ISG15 Is Counteracted by Vaccinia Virus E3 Protein and Controls the Proinflammatory Response against Viral Infection

Conjugation of ISG15 inhibits replication of several viruses. Here, using an expression system for assaying human and mouse ISG15 conjugations (ISGylations), we have demonstrated that vaccinia virus E3 protein binds and antagonizes human and mouse ISG15 modification. To study ISGylation importance in poxvirus infection, we used a mouse model that expresses deconjugating proteases. Our results indicate that ISGylation restricts in vitro replication of the vaccinia virus VVΔE3L mutant but unconjugated ISG15 is crucial to counteract the inflammatory response produced after VVΔE3L infection.     

Vaccinia virus E3 prevents sensing of Z-RNA to block ZBP1-dependent necroptosis

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Nitrosylation of ISG15 prevents the disulfide bond-mediated dimerization of ISG15 and contributes to effective ISGylation

The expression of the ubiquitin-like molecule ISG15 (UCRP) and protein modification by ISG15 (ISGylation) are strongly activated by interferon, genotoxic stress, and pathogen infection, suggesting that ISG15 plays an important role in innate immune responses. Inducible nitric-oxide synthase (iNOS) is induced by the similar stimuli as ISG15 and enhances the production of nitric oxide (NO), a pleiotropic free radical with antipathogen activity. Here, we report that cysteine residues (Cys-76 and -143 in mouse, Cys-78 in human) of ISG15 can be modified by NO, and the NO modification of ISG15 decreases the dimerization of ISG15. The mutation of the cysteine residue of ISG15 to serine improves total ISGylation. The NO synthase inhibitor S-ethylisothiourea reduces endogenous ISGylation. Furthermore, ectopic expression of iNOS enhanced total ISGylation. Together, these results suggest that nitrosylation of ISG15 enhances target protein ISGylation. This is the first report of a relationship between ISGylation and nitrosylation.     

Emerging role of ISG15 in antiviral immunity

Cells express a plethora of interferon-stimulated genes (ISGs) in response to viral infection. Among these is ISG15, a ubiquitin-like protein (UBL) that can be covalently attached to both host and viral proteins. Here we review recent advances toward understanding the role and mechanism of ISG15 modification in antiviral defense.     

Negative regulation of ISG15 E3 ligase EFP through its autoISGylation

The function of ubiquitin-like protein ISG15 and protein modification by ISG15 (ISGylation) has been an enigma for many years. Recently, the research of ISGylation has been accelerated by the identification of the enzymes involved in the ISG15 conjugation process. Our previous study identified the interferon inducible protein EFP as an ISG15 isopeptide ligase (E3) for 14-3-3σ. In this study, we show that ISG15 E3 ligase EFP can be modified by ISG15. Two ubiquitin E2 conjugating enzymes, UbcH6 and UbcH8, can support ISGylation of EFP. The Ring-finger domain of EFP is important for its ISGylation. Full-length EFP can enhance the ISGylation of Ring domain deleted EFP, indicating EFP can function as an ISG15 E3 ligase for itself. We also determined the ISGylation site of EFP and created its ISGylation resistant mutant EFP-K117R. Compared to the wild-type EFP, this mutant further increases the ISGylation of 14-3-3σ. Thus we propose that autoISGylation of EFP negatively regulates its ISG15 E3 ligase activity for 14-3-3σ.     

mHERC6 is the essential ISG15 E3 ligase in the murine system

Posttranslational protein modification by ubiquitin and ubiquitin-like modifiers (UBLs) is mediated by a hierarchical cascade of conjugating enzymes and affects multiple biological processes within the cell. Interferon-stimulated gene 15 (ISG15) is an UBL, which is strongly induced by type I Interferon and ISG15 modification was shown to play an essential role in antiviral defense. While hHERC5 is the major E3 ligase for ISG15 modification in humans, ISGylation in the murine systems at the level of E3 ligases was weakly characterized as rodent genomes lack a direct homologue of hHERC5. Here, we show that mHERC6 is strongly induced by different pathogen-associated molecular patterns (PAMPs) in a type I Interferon receptor (IFNAR1) dependent manner. We demonstrate that mHERC6 is essential for endogenous murine ISGylation and thus represents the dominant ISG15 E3 ligase in mice. In contrast to its human homologue, mHERC6 is also capable to mediate conjugation of human ISG15.     

