Skip to Main contentScienceDirectJournals BooksRegisterSign in Sign inRegisterJournals BooksHelpISG15ISG15 linked to UBE1L is transferred to UbcH8 (E2) and then to a target protein with the aid of an ISG15 E3 ligase, such as EFP and HERC5.From: Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease, 2010Related terms:InterferonUbiquitinC-TerminusInterferon Type ISTAT1Nested GeneInterferon-Stimulated GeneProteasesConjugationUbiquitinationView all TopicsDownload as PDFSet alertAbout this pageUbiquitin and Ubiquitin-like Protein ModifiersCaleb D. Swaim, ... Jon M. Huibregtse, in Methods in Enzymology, 2019AbstractISG15 is a ubiquitin-like protein (Ubl) that is expressed in response to Type 1 Interferon (IFN-α/β) signaling. Remarkably, ISG15 has three distinct biochemical activities involved in innate immune responses to viral and/or microbial infections. The canonical function of ISG15 is as a posttranslational modifier, and protein ISGylation has been demonstrated to be antiviral. A second intracellular function, independent of conjugation activity, is attenuation of IFN-α/β signaling at the interferon receptor, which appears to be important for terminating IFN responses. The third function of ISG15, and the focus of this chapter, is as an extracellular signaling molecule that promotes the secretion of Type 2 Interferon (IFN-γ) by Natural Killer (NK) cells. This function is important for control of microbial infections, including mycobacterial infections. Here, we describe methods for purification of ISG15, preparation, and culture of primary peripheral blood mononuclear cells (PBMCs) and NK-92 cells, assays for IL-12- and ISG15-dependent cytokine (IFN-γ and IL-10) secretion, and assays for initial intracellular signaling events triggered by extracellular ISG15.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687918305238Immunodeficiencies at the Interface of Innate and Adaptive ImmunityJacinta Bustamante, ... Jean-Laurent Casanova, in Clinical Immunology (Fifth Edition), 2019AR Complete ISG15 DeficiencyRecently, six patients with complete AR ISG15 deficiency have been identified (OMIM 616126).34,35 Three of the six patients suffered from clinical disease caused by BCG vaccine. The only features common to all patients were calcifications of the basal ganglia in the brain. The cellular phenotype was characterized by an impaired, but not abolished, IFN-γ production upon whole blood activation by BCG plus IL-12, as seen in patients with IL-12p40 or IL-12Rβ1 deficiency.1,34,35 The addition of recombinant ISG15 rescued the production of IFN-γ by T and NK cells of the patients. A high level of ISG mRNA was detected in all patients with ISG15 deficiency. The absence of intracellular ISG15 in the patients cells prevents the accumulation of USP18, a potent regulator of IFN-α/β signaling, and amplifies the IFN-α/β induced responses. This novel MSMD-causing gene acts like both an IFN-γ–inducing secreted cytokine and an intracellular negative regulator of IFN-α/β.34,35 These patients display not only MSMD but also a novel form of type I interferonopathy because intracellular human (but not mouse) ISG15 is paradoxically a negative regulator of IFN immunity.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780702068966000363Ubiquitin and Ubiquitin-like Protein ModifiersAnnette Aichem, ... Marcus Groettrup, in Methods in Enzymology, 20196 Concluding wordsSimilar to ubiquitin, SUMO-1/2/3, or ISG15, there are hundreds of substrates which become covalently conjugated to FAT10 in cells, but how FAT10ylation affects their function, intracellular levels, and localization has been investigated for only few of them. Moreover, FAT10 is the only known ubiquitin-like modifier which directly and efficiently targets its substrates for degradation by the 26S proteasome, but nevertheless, FAT10-mediated degradation differs mechanistically in many aspects from ubiquitin-dependent proteasomal degradation. Finally, the understanding of how FAT10 conjugation is regulated is far from complete. In spite of many interesting open questions, the number of laboratories involved in FAT10 research is still quite small. One reason for this might be that working with FAT10 is challenging as it easily precipitates both in cells and in vitro. By providing our validated experimental protocols in this chapter we hope to encourage more laboratories to investigate FAT10ylation and to circumvent the numerous pitfalls of FAT10 research we have experienced over the years.