies, some with affinity for VP5*, that afford broad protection in mice. We have mapped the epitope of one such antibody by determining the high-resolution cryo-EM structure of its antigen-binding fragment (Fab) bound to the virion of a candidate vaccine strain, CDC-9. The Fab contacts both the distal end of a VP5* β-barrel domain and the two VP8* lectin-like domains at the tip of a projecting spike. Its interactions with VP8* do not impinge on the likely receptor-binding site, suggesting that the mechanism of neutralization is at a step subsequent to initial attachment. We also examined structures of CDC-9 virions from two different stages of serial passaging. Nearly all the VP4 (cleaved to VP8*/VP5*) spikes on particles from the earlier passage (wild-type isolate) had transitioned from the "upright" conformation present on fully infectious virions to the "reversed" conformation that is probably the end state of membrane insertion, unable to mediate penetration, consistent with the very low in vitro infectivity of the wild-type isolate. About half the VP4 spikes were upright on particles from the later passage, which had recovered substantial in vitro infectivity but had acquired an attenuated phenotype in neonatal rats. A mutation in VP4 that occurred during passaging appears to stabilize the interface at the apex of the spike and could account for the greater stability of the upright spikes on the late-passage, attenuated isolate. IMPORTANCE Rotavirus live-attenuated vaccines generate broadly heterotypic protection, and B-cells isolated from adults encode antibodies that are broadly protective in mice. Determining the structural and mechanistic basis of broad protection can contribute to understanding the current limitations of vaccine efficacy in developing countries. The structure of an attenuated human rotavirus isolate (CDC-9) bound with the Fab fragment of a broadly heterotypic protective antibody shows that protection is probably due to inhibition of the conformational transition in the viral spike protein (VP4) critical for viral penetration, rather than to inhibition of receptor binding. A comparison of structures of CDC-9 virus particles at two stages of serial passaging supports a proposed mechanism for initial steps in rotavirus membrane penetration.

A non-enveloped virus requires a membrane lesion to deliver its genome into a target cell 1 . 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.

Rotaviruses, like other non-enveloped, double-strand RNA viruses, package an RNA-dependent RNA polymerase (RdRp) with each duplex of their segmented genomes. Rotavirus cell entry results in loss of an outer protein layer and delivery into the cytosol of an intact, inner capsid particle (the "double-layer particle," or DLP). The RdRp, designated VP1, is active inside the DLP; each VP1 achieves many rounds of mRNA transcription from its associated genome segment. Previous work has shown that one VP1 molecule lies close to each 5-fold axis of the icosahedrally symmetric DLP, just beneath the inner surface of its protein shell, embedded in tightly packed RNA. We have determined a high-resolution structure for the rotavirus VP1 RdRp in situ, by local reconstruction of density around individual 5-fold positions. We have analyzed intact virions ("triple-layer particles"), non-transcribing DLPs and transcribing DLPs. Outer layer dissociation enables the DLP to synthesize RNA, in vitro as well as in vivo, but appears not to induce any detectable structural change in the RdRp. Addition of NTPs, Mg 2+ , and S-adenosylmethionine, which allows active transcription, results in conformational rearrangements, in both VP1 and the DLP capsid shell protein, that allow a transcript to exit the polymerase and the particle. The position of VP1 (among the five symmetrically related alternatives) at one vertex does not correlate with its position at other vertices. This stochastic distribution of site occupancies limits long-range order in the 11-segment, double-strand RNA genome., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Bound calcium ions stabilize many nonenveloped virions. Loss of Ca 2+ from these particles appears to be a regulated part of entry or uncoating. The outer layer of an infectious rotavirus triple-layered particle (TLP) comprises a membrane-interacting protein (VP4) anchored by a Ca 2+ -stabilized protein (VP7). Membrane-coupled conformational changes in VP4 (cleaved to VP8* and VP5*) and dissociation of VP4 and VP7 accompany penetration of the double-layered inner capsid particle (DLP) into the cytosol. Removal of Ca 2+ in vitro strips away both outer layer proteins; we and others have postulated that the loss of Ca 2+ triggers molecular events in viral penetration. We have now investigated, with the aid of a fluorescent Ca 2+ sensor, the timing of Ca 2+ loss from entering virions with respect to the dissociation of VP4 and VP7. In live-cell imaging experiments, distinct fluorescent markers on the DLP and on VP7 report on outer layer dissociation and DLP release. The Ca 2+ sensor, placed on VP5*, monitors the Ca 2+ concentration within the membrane-bound vesicle enclosing the entering particle. Slow (1-min duration) loss of Ca 2+ precedes the onset of VP7 dissociation by about 2 min and DLP release by about 7 min. Coupled with our previous results showing that VP7 loss follows tight binding to the cell surface by about 5 min, these data indicate that Ca 2+ loss begins as soon as the particle has become fully engulfed within the uptake vesicle. We discuss the implications of these findings for the molecular mechanism of membrane disruption during viral entry. IMPORTANCE Nonenveloped viruses penetrate into the cytosol of the cells that they infect by disrupting the membrane of an intracellular compartment. The molecular mechanisms of membrane disruption remain largely undefined. Functional reconstitution of infectious rotavirus particles (TLPs) from RNA-containing core particles (DLPs) and the outer layer proteins that deliver them into a cell makes these important pediatric pathogens particularly good models for studying nonenveloped virus entry. We report here how the use of a fluorescent Ca 2+ sensor, covalently linked to one of the viral proteins, allows us to establish, using live-cell imaging, the timing of Ca 2+ loss from an entering particle and other molecular events in the entry pathway. Specific Ca 2+ binding stabilizes many other viruses of eukaryotes, and Ca 2+ loss appears to be a trigger for steps in penetration or uncoating. The experimental design that we describe may be useful for studying entry of other viral pathogens., (Copyright © 2018 Salgado et al.)

