干扰素刺激基因15在人类免疫缺陷病毒感染中作用的研究进展

随着有效的联合抗反转录病毒疗法(combination antiretroviral therapy,cART)的普及,人类免疫缺陷病毒(human immunodeficiency virus,HIV)感染者的生存期逐步延长。这一过程中,HIV感染者自身免疫反应对免疫系统功能的恢复也发挥了至关重要的作用。HIV感染激活干扰素信号通路,诱导干扰素刺激基因(interferon-stimulated gene,ISG)上调表达,从而发挥抗病毒作用。其中,类泛素蛋白ISG15在HIV感染者中显著上调,通过ISG化抑制HIV颗粒的出芽和释放;而HIV的非结构蛋白则通过干扰ISG化过程或结合干扰素信号通路关键分子,逆转ISG15对病毒的抑制作用。本文从ISG15的生物学特性、在不同细胞亚群中的表达、抗病毒功能及病毒逃逸机制等方面进行综述,为进一步解析ISG15在HIV感染中扮演的角色、探索如何获得以抗HIV感染宿主因子为契机的治疗策略提供了思路。     

类泛素蛋白ISG15及其在先天免疫中的作用

病毒感染和干扰素刺激高等动物细胞,均可以强烈地诱导表达干扰素刺激基因15编码的蛋白ISG15,它是最早发现的类泛素修饰蛋白.虽然针对泛素及其修饰功能已进行了广泛而深入地研究,但对于ISG15共价修饰以及它的生物学功能了解甚少,有待进一步探讨.该领域的研究近几年有所突破,发现了有关ISG15修饰的酶系统,ISG15及其修饰系统在先天免疫以及干扰素信号调节中的重要作用.简要介绍ISG15的发现历史、生化性质、基因调控特点以及ISG15修饰系统中所涉及的酶,总结目前研究ISG15及其修饰与调节先天性免疫相关过程的一些最新进展.     

Vaccinia virus E3 protein prevents the antiviral action of ISG15

The ubiquitin-like modifier ISG15 is one of the most predominant proteins induced by type I interferons (IFN). In this study, murine embryo fibroblast (MEFs) and mice lacking the gene were used to demonstrate a novel role of ISG15 as a host defense molecule against vaccinia virus (VACV) infection. In MEFs, the growth of replication competent Western Reserve (WR) VACV strain was affected by the absence of ISG15, but in addition, virus lacking E3 protein (VVDeltaE3L) that is unable to grow in ISG15+/+ cells replicated in ISG15-deficient cells. Inhibiting ISG15 with siRNA or promoting its expression in ISG15-/- cells with a lentivirus vector showed that VACV replication was controlled by ISG15. Immunoprecipitation analysis revealed that E3 binds ISG15 through its C-terminal domain. The VACV antiviral action of ISG15 and its interaction with E3 are events independent of PKR (double-stranded RNA-dependent protein kinase). In mice lacking ISG15, infection with VVDeltaE3L caused significant disease and mortality, an effect not observed in VVDeltaE3L-infected ISG15+/+ mice. Pathogenesis in ISG15-deficient mice infected with VVDeltaE3L or with an E3L deletion mutant virus lacking the C-terminal domain triggered an enhanced inflammatory response in the lungs compared with ISG15+/+-infected mice. These findings showed an anti-VACV function of ISG15, with the virus E3 protein suppressing the action of the ISG15 antiviral factor.     

Cynoglossus semilaevis ISG15: A Secreted Cytokine-Like Protein That Stimulates Antiviral Immune Response in a LRGG Motif-Dependent Manner

ISG15 is an ubiquitin-like protein that is induced rapidly by interferon stimulation. Like ubiquitin, ISG15 forms covalent conjugates with its target proteins in a process called ISGylation, which in mammals is known to play a role in antiviral immunity. In contrast to mammalian ISG15, the function of teleost ISG15 is unclear. In this study, we identified and analyzed the function of an ISG15 homologue, CsISG15, from tongue sole (Cynoglossus semilaevis). CsISG15 is composed of 162 residues and possesses two tandem ubiquitin-like domains and the highly conserved LRGG motif found in all known ISG15. Expression of CsISG15 occurred in a wide range of tissues and was upregulated in kidney and spleen by viral and bacterial infection. In vitro study with primary head kidney (HK) lymphocytes showed that megalocytivirus infection caused induction of CsISG15 expression and extracellular release of CsISG15 protein. Purified recombinant CsISG15 (rCsISG15) activated HK macrophages and enhanced the expression of immune genes in HK lymphocytes, both these effects, however, were significantly reduced when the conserved LRGG sequence was mutated to LAAG. Further study showed that the presence of rCsISG15 during megalocytivirus infection of HK lymphocytes reduced intracellular viral load, whereas antibody blocking of CsISG15 enhanced viral infection. Likewise, interference with CsISG15 expression by RNAi promoted viral infection. Taken together, these results indicate that CsISG15, a teleost ISG15, promotes antiviral immune response and that, unlike mammalian ISG15, CsISG15 exerts its immunoregulatory effect in the form of an unconjugated extracellular cytokine. In addition, these results also suggest a role for the LRGG motif other than that in protein conjugation.     