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0076687918305378Antiviral Immunity and Virus VaccinesIn Fenner s Veterinary Virology (Fifth Edition), 2017Interferon ResponsesIn 1957, Isaacs and Lindenmann reported that influenza virus-infected cells produce a nonviral protein they termed \"interferon” that can protect uninfected cells against the same (influenza virus) as well as unrelated viruses. It has since been determined that there are several types and subtypes of interferon and that these proteins are key elements of antiviral resistance at the cellular level. They also play a central role in both innate and adaptive immune responses to viral infections. A critical class of these proteins was collectively designated as type I interferon (IFN). These include IFN-α, which is encoded by several different genes in most species (eg, 14 in cattle and 27 in swine). How many of the IFN-α genes are used by any species in response to any infection event is not clearly defined. There are also 7 IFN-β genes in cattle and one in swine. In addition, IFN-τ, IFN-δ, IFN-ε, IFN-κ, and IFN-ω are also type I interferons. All of these protein hormones bind a common receptor, the IFN-α receptor (IFNAR). This cell surface protein is a heterodimer of IFNAR 1 and 2, and functions to transduce a signaling cascade of enzymes including the tyrosine and Janus kinases that induce signal transducers and activators of transcription, and interferon regulatory factors. Activation of this signaling cascade ultimately results in induction of the interferon response genes in cells (Fig. 4.1). Humans and animals with deficits in signaling pathways triggered by interferon often die of common viral diseases that are not usually fatal.Figure 4.1. Pathways of type I interferon (IFN) induction and receptor signaling. Recognition of viruses by pattern recognition receptors (PRRs) including Toll-like receptors (TLRs) and retinoic acid inducible gene (RIG-1) can lead to induction of genes encoding type I IFNs in the infected cell (left) which is mediated by several distinct signaling pathways. On the binding of type I IFNs to interferon α receptor (IFNAR) on a neighbouring uninfected cell (right) multiple downstream signaling pathways can be induced leading to a diverse range of biologic effects mediated by interferon stimulated genes (ISGs).From McNab, F., Mayer-Barber, K., Sher, A., Wack, A., O’Garra, A., 2015. Type I interferons in infectious disease. Nat. Rev. Immunol. 15, 87–103, with permission.Type II interferon, or IFN-γ, was originally reported as \"immune interferon.” This cytokine is central to many aspects of both innate and adaptive immunity and defines multiple subtypes of T lymphocytes. Type III interferon is designated as IFN-λ. In humans, these protein hormones were originally described as members of the interleukin 10 (IL-10) cytokine family because they are bound by IFN-λ receptor 1 and IL-10 receptor 2. IFN-λ1, 2, and 3 were first described as IL-29, IL-28A, and IL-28B, respectively. As with type I and type II interferons, the IFN-λs have cytokine activities in addition to their inherent antiviral action. In cattle and swine, there are 2 IFNλ genes reported to date, IFN-λ1 and IFN-λ3.Induction of type I interferon in virus-infected cells involves activation via an array of cellular receptors called pattern recognition receptors, which detect pathogen-associated molecules that are broadly specific to different classes of viruses. The binding of pathogen-associated molecules to these cellular receptors stimulates the transcription of numerous genes encoding proteins that are involved in innate and adaptive immune responses, including the activation of interferon production and secretion. Importantly, these responses