USP14 promotes K63‐linked RIG‐I deubiquitination and suppresses antiviral immune responses

Retinoic acid‐inducible gene I (RIG‐I) is a critical RNA virus sensor that initiates antiviral immune response through K63‐linked ubiquitination. In this study, we demonstrated USP14, a deubiquitinating enzyme, as a negative regulator in antiviral responses by directly deubiquitinating K63‐linked RIG‐I. USP14 knockdown significantly enhanced RIG‐I‐triggered type I IFN signaling and inhibited vesicular stomatitis virus (VSV) replication both in mouse peritoneal macrophages and THP1 cells. USP14 overexpression in HeLa cells attenuated RIG‐I‐triggered IFN‐β expression and promoted VSV replication. Besides, USP14‐specific inhibitor, IU1, increased RIG‐I‐mediated type I IFN production and antiviral responses in vitro and in vivo. In addition, USP14 could interact with RIG‐I and remove RIG‐I K63‐linked polyubiquitination chains. This article is the first to report that USP14 acts as a negative regulator in antiviral response through deubiquitinating K63‐linked RIG‐I. These findings provide insights into a potential new therapy targeting USP14 for RNA virus‐related diseases.     

RNA recognition and signal transduction by RIG-I-like receptors

Summary:  Viral infection is detected by cellular sensor molecules as foreign nucleic acids and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Recent identification of cytoplasmic viral sensors, such as retinoic acid-inducible gene-I-like receptors (RLRs), highlights their significance in the induction of antiviral innate immunity. Moreover, it is intriguing to understand how they can discriminate endogenous RNA from foreign viral RNA and initiate signaling cascades leading to the induction of type I IFNs. This review focuses on the current understanding of the molecular machinery underlying RNA recognition and subsequent signal transduction by RLRs.     

Viral infections in humans and mice with genetic deficiencies of the type I IFN response pathway

Type I IFNs are so-named because they interfere with viral infection in vertebrate cells. The study of cellular responses to type I IFNs led to the discovery of the JAK-STAT signaling pathway, which also governs the response to other cytokine families. We review here the outcome of viral infections in mice and humans with engineered and inborn deficiencies, respectively, of (i) IFNAR1 or IFNAR2, selectively disrupting responses to type I IFNs, (ii) STAT1, STAT2, and IRF9, also impairing cellular responses to type II (for STAT1) and/or III (for STAT1, STAT2, IRF9) IFNs, and (iii) JAK1 and TYK2, also impairing cellular responses to cytokines other than IFNs. A picture is emerging of greater redundancy of human type I IFNs for protective immunity to viruses in natural conditions than was initially anticipated. Mouse type I IFNs are essential for protection against a broad range of viruses in experimental conditions. These findings suggest that various type I IFN-independent mechanisms of human cell-intrinsic immunity to viruses have yet to be discovered.     

TRIM10 binds to IFN-α/β receptor 1 to negatively regulate type I IFN signal transduction

The type I interferon (IFN-I) system is important for antiviral and anticancer immunity. Prolonged activation of IFN/JAK/STAT signaling is closely associated with autoimmune diseases. TRIM10 dysfunction may be associated closely with certain autoimmune disorders. Here, we observed that the serum TRIM10 protein level is lower in patients with systemic lupus erythematosus than in healthy control subjects. We speculated the possible involvement of TRIM10-induced modulation of the IFN/JAK/STAT signaling pathway in systemic lupus erythematosus. In line with our hypothesis, TRIM10 inhibited the activation of JAK/STAT signaling pathway triggered by various stimuli. TRIM10 restricted the IFN-I/JAK/STAT signaling pathway, which was independent of its E3 ligase activity. Mechanistically, TRIM10 interacted with the intracellular domain of IFNAR1 and blocked the association of IFNAR1 with TYK2. These data suggest the possible TRIM10 suppresses IFN/JAK/STAT signaling pathway through blocking the interaction between IFNAR1 and TYK2. Targeting TRIM10 is a potential strategy for treating autoimmune diseases.     