ISG15 is critical in the control of Chikungunya virus infection independent of UbE1L mediated conjugation

Chikungunya virus (CHIKV) is a re-emerging alphavirus that has caused significant disease in the Indian Ocean region since 2005. During this outbreak, in addition to fever, rash and arthritis, severe cases of CHIKV infection have been observed in infants. Challenging the notion that the innate immune response in infants is immature or defective, we demonstrate that both human infants and neonatal mice generate a robust type I interferon (IFN) response during CHIKV infection that contributes to, but is insufficient for, the complete control of infection. To characterize the mechanism by which type I IFNs control CHIKV infection, we evaluated the role of ISG15 and defined it as a central player in the host response, as neonatal mice lacking ISG15 were profoundly susceptible to CHIKV infection. Surprisingly, UbE1L^−/− mice, which lack the ISG15 E1 enzyme and therefore are unable to form ISG15 conjugates, displayed no increase in lethality following CHIKV infection, thus pointing to a non-classical role for ISG15. No differences in viral loads were observed between wild-type (WT) and ISG15^−/− mice, however, a dramatic increase in proinflammatory cytokines and chemokines was observed in ISG15^−/− mice, suggesting that the innate immune response to CHIKV contributes to their lethality. This study provides new insight into the control of CHIKV infection, and establishes a new model for how ISG15 functions as an immunomodulatory molecule in the blunting of potentially pathologic levels of innate effector molecules during the host response to viral infection.     

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.     

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.     

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.     

Species Specificity of the NS1 Protein of Influenza B Virus: NS1 BINDS ONLY HUMAN AND NON-HUMAN PRIMATE UBIQUITIN-LIKE ISG15 PROTEINS*

Influenza B viruses, which cause a highly contagious respiratory disease every year, are restricted to humans, but the basis for this restriction had not been determined. Here we provide one explanation for this restriction: the species specificity exhibited by the NS1 protein of influenza B virus (NS1B protein). This viral protein combats a major host antiviral response by binding the interferon-α/β-induced, ubiquitin-like ISG15 protein and inhibiting its conjugation to an array of proteins. We demonstrate that the NS1B protein exhibits species-specific binding; it binds human and non-human primate ISG15 but not mouse or canine ISG15. In both transfection assays and virus-infected cells, the NS1B protein binds and relocalizes only human and non-human primate ISG15 from the cytoplasm to nuclear speckles. Human and non-human primate ISG15 proteins consist of two ubiquitin-like domains separated by a short hinge linker of five amino acids. Remarkably, this short hinge plays a large role in the species-specific binding by the NS1B protein. The hinge of human and non-human primate ISG15, which has a sequence that differs from that of other mammalian ISG15 proteins, including mouse and canine ISG15, is absolutely required for binding the NS1B protein. Consequently, the ISG15 proteins of humans and non-human primates are the only mammalian ISG15 proteins that would bind NS1B.     

病毒性肺炎患者外周血干扰素刺激基因15 mRNA表达特征的研究

通过分析病毒性肺炎患者外周血干扰素刺激基因15(interferon stimulated gene 15,ISG15)mRNA在感染状态下的特征性表达,探究ISG15与病毒性肺炎发病机制及疾病活动性之间的可能联系,为探索特异性呼吸道病毒感染的宿主生物标志物提供依据.本研究共收集157例社区获得性肺炎(communit...     

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.     

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.     

