Reexamination of the role of ubiquitin-like modifier ISG15 in the phenotype of UBP43-deficient mice

UBP43/USP18 was described as a specific protease that removes conjugated ubiquitin-like modifier ISG15 from target proteins. The severe phenotype of UBP43(-/-) mice characterized by premature death, brain cell injury, and deregulated STAT1 signaling was ascribed to an enhanced conjugation of ISG15. In contrast, no phenotypic changes were detected in ISG15(-/-) mice. To verify the role of ISG15 in the phenotype of UBP43(-/-) mice, we employed mice deficient for both ISG15 and UBP43. Here, we show that the phenotype of UBP43(-/-) mice was not rescued by the absence of ISG15, as evident from unchanged mortality, neurological symptoms, and occurrence of hydrocephalus. Also, the reported hypersensitivity of UBP43(-/-) mice to an interferon inducer, poly(I . C), was ISG15 independent. Furthermore, no evidence for a role of ISG15 in the modulation of STAT1 signaling or in the resistance against lymphocytic choriomeningitis virus and vesicular stomatitis virus was found. Presented results clearly demonstrate that the phenotypic alterations of UBP43(-/-) mice are not caused by the lack of ISG15 deconjugation and must be due to another, non-ISG15-mediated molecular mechanism.     

Ube1L and protein ISGylation are not essential for alpha/beta interferon signaling

The expression of ubiquitin-like modifier ISG15 and its conjugation to target proteins are highly induced by interferon (IFN) stimulation and during viral and bacterial infections. However, the biological significance of this modification has not been clearly understood. To investigate the function of protein modification by ISG15, we generated a mouse model deficient in UBE1L, an ISG15-activating enzyme. Ube1L-/- mice did not produce ISG15 conjugates but expressed free ISG15 normally. ISGylation has been implicated in the reproduction and innate immunity. However, Ube1L-/- mice were fertile and exhibited normal antiviral responses against vesicular stomatitis virus and lymphocytic choriomeningitis virus infection. Our results indicate that UBE1L and protein ISGylation are not critical for IFN-alpha/beta signaling via JAK/STAT activation. Moreover, using Ube1L/Ubp43 double-deficient mice, we showed that lack of UBP43, but not the increase of protein ISGylation, is related to the increased IFN signaling in Ubp43-deficient mice.     

ISG15, not just another ubiquitin-like protein

ISG15 is a ubiquitin-like protein containing two ubiquitin homology domains and becomes conjugated to a variety of proteins when cells are treated with type I interferon or lipopolysaccharide. Although ISG15 shares several common properties with those of other ubiquitin-like molecules, it is a unique member, whose expression and conjugation to target proteins are tightly regulated by specific signaling pathways, indicating it may be associated with specialized functions in innate immune system. Loss of UBP43 (USP18), a protease that specifically removes ISG15 from ISG15-modified proteins, in mice leads to decreased life span, brain cell injury, and hypersensitivity to interferon stimulation. In UBP43 deficient cells, interferon induces a prolonged Stat1 tyrosine phosphorylation and DNA binding, which result in a prolonged and enhanced activation of interferon-stimulated genes.     

The level of hepatitis B virus replication is not affected by protein ISG15 modification but is reduced by inhibition of UBP43 (USP18) expression

Hepatitis B virus (HBV) causes both acute and chronic infection of the human liver and is associated with the development of liver cirrhosis and hepatocellular carcinoma. UBP43 (USP18) is known as an ISG15-deconjugating enzyme and an inhibitor of type I IFN signaling independent of its enzyme activity. In this study, we examined the role of these two previously identified functions of UBP43 in the innate immune response to HBV viral infection. As an in vivo HBV replication model system, a replication-competent DNA construct was injected hydrodynamically into the tail veins of mice. Although the lack of ISG15 conjugation in the absence of ISG15-activating enzyme UBE1L (UBA7) did not affect the level of HBV replication, the steady-state level of HBV DNA was substantially reduced in the UBP43-deficient mice in comparison to the wild-type controls. In addition, introduction of short hairpin RNA against UBP43 resulted in substantially lower levels of HBV DNA at day 4 postinjection and higher levels of ISG mRNAs. These results suggest that HBV infection is more rapidly cleared if UBP43 expression is reduced. Furthermore, these results illustrate the therapeutic potential of modulating UBP43 levels in treating viral infection, especially for viruses sensitive to IFN signaling.     

