The Essential,Nonredundant Roles of RIG-I and MDA5 in Detecting and Controlling West Nile Virus Infection

Virus recognition and response by the innate immune system are critical components of host defense against infection. Activation of cell-intrinsic immunity and optimal priming of adaptive immunity against West Nile virus (WNV), an emerging vector-borne virus, depend on recognition by RIG-I and MDA5, two cytosolic pattern recognition receptors (PRRs) of the RIG-I-like receptor (RLR) protein family that recognize viral RNA and activate defense programs that suppress infection. We evaluated the individual functions of RIG-I and MDA5 both in vitro and in vivo in pathogen recognition and control of WNV. Lack of RIG-I or MDA5 alone results in decreased innate immune signaling and virus control in primary cells in vitro and increased mortality in mice. We also generated RIG-I^−/− × MDA5^−/− double-knockout mice and found that a lack of both RLRs results in a complete absence of innate immune gene induction in target cells of WNV infection and a severe pathogenesis during infection in vivo, similar to findings for animals lacking MAVS, the central adaptor molecule for RLR signaling. We also found that RNA products from WNV-infected cells but not incoming virion RNA display at least two distinct pathogen-associated molecular patterns (PAMPs) containing 5′ triphosphate and double-stranded RNA that are temporally distributed and sensed by RIG-I and MDA5 during infection. Thus, RIG-I and MDA5 are essential PRRs that recognize distinct PAMPs that accumulate during WNV replication. Collectively, these experiments highlight the necessity and function of multiple related, cytoplasmic host sensors in orchestrating an effective immune response against an acute viral infection.     

Pattern Recognition Receptor MDA5 Modulates CD8+ T Cell-Dependent Clearance of West Nile Virus from the Central Nervous System

Many viruses induce type I interferon responses by activating cytoplasmic RNA sensors, including the RIG-I-like receptors (RLRs). Although two members of the RLR family, RIG-I and MDA5, have been implicated in host control of virus infection, the relative role of each RLR in restricting pathogenesis in vivo remains unclear. Recent studies have demonstrated that MAVS, the adaptor central to RLR signaling, is required to trigger innate immune defenses and program adaptive immune responses, which together restrict West Nile virus (WNV) infection in vivo. In this study, we examined the specific contribution of MDA5 in controlling WNV in animals. MDA5^−/− mice exhibited enhanced susceptibility, as characterized by reduced survival and elevated viral burden in the central nervous system (CNS) at late times after infection, even though small effects on systemic type I interferon response or viral replication were observed in peripheral tissues. Intracranial inoculation studies and infection experiments with primary neurons ex vivo revealed that an absence of MDA5 did not impact viral infection in neurons directly. Rather, subtle defects were observed in CNS-specific CD8⁺ T cells in MDA5^−/− mice. Adoptive transfer into recipient MDA5^+/+ mice established that a non-cell-autonomous deficiency of MDA5 was associated with functional defects in CD8⁺ T cells, which resulted in a failure to clear WNV efficiently from CNS tissues. Our studies suggest that MDA5 in the immune priming environment shapes optimal CD8⁺ T cell activation and subsequent clearance of WNV from the CNS.     

Deficient IFN Signaling by Myeloid Cells Leads to MAVS-Dependent Virus-Induced Sepsis

