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Transcripts encoding ADAR1, a double-stranded, RNA-specific adenosine deaminase involved in the adenosine-to-inosine (A-to-I) editing of mammalian RNAs, can be alternatively spliced to produce an interferon-inducible protein isoform (p150) that is up-regulated in both cell culture and in vivo model systems in response to pathogen or interferon stimulation. In contrast to other tissues, p150 is expressed at extremely low levels in the brain and it is unclear what role, if any, this isoform may play in the innate immune response of the central nervous system (CNS) or whether the extent of editing for RNA substrates critical for CNS function is affected by its induction. To investigate the expression of ADAR1 isoforms in response to viral infection and subsequent alterations in A-to-I editing profiles for endogenous ADAR targets, we used a neurotropic strain of reovirus to infect neonatal mice and quantify A-to-I editing in discrete brain regions using a multiplexed, high-throughput sequencing strategy. While intracranial injection of reovirus resulted in a widespread increase in the expression of ADAR1 (p150) in multiple brain regions and peripheral organs, significant changes in site-specific A-to-I conversion were quite limited, suggesting that steady-state levels of p150 expression are not a primary determinant for modulating the extent of editing for numerous ADAR targets in vivo.
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.
UNLABELLED - Most viruses that replicate in the cytoplasm of host cells form neo-organelles that serve as sites of viral genome replication and particle assembly. These highly specialized structures concentrate viral replication proteins and nucleic acids, prevent the activation of cell-intrinsic defenses, and coordinate the release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in the knowledge of how these organelles form and mediate their functions. Reoviruses are nonenveloped, double-stranded RNA (dsRNA) viruses that serve as tractable experimental models for studies of dsRNA virus replication and pathogenesis. Following reovirus entry into cells, replication occurs in large cytoplasmic structures termed inclusions that fill with progeny virions. Reovirus inclusions are nucleated by viral nonstructural proteins, which in turn recruit viral structural proteins for genome replication and particle assembly. Components of reovirus inclusions are poorly understood, but these structures are generally thought to be devoid of membranes. We used transmission electron microscopy and three-dimensional image reconstructions to visualize reovirus inclusions in infected cells. These studies revealed that reovirus inclusions form within a membranous network. Viral inclusions contain filled and empty viral particles and microtubules and appose mitochondria and rough endoplasmic reticulum (RER). Immunofluorescence confocal microscopy analysis demonstrated that markers of the ER and ER-Golgi intermediate compartment (ERGIC) codistribute with inclusions during infection, as does dsRNA. dsRNA colocalizes with the viral protein σNS and an ERGIC marker inside inclusions. These findings suggest that cell membranes within reovirus inclusions form a scaffold to coordinate viral replication and assembly.
IMPORTANCE - Viruses alter the architecture of host cells to form an intracellular environment conducive to viral replication. This step in viral infection requires the concerted action of viral and host components and is potentially vulnerable to pharmacological intervention. Reoviruses form large cytoplasmic replication sites called inclusions, which have been described as membrane-free structures. Despite the importance of inclusions in the reovirus replication cycle, little is known about their formation and composition. We used light and electron microscopy to demonstrate that reovirus inclusions are membrane-containing structures and that the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment interact closely with these viral organelles. These findings enhance our understanding of the cellular machinery usurped by viruses to form inclusion organelles and complete an infectious cycle. This information, in turn, may foster the development of antiviral drugs that impede this essential viral replication step.
Immunoreceptor tyrosine-based activation motifs (ITAMs) are signaling domains located within the cytoplasmic tails of many transmembrane receptors and associated adaptor proteins that mediate immune cell activation. ITAMs also have been identified in the cytoplasmic tails of some enveloped virus glycoproteins. Here, we identified ITAM sequences in three mammalian reovirus proteins: μ2, σ2, and λ2. We demonstrate for the first time that μ2 is phosphorylated, contains a functional ITAM, and activates NF-κB. Specifically, μ2 and μNS recruit the ITAM-signaling intermediate Syk to cytoplasmic viral factories and this recruitment requires the μ2 ITAM. Moreover, both the μ2 ITAM and Syk are required for maximal μ2 activation of NF-κB. A mutant virus lacking the μ2 ITAM activates NF-κB less efficiently and induces lower levels of the downstream antiviral cytokine beta interferon (IFN-β) than does wild-type virus despite similar replication. Notably, the consequences of these μ2 ITAM effects are cell type specific. In fibroblasts where NF-κB is required for reovirus-induced apoptosis, the μ2 ITAM is advantageous for viral spread and enhances viral fitness. Conversely, in cardiac myocytes where the IFN response is critical for antiviral protection and NF-κB is not required for apoptosis, the μ2 ITAM stimulates cellular defense mechanisms and diminishes viral fitness. Together, these results suggest that the cell type-specific effect of the μ2 ITAM on viral spread reflects the cell type-specific effects of NF-κB and IFN-β. This first demonstration of a functional ITAM in a nonenveloped virus presents a new mechanism for viral ITAM-mediated signaling with likely organ-specific consequences in the host.