Nonstructural protein 2 of porcine reproductive and respiratory syndrome virus inhibits the antiviral function of interferon-stimulated gene 15

Type I interferon (alpha/beta interferon [IFN-α/β]) stimulates the expression of interferon-stimulated gene 15 (ISG15), which encodes a ubiquitin-like protein, ISG15. Free ISG15 and ISG15 conjugates function in diverse cellular pathways, particularly regulation of antiviral innate immune responses. In this study, we demonstrate that ISG15 overexpression inhibits porcine reproductive and respiratory syndrome virus (PRRSV) replication in cell culture and that the antiviral activity of interferon is reduced by inhibition of ISG15 conjugation. PRRSV nonstructural protein 2 (nsp2) was previously identified as a potential antagonist of ISG15 production and conjugation. The protein contains a papain-like protease domain (PLP2) that plays a crucial role in the proteolytic cleavage of the PRRSV replicase polyproteins. PLP2 was also proposed to belong to the ovarian tumor domain-containing superfamily of deubiquitinating enzymes (DUBs), which is capable of inhibiting ISG15 production and counteracting ISG15 conjugation to cellular proteins. To determine whether this immune antagonist function could be selectively inactivated, we engineered a panel of mutants with deletions and/or mutations at the N-terminal border of the nsp2 PLP2-DUB domain. A 23-amino-acid deletion (amino acids 402 to 424 of the ORF1a-encoded protein) largely abolished the inhibitory effect of nsp2 on ISG15 production and conjugation, but no viable recombinant virus was recovered. A 19-amino-acid deletion (amino acids 402 to 420), in combination with a downstream point mutation (S465A), partially relieved the ISG15 antagonist function and yielded a viable recombinant virus. Taken together, our data demonstrate that ISG15 and ISGylation play an important role in the response to PRRSV infection and that nsp2 is a key factor in counteracting the antiviral function of ISG15.     

Interferon-inducible ubiquitin E2, Ubc8, is a conjugating enzyme for protein ISGylation

Protein ISGylation is unique among ubiquitin-like conjugation systems in that the expression and conjugation processes are induced by specific stimuli, mainly via the alpha/beta interferon signaling pathway. It has been suggested that protein ISGylation plays a special role in the immune response, because of its interferon-signal dependency and its appearance only in higher eukaryotic organisms. Here, we report the identification of an ISG15-conjugating enzyme, Ubc8. Like other components of the protein ISGylation system (ISG15, UBE1L, and UBP43), Ubc8 is an interferon-inducible protein. Ubc8 clearly mediates protein ISGylation in transfection assays. The reduction of Ubc8 expression by small interfering RNA causes a decrease in protein ISGylation in HeLa cells upon interferon treatment. Neither UbcH7/UbcM4, the closest homologue of Ubc8 among known ubiquitin E2s, nor the small ubiquitin-like modifier E2 Ubc9 supports protein ISGylation. These findings strongly suggest that Ubc8 is a major ISG15-conjugating enzyme responsible for protein ISGylation upon interferon stimulation. Furthermore, we established an assay system to detect ISGylated target proteins by cotransfection of ISG15, UBE1L, and Ubc8 together with a target protein to be analyzed. This method provides an easy and effective way to identify new targets for the ISGylation system and will facilitate related studies.     

The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase

The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-beta. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2-/- mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling.     