may be triggered by several redundant pathways, both cytoplasmic and extracytoplasmic. One class of pattern recognition receptors are the Toll-like receptors (TLRs), so named because of their homology to the Toll genes of Drosophila. Different Toll-like receptors detect different pathogen-associated molecular patterns (PAMPs). For instance, TLR7 and TLR8 bind single-stranded RNA (ssRNA), thus detecting RNA virus infections, which then induces production of type I interferon. This is an important response to influenza and human immunodeficiency virus infections for example. In contrast, TLR3 detects double-stranded RNA (dsRNA), a critical intermediate of RNA virus genome replication that is not present in normal cells. These Toll-like receptors are predominantly located in the endosome, where they can readily detect viruses internalized after endocytosis, including viruses or their nucleic acid released from adjacent apoptotic or lysed cells. Cytosolic pathways for pathogen sensing and type I interferon induction also can occur via TLR-independent signaling involving cytoplasmic RNA helicase proteins such as retinoic acid inducible gene (RIG-1) and melanoma differentiation-associated gene 5 (MDA5). Other intracellular pathways include mitochondrial antiviral signaling protein (MAVS; also referred to as IPS-1), which mediates activation of transcription factors that induce interferon production (Fig. 4.1).Type I interferon released from virus-infected cells or activated innate response cells (see below) stimulates adjacent cells via interferon α receptor (IFNAR) binding (Fig. 4.1). This activates a signaling pathway leading to induction of the interferon response element. In mice, this results in the transcriptional activation of more than 300 interferon-stimulated genes (ISGs). In large mammals and humans it is clear that a similar group of interferon-stimulated genes is activated following binding of type I interferons to their specific receptors. Most of these genes encode proteins that regulate either signaling pathways or transcription factors that amplify interferon production, whereas others promote an antiviral state via cytoskeletal remodeling, apoptosis, posttranscriptional events (mRNA editing, splicing, degradation), or posttranslational modifications.Proteins proven to be critical to the induction of the interferon-induced antiviral state include:•ISG15, which is a ubiquitin homolog that is not constitutively expressed in cells. Addition of ubiquitin to cellular proteins is key to regulation of the innate immune response, and ISG15 apparently can exert a similar function with more than 150 target proteins in interferon-stimulated cells. Activities of ISG15 can regulate all aspects of the interferon pathway, including induction, signaling, and action.•MxGTPase is a hydrolyzing enzyme that, like ISG15, is not constitutively expressed. The enzyme is located in the smooth endoplasmic reticulum, where it affects vesicle formation, specifically targeting the viral nucleocapsid in virus-infected cells to prevent virus maturation.•The protein kinase R (PKR) pathway is constitutively expressed at only a very low level, but is quickly upregulated by IFNAR signaling. In the presence of dsRNA, the protein kinase phosphorylates elongation (translation) initiation factor eIF2α and prevents recycling of cyclic nucleotides (GDP), which in turn halts protein synthesis. This interferon-induced pathway is especially important for inhibiting replication of reoviruses, adenoviruses, vaccinia and influenza viruses, amongst many others.