Andrea J. Pruijssers

Intestinal microbiota promote enteric virus replication and systemic pathogenesis

Commensal microflora promote the pathogenesis of mucosally acquired viruses. Intestinal bacteria aid host health and limit bacterial pathogen colonization. However, the influence of bacteria on enteric viruses is largely unknown. We depleted the intestinal microbiota of mice with antibiotics before inoculation with poliovirus, an enteric virus. Antibiotic-treated mice were less susceptible to poliovirus disease and supported minimal viral replication in the intestine. Exposure to bacteria or their N-acetylglucosamine–containing surface polysaccharides, including lipopolysaccharide and peptidoglycan, enhanced poliovirus infectivity. We found that poliovirus binds lipopolysaccharide, and exposure of poliovirus to bacteria enhanced host cell association and infection. The pathogenesis of reovirus, an unrelated enteric virus, also was more severe in the presence of intestinal microbes. These results suggest that antibiotic-mediated microbiota depletion diminishes enteric virus infection and that enteric viruses exploit intestinal microbes for replication and transmission.

Antiviral immunity via RIG-I-mediated recognition of RNA bearing 5'-diphosphates.

Mammalian cells possess mechanisms to detect and defend themselves from invading viruses. In the cytosol, the RIG-I-like receptors (RLRs), RIG-I (retinoic acid-inducible gene I; encoded by DDX58) and MDA5 (melanoma differentiation-associated gene 5; encoded by IFIH1) sense atypical RNAs associated with virus infection. Detection triggers a signalling cascade via the adaptor MAVS that culminates in the production of type I interferons (IFN-α and β; hereafter IFN), which are key antiviral cytokines. RIG-I and MDA5 are activated by distinct viral RNA structures and much evidence indicates that RIG-I responds to RNAs bearing a triphosphate (ppp) moiety in conjunction with a blunt-ended, base-paired region at the 5′-end (reviewed in refs 1, 2, 3). Here we show that RIG-I also mediates antiviral responses to RNAs bearing 5′-diphosphates (5′pp). Genomes from mammalian reoviruses with 5′pp termini, 5′pp-RNA isolated from yeast L-A virus, and base-paired 5′pp-RNAs made by in vitro transcription or chemical synthesis, all bind to RIG-I and serve as RIG-I agonists. Furthermore, a RIG-I-dependent response to 5′pp-RNA is essential for controlling reovirus infection in cultured cells and in mice. Thus, the minimal determinant for RIG-I recognition is a base-paired RNA with 5′pp. Such RNAs are found in some viruses but not in uninfected cells, indicating that recognition of 5′pp-RNA, like that of 5′ppp-RNA, acts as a powerful means of self/non-self discrimination by the innate immune system.

The Early Interferon Response to Rotavirus Is Regulated by PKR and Depends on MAVS/IPS-1, RIG-I, MDA-5, and IRF3

ABSTRACT In mouse embryonic fibroblasts (MEFs), the bovine rotavirus (UK strain) but not the simian rhesus rotavirus (RRV) robustly triggers beta interferon (IFN-β) secretion, resulting in an IFN-dependent restriction of replication. We now find that both rotavirus strains trigger antiviral transcriptional responses early during infection and that both transcriptional responses and IFN-β secretion are completely abrogated in MAVS/IPS-1−/− MEFs. Replication of UK virus could be rescued in MAVS/IPS-1−/− MEFs, and synthesis of viral RNA significantly increased early during virus infection. UK virus induced IFN-β secretion and transcription of IFN-stimulated genes (ISGs) in both RIG-I−/− and MDA-5−/− MEFs, and neither receptor was essential by itself for the antiviral response to UK rotavirus. However, when receptors RIG-I and MDA-5 were depleted using RNA interference, we found that both contribute to the magnitude of the IFN response. IRF3 was found to be essential for MAVS/IPS-1-directed ISG transcription and IFN-β secretion during rotavirus infection. Interestingly, absence of the double-stranded RNA-dependent protein kinase PKR led to a profound defect in the capacity of host cells to secrete IFN-β in response to virus. Both PKR and IRF3 restricted the early replication of UK as indicated by significant increases in viral RNA in fibroblasts lacking either gene. Despite the loss in IFN-β secretion in PKR−/− MEFs, we did not observe decreased IRF3- or NF-κB-dependent early ISG transcription in these cells. Levels of transcripts encoding IFN-α4, IFN-α5, and IFN-β were high in infected PKR−/− MEFs, indicating that during rotavirus infection, PKR functions at a stage between IFN gene transcription and subsequent IFN-β secretion. These findings reveal that activation of the antiviral response by rotavirus is dependent on MAVS/IPS-1 and IRF3 and involves both RIG-I and MDA-5 and that IFN-β secretion during rotavirus infection is regulated by PKR.