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.
UNLABELLED - Mammalian reovirus binds to cell-surface glycans and junctional adhesion molecule A and enters cells by receptor-mediated endocytosis in a process dependent on β1 integrin. Within the endocytic compartment, reovirus undergoes stepwise disassembly, allowing release of the transcriptionally active viral core into the cytoplasm. To identify cellular mediators of reovirus infectivity, we screened a library of small-molecule inhibitors for the capacity to block virus-induced cytotoxicity. In this screen, reovirus-induced cell killing was dampened by several compounds known to impair microtubule dynamics. Microtubule inhibitors were assessed for blockade of various stages of the reovirus life cycle. While these drugs did not alter reovirus cell attachment or internalization, microtubule inhibitors diminished viral disassembly kinetics with a concomitant decrease in infectivity. Reovirus virions colocalize with microtubules and microtubule motor dynein 1 during cell entry, and depolymerization of microtubules results in intracellular aggregation of viral particles. These data indicate that functional microtubules are required for proper sorting of reovirus virions following internalization and point to a new drug target for pathogens that use the endocytic pathway to invade host cells.
IMPORTANCE - Screening libraries of well-characterized drugs for antiviral activity enables the rapid characterization of host processes required for viral infectivity and provides new therapeutic applications for established pharmaceuticals. Our finding that microtubule-inhibiting drugs impair reovirus infection identifies a new cell-based antiviral target.
Many viruses cause disease within an infected host after spread from an initial portal of entry to sites of secondary replication. Viruses can disseminate via the bloodstream or through nerves. Mammalian orthoreoviruses (reoviruses) are neurotropic viruses that use both bloodborne and neural pathways to spread systemically within their hosts to cause disease. Using a robust mouse model and a dynamic reverse genetics system, we have identified a viral receptor and a viral nonstructural protein that are essential for hematogenous reovirus dissemination. Junctional adhesion molecule-A (JAM-A) is a member of the immunoglobulin superfamily expressed in tight junctions and on hematopoietic cells that serves as a receptor for all reovirus serotypes. Expression of JAM-A is required for infection of endothelial cells and development of viremia in mice, suggesting that release of virus into the bloodstream from infected endothelial cells requires JAM-A. Nonstructural protein σ1s is implicated in cell cycle arrest and apoptosis in reovirus-infected cells but is completely dispensable for reovirus replication in cultured cells. Surprisingly, a recombinant σ1s-null reovirus strain fails to spread hematogenously in infected mice, suggesting that σ1s facilitates apoptosis of reovirus-infected intestinal epithelial cells. It is possible that apoptotic bodies formed as a consequence of σ1s expression lead to reovirus uptake by dendritic cells for subsequent delivery to the mesenteric lymph node and the blood. Thus, both host and viral factors are required for efficient hematogenous dissemination of reovirus. Understanding mechanisms of reovirus bloodborne spread may shed light on how microbial pathogens invade the bloodstream to disseminate and cause disease in infected hosts.
Copyright © 2013 Elsevier Inc. All rights reserved.
Many viruses invade mucosal surfaces to establish infection in the host. Some viruses are restricted to mucosal surfaces, whereas others disseminate to sites of secondary replication. Studies of strain-specific differences in reovirus mucosal infection and systemic dissemination have enhanced an understanding of viral determinants and molecular mechanisms that regulate viral pathogenesis. After peroral inoculation, reovirus strain type 1 Lang replicates to high titers in the intestine and spreads systemically, whereas strain type 3 Dearing (T3D) does not. These differences segregate with the viral S1 gene segment, which encodes attachment protein σ1 and nonstructural protein σ1s. In this study, we define genetic determinants that regulate reovirus-induced pathology following intranasal inoculation and respiratory infection. We report that two laboratory isolates of T3D, T3D(C) and T3D(F), differ in the capacity to replicate in the respiratory tract and spread systemically; the T3D(C) isolate replicates to higher titers in the lungs and disseminates, while T3D(F) does not. Two nucleotide polymorphisms in the S1 gene influence these differences, and both S1 gene products are involved. T3D(C) amino acid polymorphisms in the tail and head domains of σ1 protein influence the sensitivity of virions to protease-mediated loss of infectivity. The T3D(C) polymorphism at nucleotide 77, which leads to coding changes in both S1 gene products, promotes systemic dissemination from the respiratory tract. A σ1s-null virus produces lower titers in the lung after intranasal inoculation and disseminates less efficiently to sites of secondary replication. These findings provide new insights into mechanisms underlying reovirus replication in the respiratory tract and systemic spread from the lung.