The type I interferon (IFN) signaling response limits infection of many RNA and DNA viruses. To define key cell types that require type I IFN signaling to orchestrate immunity against West Nile virus (WNV), we infected mice with conditional deletions of the type I IFN receptor (IFNAR) gene. Deletion of the Ifnar gene in subsets of myeloid cells resulted in uncontrolled WNV replication, vasoactive cytokine production, sepsis, organ damage, and death that were remarkably similar to infection of Ifnar ^−/− mice completely lacking type I IFN signaling. In Mavs^−/−×Ifnar^−/− myeloid cells and mice lacking both Ifnar and the RIG-I-like receptor adaptor gene Mavs, cytokine production was muted despite high levels of WNV infection. Thus, in myeloid cells, viral infection triggers signaling through MAVS to induce proinflammatory cytokines that can result in sepsis and organ damage. Viral pathogenesis was caused in part by massive complement activation, as liver damage was minimized in animals lacking complement components C3 or factor B or treated with neutralizing anti-C5 antibodies. Disease in Ifnar ^−/− and CD11c Cre⁺ Ifnar ^f/f mice also was facilitated by the proinflammatory cytokine TNF-α, as blocking antibodies diminished complement activation and prolonged survival without altering viral burden. Collectively, our findings establish the dominant role of type I IFN signaling in myeloid cells in restricting virus infection and controlling pathological inflammation and tissue injury.     

Murine coronavirus mouse hepatitis virus is recognized by MDA5 and induces type I interferon in brain macrophages/microglia

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR^−/−) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-β in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-β production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-β production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-β mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-β by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.     

Rabies Virus Infection Induces Type I Interferon Production in an IPS-1 Dependent Manner While Dendritic Cell Activation Relies on IFNAR Signaling

As with many viruses, rabies virus (RABV) infection induces type I interferon (IFN) production within the infected host cells. However, RABV has evolved mechanisms by which to inhibit IFN production in order to sustain infection. Here we show that RABV infection of dendritic cells (DC) induces potent type I IFN production and DC activation. Although DCs are infected by RABV, the viral replication is highly suppressed in DCs, rendering the infection non-productive. We exploited this finding in bone marrow derived DCs (BMDC) in order to differentiate which pattern recognition receptor(s) (PRR) is responsible for inducing type I IFN following infection with RABV. Our results indicate that BMDC activation and type I IFN production following a RABV infection is independent of TLR signaling. However, IPS-1 is essential for both BMDC activation and IFN production. Interestingly, we see that the BMDC activation is primarily due to signaling through the IFNAR and only marginally induced by the initial infection. To further identify the receptor recognizing RABV infection, we next analyzed BMDC from Mda-5−/− and RIG-I−/− mice. In the absence of either receptor, there is a significant decrease in BMDC activation at 12h post infection. However, only RIG-I−/− cells exhibit a delay in type I IFN production. In order to determine the role that IPS-1 plays in vivo, we infected mice with pathogenic RABV. We see that IPS-1−/− mice are more susceptible to infection than IPS-1+/+ mice and have a significantly increased incident of limb paralysis.     

CryoEM structure of the Nipah virus nucleocapsid assembly

Nipah and its close relative Hendra are highly pathogenic zoonotic viruses, storing their ssRNA genome in a helical nucleocapsid assembly formed by the N protein, a major viral immunogen. Here, we report the first cryoEM structure for a Henipavirus RNA-bound nucleocapsid assembly, at 3.5 Å resolution. The helical assembly is stabilised by previously undefined N- and C-terminal segments, contributing to subunit-subunit interactions. RNA is wrapped around the nucleocapsid protein assembly with a periodicity of six nucleotides per protomer, in the “3-bases-in, 3-bases-out” conformation, with protein plasticity enabling non-sequence specific interactions. The structure reveals commonalities in RNA binding pockets and in the conformation of bound RNA, not only with members of the Paramyxoviridae family, but also with the evolutionarily distant Filoviridae Ebola virus. Significant structural differences with other Paramyxoviridae members are also observed, particularly in the position and length of the exposed α-helix, residues 123–139, which may serve as a valuable epitope for surveillance and diagnostics.     

LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction

LincRNA‐EPS is an important regulator in inflammation. However, the role of lincRNA‐EPS in the host response against viral infection is unexplored. Here, we show that lincRNA‐EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV‐1. Overexpression of lincRNA‐EPS facilitates viral infection, while deficiency of lincRNA‐EPS protects the host against viral infection in vitro and in vivo. LincRNA‐EPS ^−/− macrophages show elevated expression of antiviral interferon‐stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN‐β, the key upstream inducer of these ISGs, is downregulated in lincRNA‐EPS ^−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA‐EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN‐I induction. LincRNA‐EPS inhibits PKR‐STAT1‐ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA‐EPS promoting the induction of PKR‐STAT1‐dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases.     