Crystal structure of the interferon-induced ubiquitin-like protein ISG15

The biological effects of the ISG15 protein arise in part from its conjugation to cellular targets as a primary response to interferon-alpha/beta induction and other markers of viral or parasitic infection. Recombinant full-length ISG15 has been produced for the first time in high yield by mutating Cys78 to stabilize the protein and by cloning in a C-terminal arginine cap to protect the C terminus against proteolytic inactivation. The cap is subsequently removed with carboxypeptidase B to yield mature biologically active ISG15 capable of stoichiometric ATP-dependent thiolester formation with its human UbE1L activating enzyme. The three-dimensional structure of recombinant ISG15C78S was determined at 2.4-A resolution. The ISG15 structure comprises two beta-grasp folds having main chain root mean square deviation (r.m.s.d.) values from ubiquitin of 1.7 A (N-terminal) and 1.0 A (C-terminal). The beta-grasp domains pack across two conserved 3(10) helices to bury 627 A2 that accounts for 7% of the total solvent-accessible surface area. The distribution of ISG15 surface charge forms a ridge of negative charge extending nearly the full-length of the molecule. Additionally, the N-terminal domain contains an apolar region comprising almost half its solvent accessible surface. The C-terminal domain of ISG15 was superimposed on the structure of Nedd8 (r.m.s.d. = 0.84 A) bound to its AppBp1-Uba3 activating enzyme to model ISG15 binding to UbE1L. The docking model predicts several key side-chain interactions that presumably define the specificity between the ubiquitin and ISG15 ligation pathways to maintain functional integrity of their signaling.     

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 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.     

Interferon-mediated ISG15 conjugation restricts dengue virus 2 replication

ISGylation, an ubiquitin-like post-translational modification by ISG15, has been reported to participate in the interferon (IFN)-mediated antiviral response. In this study, we analyzed the functional role of ISGylation in dengue virus 2 (DENV-2) replication. Overexpression of ISG15 was found to significantly suppress the amount of extracellular infectious virus released, while intracellular viral RNA was unaffected. This effect was not observed with a conjugation-defective ISG15 mutant. In addition, extracellular virus infectivity was decreased by ISG15 overexpression. To further clarify the role of ISGylation in the anti-DENV-2 response, we depleted endogenous ISG15 by RNA interference and analyzed the virus production in the absence or presence of type-I IFN. Results showed a significant reduction in extracellular DENV-2 RNA levels for cells treated with IFN, and that these DENV-2 RNA levels could be partially restored by the ISG15 knockdown. Among various DENV-2 proteins, NS3 and NS5 were subjected to the ISGylation. These results demonstrate that IFN-inducible ISGylation suppresses DENV-2 particle release, and that ISG15 is one of the mediators of IFN-induced inhibition of DENV-2 replication. ISG15 therefore functions as a host antiviral factor against DENV-2 infection.     

Influenza B virus NS1 protein inhibits conjugation of the interferon (IFN)-induced ubiquitin-like ISG15 protein

Of the several hundred proteins induced by interferon (IFN) alpha/beta, the ubiquitin-like ISG15 protein is one of the most predominant. We demonstrate the novel way in which the function of the ISG15 protein is inhibited by influenza B virus, which strongly induces the ISG15 protein: a specific region of the influenza B virus NS1 protein, which includes part of its effector domain, blocks the covalent linkage of ISG15 to its target proteins both in vitro and in infected cells. We identify UBE1L as the E1 enzyme that catalyzes the first activation step in the conjugation of ISG15, and show that the NS1B protein inhibits this activation step in vitro. Influenza A virus employs a different strategy: its NS1 protein does not bind the ISG15 protein, but little or no ISG15 protein is produced during infection. We discuss the likely basis for these different strategies.     

