A major component of the protective antiviral host defense is contributed by the intracellular actions of the proteins encoded by interferon-stimulated genes (ISGs); among these are the ...interferon-induced proteins with tetratricopeptide repeats (IFITs), consisting of four members in human and three in mouse. IFIT proteins do not have any known enzyme activity. Instead, they inhibit virus replication by binding and regulating the functions of cellular and viral proteins and RNAs. Although all IFITs are comprised of multiple copies of the degenerate tetratricopeptide repeats, their distinct tertiary structures enable them to bind different partners and affect host-virus interactions differently. The recent use of Ifit knockout mouse models has revealed novel antiviral functions of these proteins and new insights into the specificities of ISG actions. This article focuses on human and murine IFIT1 and IFIT2 by reviewing their mechanisms of action, their critical roles in protecting mice from viral pathogenesis, and viral strategies to evade IFIT action.
STING is a nodal point for cellular innate immune response to microbial infections, autoimmunity and cancer; it triggers the synthesis of the antiviral proteins, type I interferons. Many DNA viruses, ...including Herpes Simplex Virus 1 (HSV1), trigger STING signaling causing inhibition of virus replication. Here, we report that HSV1 evades this antiviral immune response by inducing a cellular microRNA, miR-24, which binds to the 3’ untranslated region of STING mRNA and inhibits its translation. Expression of the gene encoding miR-24 is induced by the transcription factor AP1 and activated by MAP kinases in HSV1-infected cells. Introduction of exogenous miR-24 or prior activation of MAPKs, causes further enhancement of HSV1 replication in STING-expressing cells. Conversely, transfection of antimiR-24 inhibits virus replication in those cells. HSV1 infection of mice causes neuropathy and death; using two routes of infection, we demonstrated that intracranial injection of antimiR-24 alleviates both morbidity and mortality of the infected mice. Our studies reveal a new immune evasion strategy adopted by HSV1 through the regulation of STING and demonstrates that it can be exploited to enhance STING’s antiviral action.
The transcription factor IRF-3 mediates cellular antiviral response by inducing the expression of interferon and other antiviral proteins. In RNA-virus infected cells, IRF-3’s transcriptional ...activation is triggered primarily by RIG-I-like receptors (RLR), which can also activate the RLR-induced IRF-3-mediated pathway of apoptosis (RIPA). Here, we have reported that the pathway of IRF-3 activation in RIPA was independent of and distinct from the known pathway of transcriptional activation of IRF-3. It required linear polyubiquitination of two specific lysine residues of IRF-3 by LUBAC, the linear polyubiquitinating enzyme complex, which bound IRF-3 in signal-dependent fashion. To evaluate the role of RIPA in viral pathogenesis, we engineered a genetically targeted mouse, which expressed a mutant IRF-3 that was RIPA-competent but transcriptionally inert; this single-action IRF-3 could protect mice from lethal viral infection. Our observations indicated that IRF-3-mediated apoptosis of virus-infected cells could be an effective antiviral mechanism, without expression of the interferon-stimulated genes.
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•RLR-signaling activates IRF-3 to trigger RIPA, the pro-apoptotic antiviral pathway•RIPA can protect the host in the absence of antiviral gene induction•RIPA requires ubiquitination of IRF-3 on specific lysines
The antiviral effects of IRF-3 are known to involve transcription of interferon stimulated genes as well as inducing apoptosis in infected cells. Sen and colleagues report that these two antiviral pathways mediated by IRF-3 are independent and that the apoptotic pathway is sufficient to inhibit viral pathogenesis
Interferon-stimulated genes (ISGs) are induced in response to interferon expression due to viral infections. Role of these ISGs can be variable in different cells or organs. Our study highlights such ...cell-specific role of an ISG, Ddx3, which regulates the translation of mRNAs essential for interferon induction (PACT) and interferon signaling (STAT1) in a cell-specific manner. Our study also highlights the role of PACT in RNA virus-induced RLR signaling. Our study depicts how Ddx3 regulates innate immune signaling pathways in an indirect manner. Such cell-specific behavior of ISGs helps us to better understand viral pathogenesis and highlights the complexities of viral tropism and innate immune responses.