•The 2′–5′ oligoadenylate synthetase (OAS) pathway, like the PKR pathway, is constitutively expressed only at a low level. After IFNAR stimulation and in the presence of dsRNA, this enzyme produces oligoadenylates with a distinctive 2′–5′ linkage, as contrasted with the normal 3′–5′ lineage. These 2′–5′ oligoadenylates in turn activate cellular RNase that degrades RNA, which cleaves viral messenger and genomic RNA. Picornaviruses are especially susceptible to inhibition by this pathway, as is West Nile virus.In summary, type I interferon is produced after virus infection of many different types of cells, and the interferon released from these cells then induces an antiviral state in adjacent cells. In addition, cells of the innate immune system can be activated to secrete interferon by virus infection, including nonproductive infections or by their \"sensing” of viral infection, which augments the level of antiviral signaling and the local antiviral state in tissue. In many instances, this response may control, or even eliminate, a viral infection before the development of systemic infection or the occurrence of overt disease. If the virus overwhelms the early innate immune response then systemic spread occurs and disease may be detected clinically.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780128009468000040ElaD Peptidase (Escherichia coli)Hidde Ploegh, in Handbook of Proteolytic Enzymes (Third Edition), 2013Activity and SpecificityAnalysis of elaD’s specificity was performed not only for ubiquitin and derivatives but was extended to the ubiquitin homologs Nedd8 and ISG15. Both share significant sequence identity to ubiquitin, and ISG15 is even identical at the critical C-terminal region. No reactivity of elaD towards ISG15-vinylsulfone was observed, and the binding of elaD to Nedd8-vinylsulfone was significantly weaker than to the ubiquitin probe. Furthermore, elaD catalyzed hydrolysis of the C-terminal peptide bond in ubiquitin-AMC, but not in Nedd8-AMC. These features clearly distinguish elaD from the more promiscuous viral CE peptidases. Given the similarity of primary, secondary and tertiary structure among these Ubls, hydrolysis of ubiquitin by elaD reflects a highly specific interaction. With the exception of A. avenae, no bacterial strain in the dataset used encodes a homolog of ubiquitin, making it likely that the substrate of elaD is indeed eukaryotic ubiquitin. Moreover, the ortholog of elaD in Salmonella – sseL – has a virulence factor and displays deubiquitinating activity in vitro and in vivo [2]. This enzyme is encoded by all currently sequenced Salmonellae, but only present as a pseudogene in Shigellae [3]. Likewise, elaD is not essential for E. coli under laboratory conditions [4]. A comparison of the genomes of 16 sequenced E. coli strains reveals that elaD is present in the commensal E. coli strain K12, and in all intestinal pathogenic strains (EAEC, EHEC, EIEC, EPEC, ETEC), but absent in all ExPEC strains (APEC, NMEC, UPEC) (see Catic et al. [1] for a full Table).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123822192005342Coronavirus Papain-like PeptidasesKiira Ratia, ... Susan C. Baker, in Handbook of Proteolytic Enzymes (Third Edition), 2013Activity and SpecificityA soluble and active form of SARS-CoV PLpro was expressed in E. coli and purified using column chromatography [12]. This 35 kilodalton protein was evaluated for the ability to process a variety of substrates, including a FRET-based peptide representing polyprotein recognition sequences: EEdans-RELNGG↓APIKDabcyl-S. To test the de-ubiquitinating activity of the enzyme, PLpro was characterized with several fluorescent, ubiquitin (Ub)-related substrates, including full-length Ub-AMC, ISG15-AMC, and the short peptide RLRGG-AMC, representing the 5 C-terminal residues of ubiquitin and ISG15 [10,12]. All assays were performed at 25°C, in 20 mM HEPES, pH 7.5, 0.1 mg/mL BSA, and 5 mM DTT. With the exception of ISG15-AMC, none of the substrates saturated the enzyme up to the concentrations tested, and therefore pseudo first-order rate constants, kapp were reported (kapp~kcat/Km for non-saturable enzymes): EEdans-RELNGG↓APIKDabcyl-S, 0.0244±0 0003 min−1 µM−1; Ub-AMC, 4.48±0.1−1 min−1 µM−1; RLRGG-AMC, 0.61±0.01 min−1 µM−1. PLpro is considerably more active with the ISG15-AMC substrate, producing kcat and Km values of 370±16 min−1 and 2.3±0.3 µM, respectively [10].View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123822192004932How Does Vaccinia Virus Interfere With Interferon?Geoffrey L. Smith, ... Yongxu Lu, in Advances in Virus Research, 20182.3 IFN-Stimulated GenesIFNs induce the transcription of hundreds of