The Nogo Receptor NgR1 Mediates Infection by Mammalian Reovirus

Reovirus attachment protein 1 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 1 protein is also the primary target of neutralizing antibodies. Here, we present a structural and functional characterization of two neutralizing antibodies that target 1 of serotype 1 (T1) and serotype 3 (T3) reoviruses. The crystal structures revealed that each antibody engages its cognate 1 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 1 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 1, the carbohydrate-binding site of T3 1 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 1 are in closer spatial proximity on virions than on ISVPs. Our results provide direct evidence for a structural rearrangement of 1 during virion-to-ISVP conversion and contribute new information about the mechanisms of antibody-mediated neutralization of reovirus.IMPORTANCE 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 1 of reovirus. Furthermore, we characterized the neutralization efficiency, the binding affinity for 1, 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 1 protein during viral cell entry.

Section: Discussionmentioning

confidence: 99%

Structural Insights into Reovirus σ1 Interactions with Two Neutralizing Antibodies

Dietrich

Ogden

Katen

et al. 2017

“…These peptides are thought to facilitate core delivery by generating size-selective pores within the endosomal membrane (39,41,43). Following attachment to host cell receptors (13)(14)(15)(16)(17)(18), reovirus is internalized by receptor-mediated endocytosis (20)(21)(22)(23)(24). Only particles that traffic through the early and late endosomes are capable of establishing a productive infection (53).…”

Section: Discussionmentioning

confidence: 99%

“…Following attachment to proteinaceous or carbohydrate receptors, such as junctional adhesion molecule A, Ngr1, or glycans (13)(14)(15)(16)(17)(18)(19), virions are internalized by receptor-mediated endocytosis (20)(21)(22)(23)(24). Within the endosome, acid-dependent cathepsin proteases degrade the 3 outer capsid protein (24)(25)(26)(27)(28)(29)(30).…”

mentioning

confidence: 99%

The Loop Formed by Residues 340 to 343 of Reovirus μ1 Controls Entry-Related Conformational Changes

Snyder

Danthi

2017

Reovirus particles are covered with 200 1/3 heterohexamers. Following attachment to cell surface receptors, reovirus is internalized by receptor-mediated endocytosis. Within the endosome, particles undergo a series of stepwise disassembly events. First, the 3 protector protein is degraded by cellular proteases to generate infectious subviral particles (ISVPs). Second, the 1 protein rearranges into a protease-sensitive conformation to generate ISVP*s and releases two virus-encoded peptides, 1N and ⌽. The released peptides promote delivery of the genome-containing core by perforating the endosomal membrane. Thus, to establish a productive infection, virions must be stable in the environment but flexible to disassemble in response to the appropriate cellular cue. The reovirus outer capsid is stabilized by 1 intratrimer, intertrimer, and trimercore interactions. As a consequence of ISVP-to-ISVP* conversion, neighboring 1 trimers unwind and separate. Located within the 1 jelly roll ␤ barrel domain, which is a known regulator of ISVP* formation, residues 340 to 343 form a loop and have been proposed to facilitate viral entry. To test this idea, we generated recombinant reoviruses that encoded deletions within this loop (Δ341 and Δ342). Both deletions destabilized the outer capsid. Notably, Δ342 impaired the viral life cycle; however, replicative fitness was restored by an additional change (V403A) within the 1 jelly roll ␤ barrel domain. In the Δ341 and Δ342 backgrounds, V403A also rescued defects in ISVP-to-ISVP* conversion. Together, these findings reveal a new region that regulates reovirus disassembly and how perturbing a metastable capsid can compromise replicative fitness.IMPORTANCE Capsids of nonenveloped viruses are composed of protein complexes that encapsulate, or form a shell around, nucleic acid. The protein-protein interactions that form this shell must be stable to protect the viral genome but also sufficiently flexible to disassemble during cell entry. Thus, capsids adopt conformations that undergo rapid disassembly in response to a specific cellular cue. In this work, we identify a new region within the mammalian orthoreovirus outer capsid that regulates particle stability. Amino acid deletions that destabilize this region impair the viral replication cycle. Nonetheless, replicative fitness is restored by a compensatory mutation that restores particle stability. Together, this work demonstrates the critical balance between assembling virions that are stable and maintaining conformational flexibility. Any factor that perturbs this balance has the potential to block a productive infection.KEYWORDS capsid, conformational change, metastable, nonenveloped virus, reovirus, thermostability C apsids of nonenveloped and enveloped viruses serve at least two antagonistic functions. First, the protein-protein, protein-nucleic acid, and/or protein-lipid interactions that stabilize the capsid must be sufficiently strong to allow particle assembly and stability within the environment. This protection functio...