Rickettsia conorii infection stimulates the expression of ISG15 and ISG15 protease UBP43 in human microvascular endothelial cells

Rickettsia conorii, an obligate intracellular bacterium and the causative agent of Mediterranean spotted fever, preferentially infects microvascular endothelial cells of the mammalian hosts leading to onset of innate immune responses, characterized by the activation of intracellular signaling mechanisms, release of pro-inflammatory cytokines and chemokines, and killing of intracellular rickettsiae. Our recent studies have shown that interferon (IFN)-β, a cytokine traditionally considered to be involved in antiviral immunity, plays an important role in the autocrine/paracrine regulation of host defense mechanisms and control of R. conorii growth in the host endothelial cells. Here, we show that R. conorii infection induces the expression of ISG15 (an interferon-stimulated gene coding a protein of 17kD) and UBP43 (an ISG15-specific protease) at the levels of mRNA and protein and report the evidence of ISGylation of as yet unidentified target proteins in cultured human microvascular endothelium. Infection-induced expression of ISG15 and UBP43 requires intracellular replication of rickettsiae and production of IFN-β, because treatment with tetracycline and presence of an antibody capable of neutralizing IFN-β activity resulted in near complete attenuation of both responses. Inhibition of R. conorii-induced ISG15 by RNA interference results in significant increase in the extent of rickettsial replication, whereas UBP43 knockdown yields a reciprocal inhibitory effect. In tandem, these results demonstrate the stimulation of interferon-β-mediated innate immune mechanisms capable of perturbing the growth and replication of pathogenic rickettsiae and provide first evidence for ISG15-mediated post-translational modification of host cellular proteins during infection with an intracellular bacterium.     

Role of ISG15 protease UBP43 (USP18) in innate immunity to viral infection

Innate immune responses provide the host with an early protection barrier against infectious agents, including viruses, and help shape the nature and quality of the subsequent adaptive immune responses of the host. Expression of ISG15 (UCRP), a ubiquitin-like protein, and protein ISGylation are highly increased upon viral infection. We have identified UBP43 (USP18) as an ISG15 deconjugating protease. Protein ISGylation is enhanced in cells deficient in UBP43 (ref. 6). Here we have examined the role of UBP43, encoded by the gene Usp18, in innate immunity to virus infection. Usp18(-/-) mice were resistant to the fatal lymphocytic choriomeningitis and myeloencephalitis that developed in wild-type mice after intracerebral inoculation with lymphocytic choriomeningitis virus (LCMV) or vesicular stomatitis virus (VSV), respectively. Survival of Usp18(-/-) mice after intracerebral LCMV infection correlated with a severe inhibition of LCMV RNA replication and antigen expression in the brain and increased levels of protein ISGylation. Consistent with these findings, mouse embryonic fibroblasts (MEF) and bone marrow-derived macrophages from Usp18(-/-) mice showed restricted LCMV replication. Moreover, MEF from Usp18(-/-) mice showed enhanced interferon-mediated resistance to the cytopathic effect caused by VSV and Sindbis virus (SNV). This report provides the first direct evidence that the ISG15 protease UBP43 and possibly protein ISGylation have a role in innate immunity against viral infection.     

Enhanced antibacterial potential in UBP43-deficient mice against Salmonella typhimurium infection by up-regulating type I IFN signaling

ISG15 is an IFN-inducible ubiquitin-like protein and its expression and conjugation to target proteins are dramatically induced upon viral or bacterial infection. We have generated a UBP43 knockout mouse model that is lacking an ISG15-specific isopeptidase to study the biological role of the protein ISGylation system. We report that UBP43-deficient mice are hypersensitive to LPS-induced lethality and that TIR domain-containing adapter inducing IFN-beta --> IFN regulatory factor 3 --> type I IFN is the major axis to induce protein ISGylation and UBP43 expression in macrophages upon LPS treatment. In ubp43(-/-) macrophages, upon LPS treatment we detected increased expression of IFN-stimulated genes, including genes for several cytokines and chemokines involved in the innate immune response. The ubp43(-/-) mice were able to restrict the growth of Salmonella typhimurium more efficiently than wild-type mice. These results clearly demonstrate two aspects of IFN-signaling, a beneficial effect against pathogens but a detriment to the body without strict control.     