Negative regulators of the RIG‐I‐like receptor signaling pathway

Upon recognition of specific molecular patterns on microbes, host cells trigger an innate immune response, which culminates in the production of type I interferons, proinflammatory cytokines and chemokines, and restricts pathogen replication and spread within the host. At each stage of this response, there are stimulatory and inhibitory signals that regulate the magnitude, quality, and character of the response. Positive regulation promotes an antiviral state to control and eventually clear infection, whereas negative regulation dampens inflammation and prevents immune‐mediated tissue damage. An overexuberant innate response can lead to cell and tissue destruction, and the development of spontaneous autoimmunity. The retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs), RIG‐I and melanoma differentiation‐associated gene 5 (MDA5), belong to a family of cytosolic host RNA helicases that recognize distinct nonself RNA signatures and trigger innate immune responses against several RNA viruses by signaling through the essential adaptor protein mitochondrial antiviral signaling (MAVS). The RLR signaling pathway is tightly regulated to maximize antiviral immunity and minimize immune‐mediated pathology. This review highlights contemporary findings on negative regulators of the RLR signaling pathway, with specific focus on the proteins and biological processes that directly regulate RIG‐I, MDA5 and MAVS signaling function.     

Critical role of the endogenous interferon ligand–receptors in type I and type II interferons response

Separate ligand–receptor paradigms are commonly used for each type of interferon (IFN). However, accumulating evidence suggests that type I and type II IFNs may not be restricted to independent pathways. Using different cell types deficient in IFNAR1, IFNAR2, IFNGR1, IFNGR2 and IFN‐γ, we evaluated the contribution of each element of the IFN system to the activity of type I and type II IFNs. We show that deficiency in IFNAR1 or IFNAR2 is associated with impairment of type II IFN activity. This impairment, presumably resulting from the disruption of the ligand–receptor complex, is obtained in all cell types tested. However, deficiency of IFNGR1, IFNGR2 or IFN‐γ was associated with an impairment of type I IFN activity in spleen cells only, correlating with the constitutive expression of type II IFN (IFN‐γ) observed on those cells. Therefore, in vitro the constitutive expression of both the receptors and the ligands of type I or type II IFN is critical for the enhancement of the IFN activity. Any IFN deficiency can totally or partially impair IFN activity, suggesting the importance of type I and type II IFN interactions. Taken together, our results suggest that type I and type II IFNs may regulate biological activities through distinct as well as common IFN receptor complexes.     

DEAD/H BOX 3 (DDX3) helicase binds the RIG‐I adaptor IPS‐1 to up‐regulate IFN‐β‐inducing potential

Retinoic acid‐inducible gene‐I (RIG‐I)‐like receptors (RLR) are members of the DEAD box helicases, and recognize viral RNA in the cytoplasm, leading to IFN‐β induction through the adaptor IFN‐β promoter stimulator‐1 (IPS‐1) (also known as Cardif, mitochondrial antiviral signaling protein or virus‐induced signaling adaptor). Since uninfected cells usually harbor a trace of RIG‐I, other RNA‐binding proteins may participate in assembling viral RNA into the IPS‐1 pathway during the initial response to infection. We searched for proteins coupling with human IPS‐1 by yeast two‐hybrid and identified another DEAD (Asp‐Glu‐Ala‐Asp) box helicase, DDX3 (DEAD/H BOX 3). DDX3 can bind viral RNA to join it in the IPS‐1 complex. Unlike RIG‐I, DDX3 was constitutively expressed in cells, and some fraction of DDX3 is colocalized with IPS‐1 around mitochondria. The 622‐662 a.a DDX3 C‐terminal region (DDX3‐C) directly bound to the IPS‐1 CARD‐like domain, and the whole DDX3 protein also associated with RLR. By reporter assay, DDX3 helped IPS‐1 up‐regulate IFN‐β promoter activation and knockdown of DDX3 by siRNA resulted in reduced IFN‐β induction. This activity was conserved on the DDX3‐C fragment. DDX3 only marginally enhanced IFN‐β promoter activation induced by transfected TANK‐binding kinase 1 (TBK1) or I‐kappa‐B kinase‐ε (IKKε). Forced expression of DDX3 augmented virus‐mediated IFN‐β induction and host cell protection against virus infection. Hence, DDX3 is an antiviral IPS‐1 enhancer.     