ISGs. While some are upregulated by all IFNs, others are upregulated selectively by distinct IFNs. For example, IRF1 is upregulated preferentially in response to IFNγ (Der et al., 1998). This type of specificity in IFN-induced responses is critical for a highly coordinated and fine-tuned innate immune response as well as a heightened antiviral state within cells, thereby halting the spread of infection. Of the many hundreds of ISGs, well-characterized examples include ISG15, protein kinase R (PKR), and 2′-5′-oligoadenylate synthase (OAS).ISG15 is a 15-kDa protein that has sequence similarity to ubiquitin and is one of the most strongly induced genes upon viral infection (Blomstrom et al., 1986; Haas et al., 1987). ISG15 can become conjugated to both cytoplasmic and nuclear proteins via an isopeptidase bond in a process known as ISGylation. Conjugation of ISG15 to target proteins such as IRF3, RIG-I, human MxA, and PKR can increase their stability preventing their degradation (Villarroya-Beltri et al., 2017). Additionally, ISGylation through attachment of ISG15 can modulate JAK–STAT signaling (Malakhova et al., 2003).PKR becomes activated by autophosphorylation upon binding dsRNA, a product often formed during infection by both RNA and DNA viruses. Subsequently, PKR phosphorylates the eukaryotic translational initiation factor 2 alpha (eIF2α) at serine 51, thereby preventing recycling of eIF2α and preventing further protein synthesis (Meurs et al., 1990).2′-5′-OAS is also activated upon binding to dsRNA. Once activated, 2′-5′-OAS synthesizes 2′-5′-oligoadenylates using ATP as a substrate. In turn, 2′-5′-oligoadenylates activate the ribonuclease RNAaseL, which degrades both viral and cellular mRNAs as well as cellular tRNAs and rRNAs, leading to inhibition of translation (Silverman, 2007).The importance of the IFN system for protection against viruses has been demonstrated in many ways, not least by the presence of many IFN antagonists in virus genomes. This is illustrated particularly well with VACV that has been known for decades to be relatively resistant to IFN and to confer resistance to IFN to other viruses such as vesicular stomatitis virus (Thacore and Youngner, 1973; Whitaker-Dowling and Youngner, 1983). The observation that large deletions in terminal regions of the VACV genome could arise spontaneously during passage of virus in tissue culture and that this was prevented in the presence of IFN suggested that genes conferring resistance to IFN were present in these terminal regions (Paez and Esteban, 1985). We now know that VACV interferes with the production and action of IFNs in many ways.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/S0065352718300034Interferons: Cellular and Molecular Biology of Their Actions☆Dhananjaya V Kalvakolanu, ... Sudhakar Kalakonda, in Encyclopedia of Cancer (Third Edition), 2019Human IFN-Related Inborn Errors That Increase InfectionsA number of compelling studies showed genetic mutations/variations in IFN signaling pathways or downstream products enhances sensitivity to infectious pathogens (Table 5). Earliest studies showed certain humans, who do not display any overt defects in hematopoietic system, were predisposed to infection with attenuated (e.g., BCG) and environmental mycobacteria. Defined now as’Mendelian susceptibility to mycobacterial disease (MSMD)’, these rare conditions are due to genetic defects in IFN signaling pathways. Nine of the 18 genes described in MSMD mapped to critical players involved in IFN signaling such as IFNGR1, IFNGR2, STAT1, IL12B, IL12RB1, ISG15, NEMO, CYBB (codes for the gp91 subunit of the phagocyte NADPH oxidase) and IRF8. The MSMD patients do not produce IL12RB1, ISG15, NEMO and IRF8 owing to defects in IFNGR1, IFNGR2, STAT1 and IRF8 in IFN-γ signaling. Although MSMD patients do not recapitulate all the clinical features, they are also increasingly susceptible to salmonellosis, candidiasis and tuberculosis, and rarely to infections with other intramacrophagic bacteria, fungi, or parasites and some viruses. Genetic defects (heterozygotic) in the human IRF7 gene are also connected to