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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 its contributions to viral replication are unknown. Electron micrographs of σ1 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 σ1 tail organization and properties, we determined high-resolution structures of the reovirus type 1 Lang (T1L) and type 3 Dearing (T3D) σ1 tail domains. Both molecules feature extended α-helical coiled coils, with T1L σ1 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 σ1 mutations suggest that conserved residues predicted to stabilize the coiled-coil-to-body junction are essential for σ1 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 σ1 and provide insight into the stabilization of a multidomain virus attachment protein. 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 σ1 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 σ1 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 σ1 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 σ1 attachment protein.
Copyright © 2018 American Society for Microbiology.
Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease. Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.
Copyright © 2018 American Society for Microbiology.
Neurotropic viruses initiate infection in peripheral tissues prior to entry into the central nervous system (CNS). However, mechanisms of dissemination are not completely understood. We used genetically marked viruses to compare dissemination of poliovirus, yellow fever virus 17D (YFV-17D), and reovirus type 3 Dearing in mice from a hind limb intramuscular inoculation site to the sciatic nerve, spinal cord, and brain. While YFV-17D likely entered the CNS via blood, poliovirus and reovirus likely entered the CNS by transport through the sciatic nerve to the spinal cord. We found that dissemination was inefficient in adult immune-competent mice for all three viruses, particularly reovirus. Dissemination of all viruses was more efficient in immune-deficient mice. Although poliovirus and reovirus both accessed the CNS by transit through the sciatic nerve, stimulation of neuronal transport by muscle damage enhanced dissemination only of poliovirus. Our results suggest that these viruses access the CNS using different pathways.
Copyright © 2015 Elsevier Inc. All rights reserved.
UNLABELLED - Mammalian orthoreoviruses (reoviruses) are nonenveloped double-stranded RNA viruses that infect most mammalian species, including humans. Reovirus binds to cell surface glycans, junctional adhesion molecule A (JAM-A), and the Nogo-1 receptor (depending on the cell type) and enters cells by receptor-mediated endocytosis. Within the endocytic compartment, reovirus undergoes stepwise disassembly, which is followed by release of the transcriptionally active viral core into the cytoplasm. In a small-molecule screen to identify host mediators of reovirus infection, we found that treatment of cells with 5-nonyloxytryptamine (5-NT), a prototype serotonin receptor agonist, diminished reovirus cytotoxicity. 5-NT also blocked reovirus infection. In contrast, treatment of cells with methiothepin mesylate, a serotonin antagonist, enhanced infection by reovirus. 5-NT did not alter cell surface expression of JAM-A or attachment of reovirus to cells. However, 5-NT altered the distribution of early endosomes with a concomitant impairment of reovirus transit to late endosomes and a delay in reovirus disassembly. Consistent with an inhibition of viral disassembly, 5-NT treatment did not alter infection by in vitro-generated infectious subvirion particles, which bind to JAM-A but bypass a requirement for proteolytic uncoating in endosomes to infect cells. We also found that treatment of cells with 5-NT decreased the infectivity of alphavirus chikungunya virus and coronavirus mouse hepatitis virus. These data suggest that serotonin receptor signaling influences cellular activities that regulate entry of diverse virus families and provides a new, potentially broad-spectrum target for antiviral drug development.
IMPORTANCE - Identification of well-characterized small molecules that modulate viral infection can accelerate development of antiviral therapeutics while also providing new tools to increase our understanding of the cellular processes that underlie virus-mediated cell injury. We conducted a small-molecule screen to identify compounds capable of inhibiting cytotoxicity caused by reovirus, a prototype double-stranded RNA virus. We found that 5-nonyloxytryptamine (5-NT) impairs reovirus infection by altering viral transport during cell entry. Remarkably, 5-NT also inhibits infection by an alphavirus and a coronavirus. The antiviral properties of 5-NT suggest that serotonin receptor signaling is an important regulator of infection by diverse virus families and illuminate a potential new drug target.
Copyright © 2015, American Society for Microbiology. All Rights Reserved.