Prevention and cure of rotavirus infection via TLR5/NLRC4–mediated production of IL-22 and IL-18

Activators of innate immunity may have the potential to combat a broad range of infectious agents. We report that treatment with bacterial flagellin prevented rotavirus (RV) infection in mice and cured chronically RV-infected mice. Protection was independent of adaptive immunity and interferon (IFN, type I and II) and required flagellin receptors Toll-like receptor 5 (TLR5) and NOD-like receptor C4 (NLRC4). Flagellin-induced activation of TLR5 on dendritic cells elicited production of the cytokine interleukin-22 (IL-22), which induced a protective gene expression program in intestinal epithelial cells. Flagellin also induced NLRC4-dependent production of IL-18 and immediate elimination of RV-infected cells. Administration of IL-22 and IL-18 to mice fully recapitulated the capacity of flagellin to prevent or eliminate RV infection and thus holds promise as a broad-spectrum antiviral agent. Bacterial flagellin protein protects rotavirus-infected mice by stimulating cytokine production. Flagellin gives rotavirus a one-two punch Rotavirus causes gastroenteritis, which can be especially severe in infants and young children. The bacterial protein flagellin activates the innate immune system and protects mice against a variety of infectious and inflammatory agents. Zhang et al. now report that flagellin both prevented rotavirus infection in mice and cured mice chronically infected with rotavirus. It did so by activating two distinct innate immune signaling pathways, which led to cells in the infected mice producing the cytokines interleukin 22 and interleukin 18. Similar to flagellin, treating mice with both of these cytokines prevented or cured rotavirus infection. Science, this issue p. 861

Reovirus infection triggers inflammatory responses to dietary antigens and development of celiac disease

A nonpathogenic virus can promote inflammatory immunity to dietary antigens and may be linked to the development of celiac disease. Viruses compound dietary pathology Reoviruses commonly infect humans and mice asymptomatically. Bouziat et al. found that immune responses to two gut-infecting reoviruses take different paths in mice (see the Perspective by Verdu and Caminero). Both reoviruses invoked protective immune responses, but for one reovirus, when infection happened in the presence of a dietary antigen (such as gluten or ovalbumin), tolerance to the dietary antigen was lost. This was because this strain prevented the formation of tolerogenic T cells. Instead, it promoted T helper 1 immunity to the dietary antigen through interferon regulatory factor 1 signaling. Celiac disease patients also exhibited elevated levels of antibodies against reovirus. Science, this issue p. 44; see also p. 29 Viral infections have been proposed to elicit pathological processes leading to the initiation of T helper 1 (TH1) immunity against dietary gluten and celiac disease (CeD). To test this hypothesis and gain insights into mechanisms underlying virus-induced loss of tolerance to dietary antigens, we developed a viral infection model that makes use of two reovirus strains that infect the intestine but differ in their immunopathological outcomes. Reovirus is an avirulent pathogen that elicits protective immunity, but we discovered that it can nonetheless disrupt intestinal immune homeostasis at inductive and effector sites of oral tolerance by suppressing peripheral regulatory T cell (pTreg) conversion and promoting TH1 immunity to dietary antigen. Initiation of TH1 immunity to dietary antigen was dependent on interferon regulatory factor 1 and dissociated from suppression of pTreg conversion, which was mediated by type-1 interferon. Last, our study in humans supports a role for infection with reovirus, a seemingly innocuous virus, in triggering the development of CeD.

PTP-H2 and PTP-H3 from Microplitis demolitor Bracovirus Localize to Focal Adhesions and Are Antiphagocytic in Insect Immune Cells

ABSTRACT Viruses in the family Polydnaviridae are symbiotically associated with parasitoid wasps. Wasps inject polydnaviruses (PDVs) when laying an egg into their insect host, and expression of viral gene products causes several physiological alterations, including immunosuppression, that allow the wasps progeny to develop. As with other PDVs, most Microplitis demolitor bracovirus (MdBV) genes are related variants that form gene families. The largest MdBV gene family includes 13 members that encode predicted proteins related to protein tyrosine phosphatases (PTPs). Sequence analysis during the present study indicated that five PTP family members (PTP-H2, -H3, -N1, and -N2) have fully conserved catalytic domains, whereas other family members exhibited replacements, deletions, or rearrangements of amino acids considered essential for tyrosine phosphatase activity. Expression studies indicated that most MdBV PTP genes are expressed in virus-infected host insects, with transcript abundance usually being highest in hemocytes. MdBV-infected hemocytes also exhibited higher levels of tyrosine phosphatase activity than noninfected hemocytes. We produced expression constructs for four of the most abundantly expressed PTP family members and conducted functional studies with hemocyte-like Drosophila S2 cells. These experiments suggested that recombinant PTP-H2 and PTP-H3 are functional tyrosine phosphatases whereas PTP-H1 and PTP-J1 are not. PTP-H2 and -H3 localized to focal adhesions in S2 cells, and coexpression with another MdBV gene product, Glc1.8, resulted in complete inhibition of phagocytosis.