life-threatening influenza infections. Cells from these individuals do amplify the initial IFN response that leads to the production of IFN-α and IFN-λ. Apart from these, autosomal recessive defects due to TYK2 loss down regulate IFN-α/β, and IFN-λ, resulting in a hyper-susceptibility to mycobacterial, Salmonella, staphylococcal and viral infections. It should be noted that these phenotypes are likely to be more complex given the requirement of TYK2 for IL-6, IL-10, IL-12 and IL-23 signaling. Patients with autosomal recessive STAT1 deficiency are broadly susceptible to viruses, including herpes simplex virus-1 (HSV-1) infections, which may cause HSV-1 encephalitis (HSE). IL-10R2-deficient patients would be expected to be unresponsive to IFN-λ, for this receptor is not utilized by either IFN-α/β or IFN-γ. Genome-wide sequencing and association studies on chronic mucocutaneous candidiasis have identified mutations in STAT1 (in its coiled-coil domain) in some patients. These mutations were also found in patients with disseminated disease caused by other fungal pathogens such as Histoplasma capsulatum and Coccidioides immitis. Strikingly, these gain-of-function and dominant mutations promote disease, suggesting a detrimental role for IFN-α/β in fungal infections, possibly through a suppression of TH17 responses and/or an excessive differentiation of TH1 cells. Since other cytokines, for example, IFN-γ and IL-27, also employ STAT1 for signal transduction, they may also be responsible for disease promotion.Table 5. Human inborn errors that dysregulate IFN-signaling pathways and increase susceptibility to infectionsGeneInheritanceAlleleCytokinesDisease Phenotype/InfectionsSTAT1ARLOF, HPOIFN-α and IFN-β, IFN-γ, IFN- λ, IL-27mycobacteria, virusesADaLOF, HPOIFN-γmycobacteriaIFNGR1ARLOF, HPOIFN-γmycobacteriaADLOFIFN-γmycobacteriaIFNGR2ARLOF, HPOIFN-γmycobacteriaIRF7ADLOFIFN-αLife threatening influenza infectionsIRF8ADHPOIFN-γ and others?MycobacteriaIL12BARLOFIL-12, IL-23mycobacteria, Salmonella, CMCIL12RB1ARLOFIL-12, IL-23mycobacteria, Salmonella, CMCIKBKGXRHPOmultiple mycobacteriaCYBBXRHPOmycobacteriaTYK2ARLOFIFN-α, IFN-β, IFN-λ, IL-6, IL-10, IL-12, IL-23mycobacteria, salmonella, viruses, Staphylococci, atopybSTAT1ADaHPRIFN-α, IFN-β, IFN-γ, IFN-λ, IL-27cCMCIL10RB2ARLOFIL-10, IL-22, IFN-λcolitisAR, autosomal-recessive; AD, autosomal-dominant; XR, X-linked recessive; LOF, loss-of-function (null); HPO, hypomorphic; HPR, hypermorphic (gain of function); colitis, early-onset inflammatory colitis; CMC, chronic mucocutaneous candidiasis.aMutations were found in the DBD and tyrosine phosphorylation domain.belevated IgE.cSTAT1-hyperactive patients lack IL-17 T cells.Based on Casanova, J.L. et al. (2012) Immunity. 36, 515–428.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780128012383961166Genomics in Pathogenesis of CirrhosisN.A. Shackel, ... J. McHutchison, in Essentials of Genomic and Personalized Medicine, 2010Hepatitis C Virus InfectionAcute and chronic hepatitis C virus (HCV) infection has been studied using functional genomics techniques. These experiments have examined acute HCV infection in primates, as well as the sequelae of chronic infection. The results from microarray studies of acute HCV infection in the chimpanzee are intriguing. Acute HCV infection is characterized by a rapid (within 2 weeks) as well as a delayed induction (up to 6 weeks) of genes involved associated with the innate immune response (Bigger et al., 2001). Most of these genes are associated with interferon gene expression and are known as interferon response genes (IRG including ISG15, ISG16, CXCL9, CXCL10, Mx-1, stat-1, 2′5′-oligoadenylate synthetase and p27). Viral clearance appears to be associated with rapid induction of these IRG s. Overall HCV persistence appears to be associated with comparatively less induction of IRG s compared to viral clearance. Further, chronic HCV-related liver injury appears to be characterized by an IRG-associated chronic Th1 immune response, which is insufficient to clear the virus but is chronic and responsible for ongoing liver injury. The situation with interferon