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Structural and Functional Features of the Reovirus ?1 Tail.

ABSTRACT: Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein ?1, which projects from the virion surface. The ?1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from ?-spiral repeats; and a globular head with receptor-binding properties. The ?1 tail is predicted to form an ?-helical coiled coil. Although ?1 undergoes a conformational change during cell entry, the nature of this change and its contributions to viral replication are unknown. Electron micrographs of ?1 molecules released from virions identified three regions of flexibility, including one at the midpoint of the molecule, that may be involved in its structural rearrangement. To enable a detailed understanding of essential ?1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) ?1 tail domains. Both molecules feature extended ?-helical coiled coils, with T1L ?1 harboring central chloride ions. Each molecule displays a discontinuity (stutter) within the coiled coil and an unexpectedly seamless transition to the body domain. The transition region features conserved interdomain interactions and appears rigid rather than highly flexible. Functional analyses of reoviruses containing engineered ?1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for ?1 folding and encapsidation, whereas central chloride ion coordination and the stutter are dispensable for efficient replication. Together, these findings enable modeling of full-length reovirus ?1 and provide insight into the stabilization of a multidomain virus attachment protein.IMPORTANCE While it is established that different conformational states of attachment proteins of enveloped viruses mediate receptor binding and membrane fusion, less is understood about how such proteins mediate attachment and entry of nonenveloped viruses. The filamentous reovirus attachment protein ?1 binds cellular receptors; contains regions of predicted flexibility, including one at the fiber midpoint; and undergoes a conformational change during cell entry. Neither the nature of the structural change nor its contribution to viral infection is understood. We determined crystal structures of large ?1 fragments for two different reovirus serotypes. We observed an unexpectedly tight transition between two domains spanning the fiber midpoint, which allows for little flexibility. Studies of reoviruses with engineered changes near the ?1 midpoint suggest that the stabilization of this region is critical for function. Together with a previously determined structure, we now have a complete model of the full-length, elongated reovirus ?1 attachment protein.

SUBMITTER: Dietrich MH

PROVIDER: S-EPMC6026731 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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Structural and Functional Features of the Reovirus σ1 Tail.

Dietrich Melanie H MH Ogden Kristen M KM Long Jacob M JM Ebenhoch Rebecca R Thor Alexandra A Dermody Terence S TS Stehle Thilo T

Journal of virology 20180629 14

Mammalian orthoreovirus attachment to target cells is mediated by the outer capsid protein σ1, which projects from the virion surface. The σ1 protein is a homotrimer consisting of a filamentous tail, which is partly inserted into the virion; a body domain constructed from β-spiral repeats; and a globular head with receptor-binding properties. The σ1 tail is predicted to form an α-helical coiled coil. Although σ1 undergoes a conformational change during cell entry, the nature of this change and i ...[more]

PMID: 29695426

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Project description:Reovirus attachment protein σ1 engages glycan receptors and junctional adhesion molecule-A (JAM-A) and is thought to undergo a conformational change during the proteolytic disassembly of virions to infectious subvirion particles (ISVPs) that accompanies cell entry. The σ1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target σ1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate σ1 protein within the head domain via epitopes distinct from the JAM-A-binding site. Surface plasmon resonance and cell-binding assays indicated that both antibodies likely interfere with JAM-A engagement by steric hindrance. To define the interplay between the carbohydrate receptor and antibody binding, we conducted hemagglutination inhibition assays using virions and ISVPs. The glycan-binding site of T1 σ1 is located in the head domain and is partly occluded by the bound Fab in the crystal structure. The T1-specific antibody inhibited hemagglutination by virions and ISVPs, probably via direct interference with glycan engagement. In contrast to T1 σ1, the carbohydrate-binding site of T3 σ1 is located in the tail domain, distal to the antibody epitope. The T3-specific antibody inhibited hemagglutination by T3 virions but not ISVPs, indicating that the antibody- and glycan-binding sites in σ1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of σ1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus. Virus attachment proteins mediate binding to host cell receptors, serve critical functions in cell and tissue tropism, and are often targeted by the neutralizing antibody response. The structural investigation of antibody-antigen complexes can provide valuable information for understanding the molecular basis of virus neutralization. Studies with enveloped viruses, such as HIV and influenza virus, have helped to define sites of vulnerability and guide vaccination strategies. By comparison, less is known about antibody binding to nonenveloped viruses. Here, we structurally investigated two neutralizing antibodies that bind the attachment protein σ1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for σ1, and the effect of the antibodies on reovirus receptor engagement. Our analysis defines reovirus interactions with two neutralizing antibodies, allows us to propose a mechanism by which they block virus infection, and provides evidence for a conformational change in the σ1 protein during viral cell entry.