Distinct RIG-I and MDA5 signaling by RNA viruses in innate immunity

Alpha/beta interferon immune defenses are essential for resistance to viruses and can be triggered through the actions of the cytoplasmic helicases retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Signaling by each is initiated by the recognition of viral products such as RNA and occurs through downstream interaction with the IPS-1 adaptor protein. We directly compared the innate immune signaling requirements of representative viruses of the Flaviviridae, Orthomyxoviridae, Paramyxoviridae, and Reoviridae for RIG-I, MDA5, and interferon promoter-stimulating factor 1 (IPS-1). In cultured fibroblasts, IPS-1 was essential for innate immune signaling of downstream interferon regulatory factor 3 activation and interferon-stimulated gene expression, but the requirements for RIG-I and MDA5 were variable. Each was individually dispensable for signaling triggered by reovirus and dengue virus, whereas RIG-I was essential for signaling by influenza A virus, influenza B virus, and human respiratory syncytial virus. Functional genomics analyses identified cellular genes triggered during influenza A virus infection whose expression was strictly dependent on RIG-I and which are involved in processes of innate or adaptive immunity, apoptosis, cytokine signaling, and inflammation associated with the host response to contemporary and pandemic strains of influenza virus. These results define IPS-1-dependent signaling as an essential feature of host immunity to RNA virus infection. Our observations further demonstrate differential and redundant roles for RIG-I and MDA5 in pathogen recognition and innate immune signaling that may reflect unique and shared biologic properties of RNA viruses whose differential triggering and control of gene expression may impact pathogenesis and infection.     

The Pivotal Role of 5-Lipoxygenase-Derived LTB4 in Controlling Pulmonary Paracoccidioidomycosis

Leukotrienes (LTs) produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. However, studies published in the literature regarding these mediators are contradictory and it remains uncertain whether these lipid mediators play a role in host defense against the fungal pathogen Paracoccidioides brasiliensis. To determine the involvement of LTs in the host response to pulmonary infection, wild-type and LT-deficient mice by targeted disruption of the 5-lipoxygenase gene (knockout mice) were studied following intratracheal challenge with P. brasiliensis yeasts. The results showed that infection is uniformly fatal in 5-LO-deficient mice and the mechanisms that account for this phenotype are an exacerbated lung injury and higher fungal pulmonary burden. Genetic ablation or pharmacological inhibition of LTs resulted in lower phagocytosis and fungicidal activity of macrophages in vitro, suggesting that deficiency in fungal clearance seems to be secondary to the absence of activation in 5-LO^−/− macrophages. Exogenous LTB₄ restored phagocytosis and fungicidal activity of 5-LO^−/− macrophages. Moreover, P. brasiliensis killing promoted by LTB₄ was dependent on nitric oxide (NO) production by macrophages. Taken together, these results reveal a fundamental role for 5-LO-derived LTB₄ in the protective response to P. brasiliensis infection and identify relevant mechanisms for the control of fungal infection during the early stages of the host immune response.     

Global quantitative proteomic analysis profiles host protein expression in response to Sendai virus infection