Proteasomes modulate conjugation to the ubiquitin-like protein,ISG15

ISG15 is a ubiquitin-like protein that is induced by interferon and microbial challenge. Ubiquitin-like proteins are covalently conjugated to cellular proteins and may intersect the ubiquitin-proteasome system via common substrates or reciprocal regulation. To investigate the relationship between ISG15 conjugation and proteasome function, we treated interferon-induced cells with proteasome inhibitors. Surprisingly, inhibition of proteasomal, but not lysosomal, proteases dramatically enhanced the level of ISG15 conjugates. The stimulation of ISG15 conjugates occurred rapidly in the absence of protein synthesis and was most dramatic in the cytoskeletal protein fraction. Inhibition of ISG15 conjugation by ATP depletion abrogated the proteasome inhibitor-dependent increase in ISG15 conjugates, suggesting that the effect was mediated by de novo conjugation, rather than protection from proteasomal degradation or inhibition of ISG15 deconjugating activity. The increase in ISG15 conjugates did not occur through a stabilization of the ISG15 E1 enzyme, UBE1L. Furthermore, simultaneous modification of proteins by both ISG15 and ubiquitin did not account for the proteasome inhibitor-dependent increase in ISG15 conjugates. These findings provide the first evidence for a link between ISG15 conjugation and proteasome function and support a model in which proteins destined for ISG15 conjugation are proteasome-regulated.     

泛素样蛋白ISG15抗病毒机制研究进展

ISG15(Interferon stimulated gene 15,ISG15)蛋白是由干扰素诱导产生的一种泛素样蛋白分子,分子量大小约为15kD。ISG15同泛素分子相类似可以被共价结合于其他蛋白分子上,这种现象称为ISG化(ISGylation)现象。ISG化系统包括ISG15、UBE1L、UBCH8和HERC5四类蛋白分子,协同完成ISG化过程。ISG15及ISG化系统在抗病毒反应中具有重要作用。近几年对于ISG15的抗病毒作用和机制的研究已经有了很大的突破,ISG15的抗病毒作用也越来越受到人们重视,了解清楚ISG15抗病毒机制对于研制新的抗病毒药物及提出新的抗病毒策略具有重要意义。本文对ISG15在不同种病毒中的抗病毒机制研究进展进行了简要综述。     

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.     

Link between the ubiquitin conjugation system and the ISG15 conjugation system: ISG15 conjugation to the UbcH6 ubiquitin E2 enzyme

ISG15 is a ubiquitin-like protein that is upregulated on treatment with interferon. ISG15 is considered to be covalently conjugated to cellular proteins through a sequential reaction similar to that of the ubiquitin conjugation system consisting of E1/E2/E3 enzymes: UBE1L and UbcH8 have been reported to function as E1 and E2 enzymes, respectively, for ISG15 conjugation. Several cellular proteins have been identified as targets for ISG15 conjugation, but the roles of ISG15 conjugation remain unclear. In this study, we found that UbcH6 and UbcH8, E2 enzymes for ubiquitin conjugation, are covalently modified by ISG15. We also found that UbcH6 is capable of forming a thioester intermediate with ISG15 through Cys131. We determined that the Lys136 residue near the catalytic site Cys131 is the ISG15 conjugation site in UbcH6. We isolated ISG15-modified and unmodified UbcH6 proteins, and analyzed their abilities to form thioester intermediates with ubiquitin. A ubiquitin thioester intermediate was detected in the case of unmodified UbcH6, but not in that of ISG15-modified UbcH6, strongly suggesting that ISG15 conjugation to UbcH6 suppresses its ubiquitin E2 enzyme activity. Thus, we provide evidence for a link between the ubiquitin conjugation system and the ISG15 conjugation system.     

Crystal structure of human ISG15 protein in complex with influenza B virus NS1B

ISG15 (interferon-stimulated gene 15), the first ubiquitin-like protein (UBL) identified, has emerged as an important cellular antiviral factor. It consists of two UBL domains with a short linker between them. The covalent attachment of ISG15 to host and viral proteins to modify their functions, similar to ubiquitylation, is named ISGylation. Influenza B virus NS1B protein antagonizes human but not mouse ISGylation because NS1B exhibits species specificity; it only binds human and non-human primate ISG15. Previous studies have demonstrated that the N-terminal UBL domain and linker of ISG15 are required for the binding by NS1B and that the linker plays a large role in the species specificity, but the structural basis for them has not been elucidated. Here we report the crystal structure of human ISG15 in complex with NS1B at a resolution of 2.0 Å. A loop in the ISG15 N-terminal UBL domain inserts into a pocket in the NS1B dimer, forming a high affinity binding site. The nonspecific van der Waals contacts around the ISG15 linker form a low affinity site for NS1B binding. However, sequence alignment reveals that residues in the high affinity site are highly conserved in primate and non-primate ISG15. We propose that the low affinity binding around the ISG15 linker is important for the initial contact with NS1B and that the stable complex formation is largely contributed by the following high affinity interactions between ISG15 N-terminal UBL domain and NS1B. This provides a structural basis for the species-specific binding of ISG15 by the NS1B protein.