treatment of HCV infected individuals, which is aimed at viral eradication, is similar to acute infection as an immune response, characterized by a significant increase in IRG expression following treatment, is associated with a greater likelihood of a sustained long-term therapy response. Clearly, the immune response to HCV drives fibrogenesis, as interferon administration is associated with a reduction intrahepatic inflammation and fibrosis even in the absence of a long-term virological clearance following treatment.View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123749345000507Avian Immunosuppressive Diseases and ImmunoevasionKarel A. Schat, Michael A. Skinner, in Avian Immunology (Second Edition), 201416.4.4 Immunoevasion Mechanism of the Avian OrthomyxovirusesMembers of the Orthomyxoviridae family are enveloped viruses enclosing eight segments of negative-strand RNA. The best known are of the Influenzavirus A genus, mostly as a consequence of their role in human pandemics. As is now widely appreciated since the emergence of the H5N1 strain, influenza A viruses are primarily an infection of birds, originating in waterfowl, and readily passing to a wide range of birds both wild and domestic.It was by using influenza virus infection that IFN was discovered in 1957 [208]. Subsequently, it was one of the first viruses for which a resistance mechanism to IFN (activation of host protein p58) was elucidated [209]. Since 1997 and the emergence of the highly pathogenic H5N1 avian influenza virus (AIV), which is capable of causing high mortality in humans, NS1 has been identified as the major viral IFN resistance protein [210–213]. Moreover, a determinant of the virulence of H5N1 (D92E) has been located on NS1 [214]. These studies have been performed in the context of mammalian IFN responses, which is appropriate because of the clinical threat and practical because the avian IFN response is not fully understood and there is a lack of avian reagents.Perhaps because of the lack of biochemical assays and reagents, one of the few direct studies of the interaction between AIV and avian IFN was performed with live virus [215]. The study concluded that there was considerable heterogeneity within and between virus populations in their ability to induce and resist avian IFN-I. This heterogeneity was ascribed to their presence in the population of subpopulations that had packaged multiple genome segments. It was presumed that those particles that had packaged multiple segments encoding IFN resistance protein(s) displayed higher resistance, although this characteristic was not necessarily inherited. Such heterogeneity illustrates one of the potential complications of using live-recombinant influenza viruses to dissect out the role of particular molecular determinants of IFN resistance.Studies with the mammalian system have revealed that NS1 is a very complex, multi-functional protein. It has at least three major functions: (1) binding dsRNA to block IFN, (2) inhibiting host gene expression by preventing mRNA splicing and nuclear export, and (3) enhancing viral mRNA translation. All of these functions merit further investigation in mammalian and avian hosts.Resistance of influenza virus (influenza A virus unless otherwise specified) to IFN was initially believed to be primarily due to activation of a cellular inhibitor, p58(IPK), or PKR [209]. The mechanism of activation is still not known. Another mechanism involved the ubiquitin-like host protein ISG15, which is one of the most predominant proteins induced by type-I IFN [216]. Influenza B virus strongly induces ISG15, but a specific region of the influenza B virus NS1 protein (NS1B), which includes part of its effector domain, blocks the ability of ISG15 to become covalently linked to its target proteins by inhibiting its UBE1L-mediated activation. The influenza A virus NS1 protein does not bind ISG15, but inhibits its synthesis [217].Subsequently, however, NS1 was shown to play a major role in resistance to IFN. Specifically, NS1 mutants were able to replicate only in IFN-defective cell lines [211]. NS1, normally 230 