The ISG15/USP18 ubiquitin-like pathway (ISGylation system) in hepatitis C virus infection and resistance to interferon therapy

The ISG15/USP18 pathway modulates cellular functions and is important for the host innate immune response to chronic viral infections such as Hepatitis C Virus (HCV). Interferon stimulated gene 15 (ISG15) was the first ubiquitin-like protein modifier identified. As in ubiquitination, ISG15 conjugates to target proteins (ISGylation) through the sequential enzymatic action of activating E1, conjugating E2, and ligating E3 enzymes. ISGylation modulates signal transduction pathways and host anti-viral response. The ISGylation process is reversible through the action of an ISG15 protease, USP18. Ubiquitin-like specific protease 18 (USP18) has functions that are both ISG15-dependent and ISG15-independent; the importance of the ISG15/USP18 pathway to chronic HCV infection is illustrated by the consistent finding of increased levels of ISG15 and USP18 in the liver tissue of patients who do not respond to interferon-based treatments. Mechanistically, HCV seems to exploit the ISG15/USP18 pathway to promote viral replication and evade innate anti-viral immune responses.     

Covalent protein modification with ISG15 via a conserved cysteine in the hinge region

The ubiquitin-like protein ISG15 (interferon-stimulated gene of 15 kDa) is strongly induced by type I interferons and displays antiviral activity. As other ubiquitin-like proteins (Ubls), ISG15 is post-translationally conjugated to substrate proteins by an isopeptide bond between the C-terminal glycine of ISG15 and the side chains of lysine residues in the substrates (ISGylation). ISG15 consists of two ubiquitin-like domains that are separated by a hinge region. In many orthologs, this region contains a single highly reactive cysteine residue. Several hundred potential substrates for ISGylation have been identified but only a few of them have been rigorously verified. In order to investigate the modification of several ISG15 substrates, we have purified ISG15 conjugates from cell extracts by metal-chelate affinity purification and immunoprecipitations. We found that the levels of proteins modified by human ISG15 can be decreased by the addition of reducing agents. With the help of thiol blocking reagents, a mutational analysis and miRNA mediated knock-down of ISG15 expression, we revealed that this modification occurs in living cells via a disulphide bridge between the substrates and Cys78 in the hinge region of ISG15. While the ISG15 activating enzyme UBE1L is conjugated by ISG15 in the classical way, we show that the ubiquitin conjugating enzyme Ubc13 can either be classically conjugated by ISG15 or can form a disulphide bridge with ISG15 at the active site cysteine 87. The latter modification would interfere with its function as ubiquitin conjugating enzyme. However, we found no evidence for an ISG15 modification of the dynamin-like GTPases MxA and hGBP1. These findings indicate that the analysis of potential substrates for ISG15 conjugation must be performed with great care to distinguish between the two types of modification since many assays such as immunoprecipitation or metal-chelate affinity purification are performed with little or no reducing agent present.     

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.     

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.     

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.     

Viral defense, carcinogenesis and ISG15: novel roles for an old ISG

Recent studies have established that type I interferon modulates expression of large number of cellular genes. While the proteins encoded by some of these genes have a direct antiviral activity, the functions of the majority of the others have not yet been determined. One of the first identified IFN stimulated gene, encodes ubiquitin like protein ISG15 that is also expressed in response to different stress stimuli. Although it was shown that ISG15 functions as protein modifier, it has been only recently that the targets of ISG15 conjugation were identified. Recent studies have also revealed mechanism of ISG15 conjugation and its interaction with the ubiquitin conjugation pathway. This review is focused on the possible role of ISG15 in the antiviral response, regulation of cell growth and carcinogenesis.     

Stress at the intestinal surface: catecholamines and mucosa–bacteria interactions

Psychological stress has profound effects on gastrointestinal function, and investigations over the past few decades have examined the mechanisms by which neural and hormonal stress mediators act to modulate gut motility, epithelial barrier function and inflammatory states. With its cellular diversity and large commensal bacterial population, the intestinal mucosa and its overlying mucous environment constitute a highly interactive environment for eukaryotic host cells and prokaryotic bacteria. The elaboration of stress mediators, particularly norepinephrine, at this interface influences host cells engaged in mucosal protection and the bacteria which populate the mucosal surface and gut lumen. This review will address growing evidence that norepinephrine and, in some cases, other mediators of the adaptation to stress modulate mucosal interactions with enteric bacteria. Stress-mediated changes in this delicate interplay may shift the microbial colonization patterns on the mucosal surface and alter the susceptibility of the host to infection. Moreover, changes in host-microbe interactions in the digestive tract may also influence ongoing neural activity in stress-responsive brain areas.     

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.