UNLABELLED - Receptors expressed on the host cell surface adhere viruses to target cells and serve as determinants of viral tropism. Several viruses bind cell surface glycans to facilitate entry, but the contribution of specific glycan moieties to viral disease is incompletely understood. Reovirus provides a tractable experimental model for studies of viral neuropathogenesis. In newborn mice, serotype 1 (T1) reovirus causes hydrocephalus, whereas serotype 3 (T3) reovirus causes encephalitis. T1 and T3 reoviruses engage distinct glycans, suggesting that glycan-binding capacity contributes to these differences in pathogenesis. Using structure-guided mutagenesis, we engineered a mutant T1 reovirus incapable of binding the T1 reovirus-specific glycan receptor, GM2. The mutant virus induced substantially less hydrocephalus than wild-type virus, an effect phenocopied by wild-type virus infection of GM2-deficient mice. In comparison to wild-type virus, yields of mutant virus were diminished in cultured ependymal cells, the cell type that lines the brain ventricles. These findings suggest that GM2 engagement targets reovirus to ependymal cells in mice and illuminate the function of glycan engagement in reovirus serotype-dependent disease.
IMPORTANCE - Receptor utilization strongly influences viral disease, often dictating host range and target cell selection. Different reovirus serotypes bind to different glycans, but a precise function for these molecules in pathogenesis is unknown. We used type 1 (T1) reovirus deficient in binding the GM2 glycan and mice lacking GM2 to pinpoint a role for glycan engagement in hydrocephalus caused by T1 reovirus. This work indicates that engagement of a specific glycan can lead to infection of specific cells in the host and consequent disease at that site. Since reovirus is being developed as a vaccine vector and oncolytic agent, understanding reovirus-glycan interactions may allow manipulation of reovirus glycan-binding properties for therapeutic applications.
Copyright © 2015 Stencel-Baerenwald et al.
Neurotropic viruses, including mammalian reovirus, must disseminate from an initial site of replication to the central nervous system (CNS), often binding multiple receptors to facilitate systemic spread. Reovirus engages junctional adhesion molecule A (JAM-A) to disseminate hematogenously. However, JAM-A is dispensable for reovirus replication in the CNS. We demonstrate that reovirus binds Nogo receptor NgR1, a leucine-rich repeat protein expressed in the CNS, to infect neurons. Expression of NgR1 confers reovirus binding and infection of nonsusceptible cells. Incubating reovirus virions with soluble NgR1 neutralizes infectivity. Blocking NgR1 on transfected cells or primary cortical neurons abrogates reovirus infection. Concordantly, reovirus infection is ablated in primary cortical neurons derived from NgR1 null mice. Reovirus virions bind to soluble JAM-A and NgR1, while infectious disassembly intermediates (ISVPs) bind only to JAM-A. These results suggest that reovirus uses different capsid components to bind distinct cell-surface molecules, engaging independent receptors to facilitate spread and tropism.
Copyright © 2014 Elsevier Inc. All rights reserved.
UNLABELLED - Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. M cells have also been suggested as a portal for pathogen entry into the host. While virus particles have been observed in M cells, it is not known whether viruses use M cells to initiate a productive infection. Noroviruses (NoVs) are single-stranded RNA viruses that infect host organisms via the fecal-oral route. Murine NoV (MNV) infects intestinal macrophages and dendritic cells and provides a tractable experimental system for understanding how an enteric virus overcomes the intestinal epithelial barrier to infect underlying target cells. We found that replication of two divergent MNV strains was reduced in mice depleted of M cells. Reoviruses are double-stranded RNA viruses that infect hosts via respiratory or enteric routes. In contrast to MNV, reovirus infects enterocytes in the intestine. Despite differences in cell tropism, reovirus infection was also reduced in M cell-depleted mice. These data demonstrate that M cells are required for the pathogenesis of two unrelated enteric viruses that replicate in different cell types within the intestine.
IMPORTANCE - To successfully infect their hosts, pathogens that infect via the gastrointestinal tract must overcome the multilayered system of host defenses. Microfold (M) cells are specialized intestinal epithelial cells that internalize particulate antigens and aid in the establishment of immune responses to enteric pathogens. Virus particles have been observed within M cells. However, it is not known whether viruses use M cells to initiate a productive infection. To address this question, we use MNV and reovirus, two enteric viruses that replicate in different cell types in the intestine, intestinal epithelial cells for reovirus and intestinal mononuclear phagocytes for MNV. Interestingly, MNV- and reovirus-infected mice depleted of M cells showed reduced viral loads in the intestine. Thus, our work demonstrates the importance of M cells in the pathogenesis of enteric viruses irrespective of the target cell type in which the virus replicates.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
Apoptosis is a type of controlled cell death that is essential for development and tissue homeostasis. It also serves as a robust host response against infection by many viruses. The capacity of neurotropic viruses to induce apoptosis strongly correlates with virulence. However, the precise function of apoptosis in viral infection is not well understood. Reovirus is a neurotropic virus that induces apoptosis in a variety of cell types, including central nervous system neurons, leading to fatal encephalitis in newborn mice. To determine the effect of apoptosis on reovirus replication in the host, we generated two otherwise isogenic viruses that differ in a single amino acid in viral capsid protein μ1 that segregates with apoptotic capacity. Apoptosis-proficient and apoptosis-deficient viruses were compared for replication, dissemination, tropism, and tissue injury in newborn mice and for the capacity to spread to uninfected littermates. Our results indicate that apoptotic capacity enhances reovirus replication in the brain and consequent neurovirulence but reduces transmission efficiency. The replication advantage of the apoptosis-proficient strain is limited to the brain and correlates with enhanced infectivity of neurons. These studies reveal a new cell type-specific determinant of reovirus virulence.