amino acids long in influenza A virus, is encoded by virus RNA segment 8. The amino terminus (73 residues) of NS1, which dimerizes [218], is capable of binding dsRNA [219]—albeit at relatively low affinity [217]—thereby preventing activation of the dsRNA-dependent protein kinase, PKR [220]. NS1 also binds poly-adenylated RNA, inhibiting nuclear export of mRNAs [221], and a stem bulge in U6 snRNA, inhibiting pre-mRNA splicing in vitro and in vivo [221]. Both of these activities can down-regulate expression of cellular genes. They can also prevent IFN production by binding to IRF-3, blocking its kinase-mediated activation [213], and by blocking NF-κB activation [222]. Although expression of NS1 alone was reported to induce apoptosis in MDCK and HeLa cells [223], in the context of a viral infection of mammalian or avian cells, its IFN-regulatory activity makes it anti-apoptotic [224]. NS1 selectively enhances translation of viral but not cellular mRNAs by binding eIF4GI, PABP1 and the 5’ UTRs of vmRNAs [225,226].It is interesting that the region of NS1 (aa 81–131) binding eIF4GI spans the location of the known virulence mutation (aa 92) and that the region of PABP1 to which NS1 binds is not conserved evolutionarily. The C terminus, or effector domain, down-regulates formation and export of cellular mRNAs by binding to the 30 kDa subunit of CPSF and to PABII [227,228]. The dsRNA-binding activity of NS1 can be abrogated by mutating two basic residues (R38 and K41 to A). In MDCK cells, virus thus mutated [229] failed to inhibit IFN-β and replicated to lower titers. On passage, a better replicating virus emerged with an S42G mutation that did not improve dsRNA binding but had intermediate virulence in mice.Recent study of the modulation of avian IFN by AIV has focused on the role of NS1 and of PB2. There are many difficulties with studying the induction of IFN responses in vivo, not least in standardizing the nature of the inducing virus; this can depend on the strain and on its passage history. Penski et al. [230] showed that NS1 deletion mutants of HPAIV (H5N1 and H7N7) induced higher levels of IFN β in CEFs than did parental virus. Liniger et al. [231] showed that NS1 from HPAIV also controls IFN β expression in the chicken macrophage HD-11 cell line. However, NS1 deletion mutants of HPAIV still manage to control IFN β levels in infected chickens [230], suggesting that other mechanisms are involved. Liniger et al. [231] demonstrated that HPAIV PB2 could block induction of IFN β expression in the immortalized chicken fibroblast DF-1 cell line when induced by over-expression of the chicken homologs of VISA/CARDIF or mda-5.The role of an ESEV motif found at the C-terminus of HPAIV NS1 proteins, but not in seasonal human AIV (which bear the motif RSKV), has been investigated by Zielecki et al. [232] The motif represents a ligand for PDZ domains of cellular proteins. It attenuates virus replication in human, murine, and duck cells but not in CEFs. Nevertheless, it appears to have little influence on virulence in either mice or chickens.The possibility of using NS1 mutant AIV as live-attenuated vaccines was explored by Steel et al. [233] Viruses with internal deletions of NS1 stimulated higher levels of IFN β expression in mice and were attenuated in chickens. They also stimulated protective immunity against HPAIV challenge (the mutants also contained an E residue at position of PB2 to restrict replication and virulence in mammals).View chapterPurchase bookRead full chapterURL: https://www.sciencedirect.com/science/article/pii/B9780123969651000169Recommended publicationsInfo iconBiochemical and Biophysical Research CommunicationsJournalCytokineJournalJournal of Molecular BiologyJournalCell ReportsJournalBrowse books and journalsAbout ScienceDirectRemote accessShopping cartAdvertiseContact and supportTerms and conditionsPrivacy policyWe use cookies to help provide and enhance our service and tailor content and ads. By continuing you agree to the use of cookies.Copyright © 2021 Elsevier B.V. or its licensors or contributors. 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