“…Whereas serotype 1 (T1) 1 engages GM2, T3 1 engages glycans that terminate in sialic acid (11)(12)(13)(14). Two other cell surface-localized host molecules, ␤1 integrin(s) and Ngr1, have also been implicated in facilitating reovirus entry and infection (15,16). Whether ␤1 integrin interacts with viral components is not known.…”

mentioning

confidence: 99%

“…Whether ␤1 integrin interacts with viral components is not known. Though Ngr1 has been demonstrated to interact directly with virus particles (16), viral structures or proteins that participate in the interaction with Ngr1 remain to be identified.…”

mentioning

confidence: 99%

Reovirus μ1 Protein Affects Infectivity by Altering Virus-Receptor Interactions

Thete

Snyder

Mainou

et al. 2016

Proteins that form the reovirus outer capsid play an active role in the entry of reovirus into host cells. Among these, the 1 protein mediates attachment of reovirus particles to host cells via interaction with cell surface glycans or the proteinaceous receptor junctional adhesion molecule A (JAM-A). The 1 protein functions to penetrate the host cell membrane to allow delivery of the genome-containing viral core particle into the cytoplasm to initiate viral replication. We demonstrate that a reassortant virus that expresses the M2 gene-encoded 1 protein derived from prototype strain T3D in an otherwise prototype T1L background (T1L/T3DM2) infects cells more efficiently than parental T1L. Unexpectedly, the enhancement in infectivity of T1L/T3DM2 is due to its capacity to attach to cells more efficiently. We present genetic data implicating the central region of 1 in altering the cell attachment property of reovirus. Our data indicate that the T3D 1-mediated enhancement in infectivity of T1L is dependent on the function of 1 and requires the expression of JAM-A. We also demonstrate that T1L/T3DM2 utilizes JAM-A more efficiently than T1L. These studies revealed a previously unknown relationship between two nonadjacent reovirus outer capsid proteins, 1 and 1. IMPORTANCEHow reovirus attaches to host cells has been extensively characterized. Attachment of reovirus to host cells is mediated by the 1 protein, and properties of 1 influence the capacity of reovirus to target specific host tissues and produce disease. Here, we present new evidence indicating that the cell attachment properties of 1 are influenced by the nature of 1, a capsid protein that does not physically interact with 1. These studies could explain the previously described role for 1 in influencing reovirus pathogenesis. These studies are also of broader significance because they highlight an example of how genetic reassortment between virus strains could produce phenotypes that are distinct from those of either parent.A ttachment of virus is the first step in the infection of host cells. Cell attachment occurs via interactions of viral attachment factors with host cell receptors. For enveloped viruses, viral glycoproteins embedded in the lipid membrane serve as attachment factors (1). For nonenveloped viruses, specific structural features on the capsid or sequences within the exposed portion of the viral structural proteins bind host receptors (1). Mutations within the receptor-binding site can alter the efficiency with which virus attaches to host cells and consequently modulate the capacity of the virus to establish infection. In viral systems where capsids are formed from multiple structural proteins, these proteins fit together in a precise geometric arrangement. Thus, changes to the properties of one capsid protein can influence the function of other capsid proteins. In this report, we highlight one such example by demonstrating a previously unknown functional relationship between two nonadjacent viral capsid proteins of mammalian orthoreo...