Antiviral responses induced by the TLR3 pathway

Antiviral responses are successively induced in virus‐infected animals, and include primary innate immune responses such as type I interferon (IFN) and cytokine production, secondary natural killer (NK) cell responses, and final cytotoxic T lymphocyte (CTL) responses and antibody production. The endosomal Toll‐like receptors (TLRs) and cytoplasmic RIG‐I‐like receptors (RLRs), which recognize viral nucleic acids, are responsible for virus‐induced type I IFN production. RLRs are expressed in most tissues and cells and are primarily implicated in innate immune responses against various viruses through type I IFN production, whereas nucleic acid‐sensing TLRs, TLRs 3, 7, 8 and 9, are expressed on the endosomal membrane of dendritic cells (DCs) and play distinct roles in antiviral immunity. TLR3 recognizes viral double‐stranded RNA taken up into the endosome and serves to protect the host against viral infection by the induction of a range of responses including type I IFN production and DC‐mediated activation of NK cells and CTLs, although the deteriorative role of TLR3 has also been reported in some virus infections. Here, we review the current knowledge on the role of TLR3 during viral infection, and the current understanding of the TLR3‐signalling cascade that operates via the adaptor protein TICAM‐1 (also called TRIF). Copyright © 2011 John Wiley & Sons, Ltd.     

Links between recognition and degradation of cytoplasmic viral RNA in innate immune response

Recognition and degradation of viral RNA are essential for antiviral innate immune responses. Cytoplasmic viral RNA is recognized by retinoic acid‐inducible gene I (RIG‐I)‐like receptors, which trigger type I interferon (IFN) production. Secreted type I IFN activates ubiquitously expressed type I IFN receptor and induces IFN‐stimulated genes (ISGs). To suppress viral replication, several nucleases degrade viral RNA. RNase L is an ISG with endonuclease activity that degrades viral RNA, producing small RNA that activates RIG‐I, resulting in the amplification of type I IFN production. Moreover, recent studies have elucidated novel links between viral RNA recognition and degradation. The RNA exosome is a protein complex that includes nucleases and is essential for host and viral RNA decay. Although the small RNAs produced by the RNA exosome do not activate RIG‐I, several accessory factors of the RNA exosome promote RIG‐I activation. Zinc‐finger antiviral protein (ZAP) is an accessory factor that recognizes viral RNA and promotes viral RNA degradation via the RNA exosome. ZAPS is an alternative splicing form of ZAP and promotes RIG‐I oligomerization and ATPase activity, resulting in RIG‐I activation. DDX60 is another cofactor involved in the viral RNA degradation via the RNA exosome. The DDX60 protein promotes RIG‐I signaling in a cell‐type specific manner. These observations imply that viral RNA degradation and recognition are linked to each other. In this review, I discuss the links between recognition and degradation of viral RNA. Copyright © 2015 John Wiley & Sons, Ltd.     

Type I interferons increase host susceptibility to Trypanosoma cruzi infection

Trypanosoma cruzi, the protozoan parasite that causes human Chagas' disease, induces a type I interferon (IFN) (IFN-α/β) response during acute experimental infection in mice and in isolated primary cell types. To examine the potential impact of the type I IFN response in shaping outcomes in experimental T. cruzi infection, groups of wild-type (WT) and type I IFN receptor-deficient (IFNAR(-/-)) 129sv/ev mice were infected with two different T. cruzi strains under lethal and sublethal conditions and several parameters were measured during the acute stage of infection. The results demonstrate that type I IFNs are not required for early host protection against T. cruzi. In contrast, under conditions of lethal T. cruzi challenge, WT mice succumbed to infection whereas IFNAR(-/-) mice were ultimately able to control parasite growth and survive. T. cruzi clearance in and survival of IFNAR(-/-) mice were accompanied by higher levels of IFN-γ production by isolated splenocytes in response to parasite antigen. The suppression of IFN-γ in splenocytes from WT mice was independent of IL-10 levels. While the impact of type I IFNs on the production of IFN-γ and other cytokines/chemokines remains to be fully determined in the context of T. cruzi infection, our data suggest that, under conditions of high parasite burden, type I IFNs negatively impact IFN-γ production, initiating a detrimental cycle that contributes to the ultimate failure to control infection. These findings are consistent with a growing theme in the microbial pathogenesis field in which type I IFNs can be detrimental to the host in a variety of nonviral pathogen infection models.     