Infectious rotavirus particles are triple-layered, icosahedral assemblies. The outer layer proteins, VP4 (cleaved to VP8* and VP5*) and VP7, surround a transcriptionally competent, double-layer particle (DLP), which they deliver into the cytosol. During entry of rhesus rotavirus, VP8* interacts with cell surface gangliosides, allowing engulfment into a membrane vesicle by a clathrin-independent process. Escape into the cytosol and outer-layer shedding depend on interaction of a hydrophobic surface on VP5* with the membrane bilayer and on a large-scale conformational change. We report here experiments that detect the fate of released DLPs and their efficiency in initiating RNA synthesis. By replacing the outer layer with fluorescently tagged, recombinant proteins and also tagging the DLP, we distinguished particles that have lost their outer layer and entered the cytosol (uncoated) from those still within membrane vesicles. We used fluorescent in situ hybridization with probes for nascent transcripts to determine how soon after uncoating transcription began and what fraction of the uncoated particles were active in initiating RNA synthesis. We detected RNA synthesis by uncoated particles as early as 15 min after adding virus. The uncoating efficiency was 20 to 50%; of the uncoated particles, about 10 to 15% synthesized detectable RNA. In the format of our experiments, about 10% of the added particles attached to the cell surface, giving an overall ratio of added particles to RNA-synthesizing particles of between 250:1 and 500:1, in good agreement with the ratio of particles to focus-forming units determined by infectivity assays. Thus, RNA synthesis by even a single, uncoated particle can initiate infection in a cell. IMPORTANCE The pathways by which a virus enters a cell transform its packaged genome into an active one. Contemporary fluorescence microscopy can detect individual virus particles as they enter cells, allowing us to map their multistep entry pathways. Rotaviruses, like most viruses that lack membranes of their own, disrupt or perforate the intracellular, membrane-enclosed compartment into which they become engulfed following attachment to a cell surface, in order to gain access to the cell interior. The properties of rotavirus particles make it possible to determine molecular mechanisms for these entry steps. In the work described here, we have asked the following question: what fraction of the rotavirus particles that penetrate into the cell make new viral RNA? We find that of the cell-attached particles, between 20 and 50% ultimately penetrate, and of these, about 10% make RNA. RNA synthesis by even a single virus particle can initiate a productive infection., (Copyright © 2017 Salgado et al.)

Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼ 700 Å inner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration.

Here, we report that the approach of metal-templated ligand synthesis can be applied to construct a dimeric protein assembly ((BMOE)RIDC1(2)), which is stabilized by noncovalent interactions and flexible covalent cross-linkers around the Zn templates. Despite its flexibility, (BMOE)RIDC1(2) selectively binds Zn(II) over other divalent metals and undergoes dimerization upon metal binding. Such simultaneous fulfillment of plasticity and selectivity is a hallmark of cellular signaling events that involve ligand/metal-induced protein dimerization., (© 2011 American Chemical Society)

Selective binding by metalloproteins to their cognate metal ions is essential to cellular survival. How proteins originally acquired the ability to selectively bind metals and evolved a diverse array of metal-centered functions despite the availability of only a few metal-coordinating functionalities remains an open question. Using a rational design approach (Metal-Templated Interface Redesign), we describe the transformation of a monomeric electron transfer protein, cytochrome cb(562), into a tetrameric assembly ((C96)RIDC-1(4)) that stably and selectively binds Zn(2+) and displays a metal-dependent conformational change reminiscent of a signaling protein. A thorough analysis of the metal binding properties of (C96)RIDC-1(4) reveals that it can also stably harbor other divalent metals with affinities that rival (Ni(2+)) or even exceed (Cu(2+)) those of Zn(2+) on a per site basis. Nevertheless, this analysis suggests that our templating strategy simultaneously introduces an increased bias toward binding a higher number of Zn(2+) ions (four high affinity sites) versus Cu(2+) or Ni(2+) (two high affinity sites), ultimately leading to the exclusive selectivity of (C96)RIDC-1(4) for Zn(2+) over those ions. More generally, our results indicate that an initial metal-driven nucleation event followed by the formation of a stable protein architecture around the metal provides a straightforward path for generating structural and functional diversity.