Sendai virus (SeV) is an enveloped nonsegmented negative‐strand RNA virus that belongs to the genus Respirovirus of the Paramyxoviridae family. As a model pathogen, SeV has been extensively studied to define the basic biochemical and molecular biologic properties of the paramyxoviruses. In addition, SeV‐infected host cells were widely employed to uncover the mechanism of innate immune response. To identify proteins involved in the SeV infection process or the SeV‐induced innate immune response process, system‐wide evaluations of SeV–host interactions have been performed. cDNA microarray, siRNA screening and phosphoproteomic analysis suggested that multiple signaling pathways are involved in SeV infection process. Here, to study SeV–host interaction, a global quantitative proteomic analysis was performed on SeV‐infected HEK 293T cells. A total of 4699 host proteins were quantified, with 742 proteins being differentially regulated. Bioinformatics analysis indicated that regulated proteins were mainly involved in “interferon type I (IFN‐I) signaling pathway” and “defense response to virus,” suggesting that these processes play roles in SeV infection. Further RNAi‐based functional studies indicated that the regulated proteins, tripartite motif (TRIM24) and TRIM27, affect SeV‐induced IFN‐I production. Our data provided a comprehensive view of host cell response to SeV and identified host proteins involved in the SeV infection process or the SeV‐induced innate immune response process.     

Isolation and Characterization of a Novel Single-Stranded RNA Virus Infecting the Bloom-Forming Diatom Rhizosolenia setigera

A novel single-stranded RNA (ssRNA) virus specifically infecting the bloom-forming diatom Rhizosolenia setigera (R. setigera RNA virus [RsRNAV]) was isolated from Ariake Sea, Japan. Viral replication occurred within the cytoplasm, and the virus particle was icosahedral, lacked a tail, and was 32 nm in diameter on average. The major nucleic acid extracted from the RsRNAV particles was an ssRNA molecule 11.2 kb in length, although smaller RNA molecules (0.6, 1.2, and 1.5 kb) were occasionally observed. The major structural proteins of RsRNAV were 41.5, 41.0, and 29.5 kDa. Inter- and intraspecies host specificity tests revealed that RsRNAV is not only species specific but also strain specific and that its intraspecies host specificity is diverse among virus clones. The latent period of RsRNAV was 2 days, and the burst sizes were 3,100 and 1,010 viruses per host cell when viruses were inoculated into the host culture at the exponential and stationary growth phases, respectively, at 15°C under a 12-h-12-h light-dark cycle of ca. 110 μmol of photons m⁻² s⁻¹ with cool white fluorescent illumination. To our knowledge, this is the first report describing the biological properties of a virus infecting a diatom. Further studies on RsRNAV will be helpful in understanding the ecological relationship between diatoms and viruses in nature.     

Extracellular Matrix Protein Lumican Promotes Clearance and Resolution of Pseudomonas aeruginosa Keratitis in a Mouse Model

Lumican is an extracellular protein that associates with CD14 on the surface of macrophages and neutrophils, and promotes CD14-TLR4 mediated response to bacterial lipopolysaccharides (LPS). Lumican-deficient (Lum ^−/−) mice and macrophages are impaired in TLR4 signals; raising the possibility that lumican may regulate host response to live bacterial infections. In a recent study we showed that in vitro Lum ^−/− macrophages are impaired in phagocytosis of gram-negative bacteria and in a lung infection model the Lum ^−/− mice showed poor survival. The cornea is an immune privileged barrier tissue that relies primarily on innate immunity to protect against ocular infections. Lumican is a major component of the cornea, yet its role in counteracting live bacteria in the cornea remains poorly understood. Here we investigated Pseudomonas aeruginosa infections of the cornea in Lum ^−/− mice. By flow cytometry we found that 24 hours after infection macrophage and neutrophil counts were lower in the cornea of Lum ^−/− mice compared to wild types. Infected Lum ^−/− corneas showed lower levels of the leukocyte chemoattractant CXCL1 by 24–48 hours of infection, and increased bacterial counts up to 5 days after infection, compared to Lum^+/− mice. The pro-inflammatory cytokine TNF-α was comparably low 24 hours after infection, but significantly higher in the Lum ^−/− compared to Lum ^+/− infected corneas by 2–5 days after infection. Taken together, the results indicate that lumican facilitates development of an innate immune response at the earlier stages of infection and lumican deficiency leads to poor bacterial clearance and resolution of corneal inflammation at a later stage.