Project description:Structural metastability of viral capsids is pivotal for viruses to survive in harsh environments and to undergo timely conformational changes required for cell entry. Mammalian orthoreovirus (reovirus) is a model to study capsid metastability. Following initial disassembly of the reovirus particle mediated by proteases, a metastable intermediate called the infectious subvirion particle (ISVP) is generated. Using a σ1 monoreassortant virus, we recently showed that σ1 properties affect its encapsidation on particles and the metastability of ISVPs. How metastability is impacted by σ1 and whether the lower encapsidation level of σ1 is connected to this property is unknown. To define a correlation between encapsidation of σ1 and ISVP stability, we generated mutant viruses with single amino acid polymorphisms in σ1 or those that contain chimeric σ1 molecules composed of σ1 portions from type 1 and type 3 reovirus strains. We found that under most conditions where σ1 encapsidation on the particle was lower, ISVPs displayed lower stability. Characterization of mutant viruses selected for enhanced stability via a forward genetic approach also revealed that in some cases, σ1 properties influence stability without influencing σ1 encapsidation. These data indicate that σ1 can also influence ISVP stability independent of its level of incorporation. Together, our work reveals an underappreciated effect of the σ1 attachment protein on the properties of the reovirus capsid. IMPORTANCE Reovirus particles are comprised of eight proteins. Among them, the reovirus σ1 protein functions engages cellular receptors. σ1 also influences the stability of an entry intermediate called ISVP. Here, we sought to define the basis of the link between σ1 properties and stability of ISVPs. Using variety of mutant strains, we determined that when virus preparations contain particles with a high amount of encapsidated σ1, ISVP stability is higher. Additionally, we identified portions of σ1 that impact its encapsidation and consequently the stability of ISVPs. We also determined that in some cases, σ1 properties alter stability of ISVPs without affecting encapsidation. This work highlights that proteins of these complex particles are arranged in an intricate, interconnected manner such that changing the properties of these proteins has a profound impact on the remainder of the particle.

Project description:Mammalian reovirus (MRV) strain type 3 Dearing (T3D) is a naturally occurring oncolytic virus that has been developed as a potential cancer therapeutic. However, MRV treatment cannot be applied to cancer cells expressing low levels of junctional adhesion molecule A (JAM-A), which is the entry receptor of MRV. In this study, we developed a reverse genetics system for MRV strain T3D-L, which showed high oncolytic potency. To modify the cell tropism of MRV, an arginine-glycine-aspartic acid (RGD) peptide with an affinity to integrin was inserted at the C terminus or loop structures of the viral cell attachment protein σ1. The recombinant RGD σ1-modified viruses induced remarkable cell lysis in human cancer cell lines with marginal JAM-A expression and in JAM-A knockout cancer cell lines generated by a CRISPR/Cas9 system. Pretreatment of cells with anti-integrin antibody decreased cell death caused by the RGD σ1-modified virus, suggesting the infection to the cells was via a specific interaction with integrin αV. By using mouse models, we assessed virulence of the RGD σ1-modified viruses in vivo This system will open new avenues for the use of genetically modified oncolytic MRV for use as a cancer therapy.IMPORTANCE Oncolytic viruses kill tumors without affecting normal cells. A variety of oncolytic viruses are used as cancer therapeutics. Mammalian reovirus (MRV), which belongs to the genus Orthoreovirus, family Reoviridae, is one such natural oncolytic virus. The anticancer effects of MRV are being evaluated in clinical trials. Unlike other oncolytic viruses, MRV has not been genetically modified for use as a cancer therapeutic in clinical trials. Here, we used a reverse genetic approach to introduce an integrin-affinity peptide sequence into the MRV cell attachment protein σ1 to alter the natural tropism of the virus. The recombinant viruses were able to infect cancer cell lines expressing very low levels of the MRV entry receptor, junctional adhesion molecule A (JAM-A), and cause tumor cell death while maintaining its original tropism via JAM-A. This is a novel report of a genetically modified oncolytic MRV by introducing a peptide sequence into σ1.

Project description:The reovirus attachment protein σ1 mediates cell attachment and receptor binding and is thought to undergo conformational changes during viral disassembly. σ1 is a trimeric filamentous protein with an α-helical coiled-coil tail, a triple-β-spiral body, and a globular head. At the trimer interface, the head domain features an unusual and conserved aspartic acid cluster, which forms the only significant intratrimer interactions in the head and must be protonated to allow trimer formation. To define the role of pH on σ1 stability and conformation, we tested its domains over a wide range of pH values. We show that all domains of σ1 are remarkably thermostable, even at the low pH of the stomach. We determined the optimal pH for stability to be between pHs 5 and 6, a value close to the pH of the endosome and of the jejunum. The σ1 head is stable at acidic and neutral pH but detrimerizes at basic pH. When Asp345 in the aspartic acid cluster is mutated to asparagine (D345N), the σ1 head loses stability at low pH and is more prone to detrimerize. Although the D345N mutation does not affect σ1 binding affinity for the JAM-A receptor, the overall binding stoichiometry is reduced by one-third. The additional replacement of the neighboring His349 with alanine disrupts inner trimer surface interactions, leading to a less thermostable and monomeric σ1 D345N head that fails to bind the JAM-A receptor. When the body is expressed together with the head domain, the thermostability is restored and the stoichiometry of the binding to JAM-A receptor is preserved. Our results confirm a fundamental role of the aspartic acid cluster as a pH-dependent molecular switch controlling trimerization and enhancing thermostability of σ1, which represent essential requirements to accomplish reovirus infection and entry and might be common mechanisms among other enteric viruses. IMPORTANCE Enteric viruses withstand the highly acidic environment of the stomach during transmission, and many of them use low pH as a trigger for conformational changes associated with entry. For many nonenveloped viruses, the structural basis of these effects is not clear. We have investigated the stability of the reovirus attachment protein σ1 over a range of pHs and find it to be remarkably thermostable, especially at low pH. We identify a role for the aspartic acid cluster in maintaining σ1 thermostability, trimeric organization, and binding to JAM-A receptor especially at the gastric pH reovirus has to withstand while passing the stomach. The understanding of monomer-trimer dynamics within σ1 enhances our knowledge of reovirus entry and has implications for stability and transmission of other enteric viruses.

Dataset Information

Structural and Functional Features of the Reovirus ?1 Tail.

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Structural and Functional Features of the Reovirus σ1 Tail.

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