Hepatitis C virus NS2 protease inhibits host cell antiviral response by inhibiting IKKε and TBK1 functions

Hepatitis C virus (HCV) encodes for several proteins that can interfere with host cell signaling and antiviral response. Previously, serine protease NS3/4A was shown to block host cell interferon (IFN) production by proteolytic cleavage of MAVS and TRIF, the adaptor molecules of the RIG‐I and TLR3 signaling pathways, respectively. This study shows that another HCV protease, NS2 can interfere efficiently with cytokine gene expression. NS2 and its proteolytically inactive mutant forms were able to inhibit type I and type III IFN, CCL5 and CXCL10 gene promoters activated by Sendai virus infection. However, the CXCL8 gene promoter was not inhibited by NS2. In addition, constitutively active RIG‐I (ΔRIG‐I), MAVS, TRIF, IKKε, and TBK1‐induced activation of IFN‐β promoter was inhibited by NS2. Cotransfection experiments with IKKε or TBK1 together with interferon regulatory factor 3 (IRF3) and HCV expression constructs revealed that NS2 in a dose‐dependent manner inhibited IKKε and especially TBK1‐induced IRF3 phosphorylation. GST pull‐down experiments with GST‐NS2 and in vitro‐translated and cell‐expressed IKKε and TBK1 demonstrated direct physical interactions of the kinases with NS2. Further evidence that the IKKε/TBK1 kinase complex is the target for NS2 was obtained from the observation that the constitutively active form of IRF3 (IRF3‐5D) activated readily IFN‐β promoter in the presence of NS2. The present study identified HCV NS2 as a potent interferon antagonist, and describes an explanation of how NS2 downregulates the major signaling pathways involved in the development of host innate antiviral responses. J. Med. Virol. 85:71–82, 2012. © 2012 Wiley Periodicals, Inc.     

Orchestrating the interferon antiviral response through the mitochondrial antiviral signaling (MAVS) adapter

Sensing of RNA virus infection by the RIG-I-like receptors (RLRs) engages a complex signaling cascade that utilizes the mitochondrial antiviral signaling (MAVS) adapter protein to orchestrate the innate host response to pathogen, ultimately leading to the induction of antiviral and inflammatory responses mediated by type I interferon (IFN) and NF-κB pathways. MAVS is localized to the outer mitochondrial membrane, and has been associated with peroxisomes, the endoplasmic reticulum and autophagosomes, where it coordinates signaling events downstream of RLRs. MAVS not only plays a pivotal role in the induction of antiviral and inflammatory pathways but is also involved in the coordination of apoptotic and metabolic functions. This review summarizes recent findings related to the MAVS adapter and its essential role in the innate immune response to RNA viruses.     

Hypervirulent M. tuberculosis W/Beijing strains upregulate type I IFNs and increase expression of negative regulators of the Jak-Stat pathway.

The role of type I interferons (IFNs) in the host response to bacterial infections is controversial. Here, we examined the role of IFN-alpha/beta in the murine response to infection with Mycobacterium tuberculosis, using wildtype mice, mice with impaired signaling through the type I IFN receptor (IFNAR), and mice treated to reduce levels of type I IFNs. In this study, we used virulent clinical isolates of M. tuberculosis, including HN878, W4, and CDC1551. Our results indicate that higher levels of type I IFNs are induced by the HN878 and W4 strains. Induction of type I IFNs was associated with lower levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin- 12 (IL-12) and reduced T cell activation, and associated with decreased survival of the mice infected with HN878 or W4 relative to infection with CDC1551. Infection of mice with HN878 and W4 was also associated with relatively higher levels of mRNA for a number of negative regulators of the Jak-Stat signaling pathway, such as suppressors of cytokine signaling (SOCS) 1, 4, and 5, CD45, protein inhibitor of activated Stat1 (PIAS1), protein tyrosine phosphatase nonreceptor type 1 (Ptpn1), and protein tyrosine phosphatase nonreceptor type substrate 1 (Ptpns1). Taken together, these results suggest that increased type I IFNs may be deleterious for survival of M. tuberculosis-infected mice in association with reduced Th1 immunity.     

NLRC5 interacts with RIG‐I to induce a robust antiviral response against influenza virus infection

The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG‐I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN‐β expression. Influenza virus leads to induction of IFN‐β that drives RIG‐I and NLRC5 expression in host cells. Our results suggest that NLRC5 extends and stabilizes influenza virus induced RIG‐I expression and delays expression of the viral inhibitor protein NS1. We show that NS1 binds to NLRC5 to suppress its function. Interaction domain mapping revealed that NLRC5 interacts with RIG‐I via its N‐terminal death domain and that NLRC5 enhanced antiviral activity in an leucine‐rich repeat domain independent manner. Taken together, our findings identify a novel role for NLRC5 in RIG‐I‐mediated antiviral host responses against influenza virus infection, distinguished from the role of NLRC5 in MHC class I gene regulation.     