Proteins are nature's premier building blocks for constructing sophisticated nanoscale architectures that carry out complex tasks and chemical transformations. Some 70%-80% of all proteins are thought to be permanently oligomeric; that is, they are composed of multiple proteins that are held together in precise spatial organization through noncovalent interactions. Although it is of great fundamental interest to understand the physicochemical basis of protein self-assembly, the mastery of protein-protein interactions (PPIs) would also allow access to novel biomaterials with nature's favorite and most versatile building block. In this Account, we describe a new approach we have developed with this possibility in mind, metal-directed protein self-assembly (MDPSA), which utilizes the strength, directionality, and selectivity of metal-ligand interactions to control PPIs. At its core, MDPSA is inspired by supramolecular coordination chemistry, which exploits metal coordination for the self-assembly of small molecules into discrete, more-or-less predictable higher order structures. Proteins, however, are not exactly small molecules or simple metal ligands: they feature extensive, heterogeneous surfaces that can interact with each other and with metal ions in unpredictable ways. We begin by first describing the challenges of using entire proteins as molecular building blocks. We follow with an examination of our work on a model protein (cytochrome cb(562)), highlighting challenges toward establishing ground rules for MDPSA as well as progress in overcoming these challenges. Proteins are also nature's metal ligands of choice. In MDPSA, once metal ions guide proteins into forming large assemblies, they are by definition embedded within extensive interfaces formed between protein surfaces. These complex surfaces make an inorganic chemist's life somewhat difficult, yet they also provide a wide platform to modulate the metal coordination environment through distant, noncovalent interactions, exactly as natural metalloproteins and enzymes do. We describe our computational and experimental efforts toward restructuring the noncovalent interaction network formed between proteins surrounding the interfacial metal centers. This approach, of metal templating followed by the redesign of protein interfaces (metal-templated interface redesign, MeTIR), not only provides a route to engineer de novo PPIs and novel metal coordination environments but also suggests possible parallels with the evolution of metalloproteins.

The enzyme alpha-galactosidase (alpha-GAL, also known as alpha-GAL A; E.C. 3.2.1.22) is responsible for the breakdown of alpha-galactosides in the lysosome. Defects in human alpha-GAL lead to the development of Fabry disease, a lysosomal storage disorder characterized by the buildup of alpha-galactosylated substrates in the tissues. alpha-GAL is an active target of clinical research: there are currently two treatment options for Fabry disease, recombinant enzyme replacement therapy (approved in the United States in 2003) and pharmacological chaperone therapy (currently in clinical trials). Previously, we have reported the structure of human alpha-GAL, which revealed the overall structure of the enzyme and established the locations of hundreds of mutations that lead to the development of Fabry disease. Here, we describe the catalytic mechanism of the enzyme derived from x-ray crystal structures of each of the four stages of the double displacement reaction mechanism. Use of a difluoro-alpha-galactopyranoside allowed trapping of a covalent intermediate. The ensemble of structures reveals distortion of the ligand into a (1)S(3) skew (or twist) boat conformation in the middle of the reaction cycle. The high resolution structures of each step in the catalytic cycle will allow for improved drug design efforts on alpha-GAL and other glycoside hydrolase family 27 enzymes by developing ligands that specifically target different states of the catalytic cycle. Additionally, the structures revealed a second ligand-binding site suitable for targeting by novel pharmacological chaperones.

Metal coordination is a key structural and functional component of a large fraction of proteins. Given this dual role we considered the possibility that metal coordination may have played a templating role in the early evolution of protein folds and complexes. We describe here a rational design approach, Metal Templated Interface Redesign (MeTIR), that mimics the time course of a hypothetical evolutionary pathway for the formation of stable protein assemblies through an initial metal coordination event. Using a folded monomeric protein, cytochrome cb(562), as a building block we show that its non-self-associating surface can be made self-associating through a minimal number of mutations that enable Zn coordination. The protein interfaces in the resulting Zn-directed, D(2)-symmetrical tetramer are subsequently redesigned, yielding unique protein architectures that self-assemble in the presence or absence of metals. Aside from its evolutionary implications, MeTIR provides a route to engineer de novo protein interfaces and metal coordination environments that can be tuned through the extensive noncovalent bonding interactions in these interfaces.

We describe the metal-dependent self-assembly of symmetrical protein homooligomers from protein building blocks that feature appropriately engineered metal-chelating motifs on their surfaces. Crystallographic studies indicate that the same four-helix-bundle protein construct, MBPC-1, can self-assemble into C(2) and C(3) symmetrical assemblies dictated by Cu(II) and Ni(II) coordination, respectively. The symmetry inherent in metal coordination can thus be directly applied to biological self-assembly.

We have previously demonstrated that non-self-associating protein building blocks can oligomerize to form discrete supramolecular assemblies under the control of metal coordination. We show here that secondary interactions (salt bridges and hydrogen bonds) can be critical in guiding the metal-induced self-assembly of proteins. Crystallographic and hydrodynamic measurements on appropriately engineered cytochrome cb562 variants pinpoint the importance of a single salt-bridging arginine side chain in determining whether the protein monomers form a discrete Zn-induced tetrameric complex or heterogeneous aggregates. The combined ability to direct PPIs through metal coordination and secondary interactions should provide the specificity required for the construction of complex protein superstructures and the selective control of cellular processes that involve protein-protein association reactions.