The interferon-inducible ubiquitin-protein isopeptide ligase (E3) EFP also functions as an ISG15 E3 ligase

The expression of the ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) are strongly activated by interferons. Accordingly, ISG15 expression and protein ISGylation are strongly activated upon viral and bacterial infections and during other stress conditions, suggesting important roles for the ISG15 system in innate immune responses. Here, we report the identification of the ubiquitin-protein isopeptide ligase (E3) EFP (estrogen-responsive finger protein) as the ISG15 E3 ligase for 14-3-3sigma protein. Like other known components of the protein ISGylation system (ISG15, UBE1L, UBP43, and UBC8), EFP is also an interferon-inducible protein. Expression of EFP small interfering RNA decreased the ISGylation of 14-3-3sigma in the 293T cell ISGylation system as well as in MCF-7 cells upon interferon treatment. Furthermore, the ISGylation enzyme activity of EFP was RING domain-dependent. These findings indicate that EFP is an ISG15 E3 ligase for 14-3-3sigma in vivo. The fact that both UBC8 and EFP are common components in the ubiquitin and ISG15 conjugation pathways suggests a mechanism whereby a limited set of enzymes accomplishes diverse post-translational modifications of their substrates in response to changes in environmental stimulations.     

The Antiviral Activities of ISG15

Post-translational protein modification is an important strategy for the regulation of the cell proteome independent of the need for new gene expression. Ubiquitin and ubiquitin-like modifiers mediate the regulation of protein levels, signaling pathways, vesicular trafficking, and many other cellular processes through their covalent conjugation to proteins. Interferon stimulated gene 15 (ISG15) is a ubiquitin-like modifier induced by type I interferon. In addition to conjugating to potentially hundreds of target proteins, ISG15 can be found in an unconjugated form both inside of the cell and released from interferon stimulated cells into the extracellular environment. Due to its robust expression after type I interferon stimulation and the broad panel of proteins that it targets, ISG15 has drawn much attention as a potential regulator of the immune response and has been shown to mediate protection in a number of different viral infection models. Here we will review the current state of the field of ISG15, the viruses against which ISG15 mediates protection, and the mechanisms by which ISG15 exerts antiviral activity.     

Microarray analysis reveals that Type I interferon strongly increases the expression of immune-response related genes in Ubp43 (Usp18) deficient macrophages

Type I interferon (IFN) contributes significantly to innate immune responses to pathogen infections in macrophages. Our previous studies demonstrate that Ubp43, an ISG15-specific isopeptidase, is highly expressed in macrophages and noncatalytically inhibits Type I IFN signaling. To understand the effect of Type I IFN and Ubp43 in macrophage activation, we analyzed the expression of IFN-beta stimulated genes in wild-type and Ubp43(-/-) bone marrow derived macrophages (BMMs). Here, we show that Ubp43 regulates IFN-beta stimulated genes at genome level. IFN hypersensitivity of Ubp43(-/-) BMMs resulted in the identification of 749 unique genes that are upregulated by IFN-beta, including a large group of previously unidentified IFN-stimulated genes. Functional analyses of these genes showed that Type I IFN strongly induced the expression of a group of immune response related genes, including genes for antigen presentation, antiviral responses, and chemokine and cytokine production. These results provide excellent biochemical support for the high resistance of viral and bacterial infection of Ubp43 knockout mice, suggesting that Ubp43 is a potential therapeutic target for the enhancement of immune responses against infections.     

ISG15 Regulates Peritoneal Macrophages Functionality against Viral Infection

Upon viral infection, the production of type I interferon (IFN) and the subsequent upregulation of IFN stimulated genes (ISGs) generate an antiviral state with an important role in the activation of innate and adaptive host immune responses. The ubiquitin-like protein (UBL) ISG15 is a critical IFN-induced antiviral molecule that protects against several viral infections, but the mechanism by which ISG15 exerts its antiviral function is not completely understood. Here, we report that ISG15 plays an important role in the regulation of macrophage responses. ISG15−/− macrophages display reduced activation, phagocytic capacity and programmed cell death activation in response to vaccinia virus (VACV) infection. Moreover, peritoneal macrophages from mice lacking ISG15 are neither able to phagocyte infected cells nor to block viral infection in co-culture experiments with VACV-infected murine embryonic fibroblast (MEFs). This phenotype is independent of cytokine production and secretion, but clearly correlates with impaired activation of the protein kinase AKT in ISG15 knock-out (KO) macrophages. Altogether, these results indicate an essential role of ISG15 in the cellular immune antiviral response and point out that a better understanding of the antiviral responses triggered by ISG15 may lead to the development of therapies against important human pathogens.     