The interferon-induced tetratricopeptide repeat protein (Ifit2) protects mice from lethal neurotropic viruses. Neurotropic coronavirus MHV-RSA59 infection of Ifit2-/- mice caused pronounced morbidity ...and mortality accompanied by rampant virus replication and spread throughout the brain. In spite of the higher virus load, induction of many cytokines and chemokines in the brains of infected Ifit2-/- mice were similar to that in wild-type mice. In contrast, infected Ifit2-/- mice revealed significantly impaired microglial activation as well as reduced recruitment of NK1.1 T cells and CD4 T cells to the brain, possibly contributing to the lack of viral clearance. These two deficiencies were associated with a lower level of microglial expression of CX3CR1, the receptor of the CX3CL1 (Fractalkine) chemokine, which plays a critical role in both microglial activation and leukocyte recruitment. The above results uncovered a new potential role of an interferon-induced protein in immune protection.
Mammalian innate immune response to virus infection is meditated by many cell-intrinsic pathways. RNA viruses, such as Sendai virus, which replicate in the cytoplasm, trigger the RIG-I-like receptor ...pathway, which activates the transcription factor, IRF3. Activated IRF3 translocates to the nucleus and induces transcription of the genes which encode interferons, the major antiviral cytokines. Interestingly, IRF3 activates another interferon-independent antiviral pathway, called RIG-I induced pathway of apoptosis (RIPA). For activating RIPA, IRF3 translocates from the cytoplasm to mitochondria. RIPA requires linear polyubiquitination of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 binds to Bax and translocates it to mitochondria causing the release of Cytochrome C, activation of caspases and apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, inhibits RIPA by deubiquitinating IRF3. Ablation of Otulin expression enhanced RIPA and its overexpression inhibited RIPA. In virus-infected cells, to overcome Otulin-mediated inhibition, RIPA actively degrades Otulin. This degradation required sequential actions of RIPA-activated Caspase 3 and proteasomes. Caspase 3 cleaved Otulin at D31; the D31A mutant was not cleaved at all. The caspase-cleaved fragment was totally degraded by proteasomes, which was preceded by its K48-linked ubiquitination. Mass spectrometric analysis of Otulin identified K64 and K197 as the ubiquitinated residues. Otulin interacted with LUBAC after virus infection and the E3-ubiquitin ligase, HOIP, a component of LUBAC, ubiquitinated Otulin to trigger its proteasome-mediated degradation. To assess the impact of Otulin degradation on RIPA-mediated antiviral action, we expressed, in Otulin-ablated cells, a non-degradable mutant of Otulin, in which D31, K64 and K197 had been mutated. The cells expressing the Otulin mutant were less susceptible to virus-induced apoptosis, because RIPA was less active, and consequently virus replication was more robust. Thus, our study has revealed an important positive feedback loop of RIPA.
Abstract There is a wealth of knowledge about how different Ser/Thr protein kinases participate in Toll-like receptor (TLR) signaling. In many cases, we know the identities of the Ser/Thr residues of ...various components of the TLR-signaling pathways that are phosphorylated, the functional consequences of the phosphorylation and the responsible protein kinases. In contrast, the analysis of Tyr-phosphorylation of TLRs and their signaling proteins is currently incomplete, because several existing analyses are not systematic or they do not rely on robust experimental data. Nevertheless, it is clear that many TLRs require, for signaling, ligand-dependent phosphorylation of specific Tyr residues in their cytoplasmic domains; the list includes TLR2, TLR3, TLR4, TLR5, TLR8 and TLR9. In this article, we discuss the current status of knowledge of the effect of Tyr-phosphorylation of TLRs and their signaling proteins on their biochemical and biological functions, the possible identities of the relevant protein tyrosine kinases (PTKs) and the nature of regulations of PTK-mediated activation of TLR signaling pathways.
Double-stranded (ds) RNA has diverse roles in host defense and disease prevention. dsRNA, produced by viral replication, elicits strong antiviral responses in host; similar protective responses can ...also be triggered by cellular dsRNA produced by necrotic, apoptotic, or otherwise stressed, uninfected cells. dsRNA is recognized in the cell by a large family of dsRNA-binding proteins, among which are the pattern recognition receptors (PRRs), toll-like receptor 3 (TLR3), and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs). TLR3 signals from the endosomal membrane where it senses extracellular dsRNA that has been endocytosed, whereas RLRs signal from the cytoplasm using a mitochondrial adaptor protein. In this review, we will summarize the signaling pathways used by these 2 PRRs, which lead to the activation of specific transcription factors and the induction of many proinflammatory and antiviral genes. However, it is becoming increasingly clear that all host responses are not mediated by the products of these induced genes; signal-dependent post-translational modifications of existing proteins can also profoundly change cellular properties. We will discuss how Src activation by TLR3 changes cell migration, adhesion, and proliferation rates and how IRF-3 activation by RLR triggers a gene induction-independent pro-apoptotic pathway that provides strong antiviral protection.
The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes ...(ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN's protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7's antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action.
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