Bid regulates the pathogenesis of neurotropic reovirus.

Reovirus infection leads to apoptosis in both cultured cells and the murine central nervous system (CNS). NF-κB-driven transcription of proapoptotic cellular genes is required for the effector phase of the apoptotic response. Although both extrinsic death-receptor signaling pathways and intrinsic pathways involving mitochondrial injury are implicated in reovirus-induced apoptosis, mechanisms by which either of these pathways are activated and their relationship to NF-κB signaling following reovirus infection are unknown. The proapoptotic Bcl-2 family member, Bid, is activated by proteolytic cleavage following reovirus infection. To understand how reovirus integrates host signaling circuits to induce apoptosis, we examined proapoptotic signaling following infection of Bid-deficient cells. Although reovirus growth was not affected by the absence of Bid, cells lacking Bid failed to undergo apoptosis. Furthermore, we found that NF-κB activation is required for Bid cleavage and subsequent proapoptotic signaling. To examine the functional significance of Bid-dependent apoptosis in reovirus disease, we monitored fatal encephalitis caused by reovirus in the presence and absence of Bid. Survival of Bid-deficient mice was significantly enhanced in comparison to wild-type mice following either peroral or intracranial inoculation of reovirus. Decreased reovirus virulence in Bid-null mice was accompanied by a reduction in viral yield. These findings define a role for NF-κB-dependent cleavage of Bid in the cell death program initiated by viral infection and link Bid to viral virulence.

Utilization of Sialylated Glycans as Coreceptors Enhances the Neurovirulence of Serotype 3 Reovirus

ABSTRACT Mammalian reoviruses display serotype-specific patterns of tropism and disease in the murine central nervous system (CNS) attributable to polymorphisms in viral attachment protein σ1. While all reovirus serotypes use junctional adhesion molecule-A as a cellular receptor, they differ in their utilization of carbohydrate coreceptors. This observation raises the possibility that carbohydrate binding by σ1 influences reovirus pathology in the CNS. In this study, we sought to define the function of carbohydrate binding in reovirus neuropathogenesis. Newborn mice were inoculated intramuscularly with wild-type strain type 3 Dearing (T3D) and T3D-σ1R202W, a point mutant T3D derivative that does not bind sialic acid (SA). Infected mice were monitored for survival, and viral loads at the sites of primary and secondary replication were quantified. Fewer mice inoculated with the wild-type virus survived in comparison to those inoculated with the mutant virus. The wild-type virus also produced higher titers in the spinal cord and brain at late times postinoculation but lower titers in the liver in comparison to those produced by the mutant virus. In addition, the wild-type virus was more virulent and produced higher titers in the brain than the mutant following intracranial inoculation. These animal infectivity studies suggest that T3D-σ1R202W harbors a defect in neural growth. Concordantly, compared with the wild-type virus, the mutant virus displayed a decreased capacity to infect and replicate in primary cultures of cortical neurons, a property dependent on cell surface SA. These results suggest that SA binding enhances the kinetics of reovirus replication in neural tissues and highlight a functional role for sialylated glycans as reovirus coreceptors in the CNS.

Interferon Regulatory Factor 3 Attenuates Reovirus Myocarditis and Contributes to Viral Clearance

ABSTRACT Apoptosis is a pathological hallmark of encephalitis and myocarditis caused by reovirus in newborn mice. In cell culture models, the antiviral transcription factor interferon regulatory factor 3 (IRF-3) enhances reovirus-induced apoptosis following activation via retinoic acid inducible gene I and interferon promoter-stimulating factor 1. To determine the role of IRF-3 in reovirus disease, we infected newborn IRF-3+/+ and IRF-3−/− mice perorally with mildly virulent strain type 1 Lang (T1L) and fully virulent strain type 3 SA+ (T3SA+) and monitored infected animals for survival. Both wild-type and IRF-3−/− mice succumbed with equivalent frequencies to infection with T3SA+. However, the absence of IRF-3 was associated with significantly decreased survival rates following infection with T1L. The two virus strains achieved similar peak titers in IRF-3+/+ and IRF-3−/− mice in the intestine, brain, heart, liver, and spleen. However, by day 12 postinoculation, titers in all organs examined were 10- to 100-fold higher in IRF-3−/− mice than those in wild-type mice. Increased titers were associated with marked pathological changes in all organs examined, especially in the heart, where absence of IRF-3 resulted in severe myocarditis. Cellular and humoral immune responses were equivalent in wild-type and IRF-3−/− animals, suggesting that IRF-3 functions independently of the adaptive immune response to enhance reovirus clearance. Thus, IRF-3 serves to facilitate virus clearance and prevent tissue injury in response to reovirus infection.

Apoptosis Induction Influences Reovirus Replication and Virulence in Newborn Mice

ABSTRACT 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.