Enterovirus 71 blocks selectively type I interferon production through the 3C viral protein in mice

Type I interferons (IFNs) represent an essential innate defense mechanism for controlling enterovirus 71 (EV 71) infection. Mice inoculated with EV 71 produced a significantly lower amount of type I IFNs than those inoculated with poly (I:C), adenovirus type V, or coxsackievirus B3 (CB3). EV 71 infection, however, mounted a proinflammatory response with a significant increase in the levels of serum and brain interleukin (IL)‐6, monocyte chemoattractant protein‐1, tumor necrosis factor, and IFN‐γ. EV 71 infection abolished both poly (I:C)‐ and CB3‐induced type I IFN production of mice. Such effect was not extended to other enteroviruses including coxsackievirus A24, B2, B3, and echovirus 9, as mice infected with these viruses retained type I IFN responsiveness upon poly (I:C) challenge. In addition, EV 71‐infected RAW264.7 cells produced significantly lower amount of type I IFNs than non‐infected cells upon poly (I:C) stimulation. The inhibitory effect of EV 71 on type I IFN production was attributed to the viral protein 3C, which was confirmed using over‐expression systems in both mice and RAW264.7 cells. The 3C over‐expression, however, did not interfere with poly (I:C)‐induced proinflammatory cytokine production. These findings indicate that EV 71 can hamper the host innate defense by blocking selectively type I IFN synthesis through the 3C viral protein. J. Med. Virol. 84:1779–1789, 2012. © 2012 Wiley Periodicals, Inc.     

PolyI:C plus IL‐2 or IL‐12 induce IFN‐γ production by human NK cells via autocrine IFN‐β

NK lymphocytes and type I IFN (IFN‐α/β) are major actors of the innate anti‐viral response that also influence adaptive immune responses. We evaluated type I IFN production by human NK cells in response to polyI:C, a potent type I IFN‐inducing TLR3 agonist. PolyI:C plus IL‐2/IL‐12 induced IFN‐β (but not IFN‐α) mRNA expression and protein production by highly pure human NK cells and by the human NK cell line NK92. Neutralizing anti‐IFNAR1 or anti‐IFN‐β Ab prevented the production of IFN‐γ induced by polyI:C plus IL‐2/IL‐12. Similarly, IFN‐γ production induced by polyI:C plus IL‐12 was reduced in NK cells isolated from IFNAR1^?/? compared with WT mice. The ability of polyI:C plus IL‐12 to induce IFN‐γ production was related to an increase of TLR3, Mda5 and IFNAR expression and by an increase of STAT1 and STAT4 phosphorylation. Collectively, these data demonstrate that NK cells, in response to polyI:C plus IL‐2/IL‐12, produce IFN‐β that induce, in an autocrine manner, the production of IFN‐γ and thereby highlight that NK cells may control the outcome of protective or injurious immune responses through type I IFN secretion.     

Recognition of viral nucleic acids in innate immunity

Viral infections are detected by sensor molecules, which initiate innate antiviral responses, including the activation of type I interferons (IFNs) and proinflammatory cytokines. These cytokines are responsible for not only inhibiting viral replication in infected cells but also regulating the induction of adaptive immunity, leading to the swift eradication of viruses. Recent advances in the identification of pathogen receptors in the innate immune system have revealed that distinct types of sensors play a role in the detection of viral nucleic acids in different ways; Toll‐like receptors (TLRs), which detect viral DNA or RNA in endosomal compartments in immune cells, retinoic acid inducible gene‐I (RIG‐I)‐like receptors (RLRs), which recognise viral RNA in the cytoplasm, and DNA sensors, which detect cytoplasmic viral DNA. Since these sensors have to exclusively recognise viral infections, it is intriguing to understand how they distinguish self nucleic acids from foreign viral ones. Here, we review the current knowledge of the recognition of viral nucleic acids by these sensor molecules and the signal transduction machinery. Copyright © 2009 John Wiley & Sons, Ltd.