UBP43 (USP18) specifically removes ISG15 from conjugated proteins.

UBP43 shows significant homology to well characterized ubiquitin-specific proteases and previously was shown to hydrolyze ubiquitin-beta-galactosidase fusions in Escherichia coli. In our assays, the activity of UBP43 toward Ub fusions was undetectable in vitro directing us to investigate the possibility of Ub-like proteins such as SUMO, Nedd8, and ISG15 as probable substrates. We consequently demonstrate that UBP43 can efficiently cleave only ISG15 fusions including native ISG15 conjugates linked via isopeptide bonds. In addition to commonly used methods we introduce a new experimental design featuring ISG15-UBP43 fusion self-processing. Deletion of the UBP43 gene in mouse leads to a massive increase of ISG15 conjugates in tissues indicating that UBP43 is a major ISG15-specific protease. UBP43 is the first bona fide ISG15-specific protease reported. Both ISG15 and UBP43 genes are known to be strongly induced by interferon, genotoxic stress, and viral infection. We postulate that UBP43 is necessary to maintain a critical cellular balance of ISG15-conjugated proteins in both healthy and stressed organisms.     

ISG15 and immune diseases

ISG15, the product of interferon (IFN)-stimulated gene 15, is the first identified ubiquitin-like protein, consisting of two ubiquitin-like domains. ISG15 is synthesized as a precursor in certain mammals and, therefore, needs to be processed to expose the C-terminal glycine residue before conjugation to target proteins. A set of three-step cascade enzymes, an E1 enzyme (UBE1L), an E2 enzyme (UbcH8), and one of several E3 ligases (e.g., EFP and HERC5), catalyzes ISG15 conjugation (ISGylation) of a specific protein. These enzymes are unique among the cascade enzymes for ubiquitin and other ubiquitin-like proteins in that all of them are induced by type I IFNs or other stimuli, such as exposure to viruses and lipopolysaccharide. Mass spectrometric analysis has led to the identification of several hundreds of candidate proteins that can be conjugated by ISG15. Some of them are type I IFN-induced proteins, such as PKR and RIG-I, and some are the key regulators that are involved in IFN signaling, such as JAK1 and STAT1, implicating the role of ISG15 and its conjugates in type I IFN-mediated innate immune responses. However, relatively little is known about the functional significance of ISG15 induction due to the lack of information on the consequences of its conjugation to target proteins. Here, we describe the recent progress made in exploring the biological function of ISG15 and its reversible modification of target proteins and thus in their implication in immune diseases.     

HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells

Type I interferon (IFN) stimulates expression and conjugation of the ubiquitin-like modifier IFN-stimulated gene 15 (ISG15), thereby restricting replication of a wide variety of viruses. Conjugation of ISG15 is critical for its antiviral activity in mice. HECT domain and RCC1-like domain containing protein 5 (HerC5) mediates global ISGylation in human cells, whereas its closest relative, HerC6, does not. So far, the requirement of HerC5 for ISG15-mediated antiviral activity has remained unclear. One of the main obstacles to address this issue has been that no HerC5 homologue exists in mice, hampering the generation of a good knock-out model. However, mice do express a homologue of HerC6 that, in contrast to human HerC6, can mediate ISGylation.Here we report that the mouse HerC6 N-terminal RCC1-like domain (RLD) allows ISG15 conjugation when replacing the corresponding domain in the human HerC6 homologue. In addition, sequences in the C-terminal HECT domain of mouse HerC6 also appear to facilitate efficient ISGylation. Mouse HerC6 paralleled human HerC5 in localization and IFN-inducibility. Moreover, HerC6 knock-down in mouse cells abolished global ISGylation, whereas its over expression enhanced the IFNβ promoter and conferred antiviral activity against vesicular stomatitis virus and Newcastle disease virus. Together these data indicate that HerC6 is likely the functional counterpart of human HerC5 in mouse cells, suggesting that HerC6(-/-) mice may provide a feasible model to study the role of human HerC5 in antiviral responses.