Reovirus nonstructural protein σ1s is implicated in cell cycle arrest at the G2/M boundary and induction of apoptosis. However, the contribution of σ1s to these effects in an otherwise isogenic viral background has not been defined. To evaluate the role of σ1s in cell cycle arrest and apoptosis, we used reverse genetics to generate a σ1s-null reovirus. Following infection with wild-type virus, we observed an increase in the percentage of cells in G2/M, whereas the proportion of cells in G2/M following infection with the σ1s-null mutant was unaffected. Similarly, we found that the wild-type virus induced substantially greater levels of apoptosis than the σ1s-null mutant. These data indicate that σ1s is required for both reovirus-induced cell cycle arrest and apoptosis. To define sequences in σ1s that mediate these effects, we engineered viruses encoding C-terminal σ1s truncations by introducing stop codons in the σ1s open reading frame. We also generated viruses in which charged residues near the σ1s amino terminus were replaced individually or as a cluster with nonpolar residues. Analysis of these mutants revealed that amino acids 1 to 59 and the amino-terminal basic cluster are required for induction of both cell cycle arrest and apoptosis. Remarkably, viruses that fail to induce cell cycle arrest and apoptosis also are attenuated in vivo. Thus, identical sequences in σ1s are required for reovirus-induced cell cycle arrest, apoptosis, and pathogenesis. Collectively, these findings provide evidence that the σ1s-mediated properties are genetically linked and suggest that these effects are mechanistically related.
Mammalian orthoreoviruses (reoviruses) are members of the Reoviridae. Reoviruses contain 10 double-stranded (ds) RNA gene segments enclosed in two concentric protein shells, called outer capsid and core. These viruses serve as a versatile experimental system for studies of viral replication events at the virus-cell interface, including engagement of cell-surface receptors, internalization and disassembly, and activation of the innate immune response, including NF-κB-dependent cellular signaling pathways. Reoviruses also provide a model system for studies of virus-induced apoptosis and organ-specific disease in vivo.Reoviruses attach to host cells via the filamentous attachment protein, σ1. The σ1 protein of all reovirus serotypes engages junctional adhesion molecule-A (JAM-A), an integral component of intercellular tight junctions. The σ1 protein also binds to cell-surface carbohydrate, with the type of carbohydrate bound varying by serotype. Following attachment to JAM-A and carbohydrate, reovirus internalization is mediated by β1 integrins, most likely via clathrin-dependent endocytosis. In the endocytic compartment, reovirus outer-capsid protein σ3 is removed by acid-dependent cysteine proteases in most cell types. Removal of σ3 results in the exposure of a hydrophobic conformer of the viral membrane-penetration protein, μ1, which pierces the endosomal membrane and delivers transcriptionally active reovirus core particles into the cytoplasm.Reoviruses induce apoptosis in both cultured cells and infected mice. Perturbation of reovirus disassembly using inhibitors of endosomal acidification or protease activity abrogates apoptosis. The μ1-encoding M2 gene is genetically linked to strain-specific differences in apoptosis-inducing capacity, suggesting a function for μ1 in induction of death signaling. Reovirus disassembly leads to activation of transcription factor NF-κB, which modulates apoptotic signaling in numerous types of cells. Inhibition of NF-κB nuclear translocation using either pharmacologic agents or expression of transdominant forms of IκB blocks reovirus-induced apoptosis, suggesting an essential role for NF-κB activation in the death response. Multiple effector pathway s downstream of NF-κB-directed gene expression execute reovirus-induced cell death. This chapter will focus on the mechanisms by which reovirus attachment and disassembly activate NF-κB and stimulate the cellular proapoptotic machinery.