The host response to viral infection includes the induction of type I interferons and the subsequent upregulation of hundreds of interferon-stimulated genes. Ubiquitin-like protein ISG15 is an interferon-induced protein that has been implicated as a central player in the host antiviral response. Over the past 15 years, efforts to understand how ISG15 protects the host during infection have revealed that its actions are diverse and pathogen-dependent. In this Review, we describe new insights into how ISG15 directly inhibits viral replication and discuss the recent finding that ISG15 modulates the host damage and repair response, immune response and other host signalling pathways. We also explore the viral immune-evasion strategies that counteract the actions of ISG15. These findings are integrated with a discussion of the recent identification of ISG15-deficient individuals and a cellular receptor for ISG15 that provides new insights into how ISG15 shapes the host response to viral infection.
During pathogen invasion, the host elicits various defence mechanisms to protect the host. Type I interferons have a central role in regulating this response through the induction of hundreds of interferon-stimulated genes (ISGs) (reviewed in ref.1). Among these ISGs, ubiquitin-like protein ISG15 is one of the most strongly2 and rapidly3 induced, and recent work has shown that it can directly inhibit viral replication and modulate host immunity.
ISG15 is a member of the ubiquitin family, which includes ubiquitin and ubiquitin-like modifiers (Ubls). Ubiquitin and Ubls are involved in the regulation of a variety of cellular activities, including protein stability, intracellular trafficking, cell cycle control and immune modulation. Whereas some Ubls are constitutively present in the host cell, others are induced by different stimuli. ISG15 and the members of the enzymatic cascade that mediate ISG15 conjugation (ISGylation) are strongly induced by type I interferons3. ISG15 can be covalently conjugated onto target proteins via an enzymatic cascade, yet the fate of these modified proteins is still largely unknown. In addition, ISG15 exists as an unconjugated protein that has been reported to function as a cytokine and can also interact with various intracellular protein partners. Progress has been made in defining some of the mechanisms by which ISGylation of both viral and host proteins inhibit viral replication and the viral evasion strategies that have evolved to circumvent ISG15, yet recent advances in the field have highlighted the complexity of this pathway. There is increasing evidence that unconjugated ISG15 can regulate viral replication and host responses through both non-covalent protein interactions and its action as a cytokine. Human genetic evidence from ISG15-null patients has revealed the importance of ISG15 in the regulation of the type I interferon response4. ISG15-null patients display type I interferon autoinflammation and no apparent increase in susceptibility to viral infection, as compared with ISG15-deficient mice, indicating functional diversity between species5.
In this Review, we discuss the basic biology and characteristics of ISG15 and the ISGylation pathway, how ISG15 regulates viral replication, the immunomodulatory properties of ISG15 and the viral evasion strategies that have evolved to circumvent ISG15.
ISG15 and the ISGylation pathway
ISG15, first identified from the study of type I interferon-treated cells6,7, is composed of two ubiquitin-like domains that have ~30% amino acid sequence homology to ubiquitin, linked by a short hinge region7,8,9,10. In addition to being strongly induced by type I interferons, ISG15 is also induced by viral and bacterial infections11,12, lipopolysaccharide (LPS)13, retinoic acid14 or certain genotoxic stressors15, indicating that the expression of ISG15 represents a host response to pathogenic stimuli.
ISG15 exists as a 17 kDa precursor protein that is rapidly processed into its mature 15 kDa form via protease cleavage to expose a carboxy-terminal LRLRGG motif11,16. ISG15 is covalently conjugated to target proteins through this motif by a three-step process known as ISGylation (reviewed in ref.17) (Fig. 1). To date, hundreds of putative targets of ISGylation have been identified using mass spectrometry; however, only a subset of these has been validated18,19 (Table 1). The functional consequence of ISGylation is still poorly understood. Unlike ubiquitylation, ISGylation does not appear to directly target proteins for proteasome-mediated degradation20. Studies of specific ISGylated proteins have found that ISG15 can compete with ubiquitin for ubiquitin binding sites on a protein, thereby indirectly regulating protein degradation21. In addition, ISG15-ubiquitin mixed chains have been observed and may negatively regulate the turnover of ubiquitylated proteins22. However, for every ISGylated protein studied, only a small fraction of the total pool of a protein is modified by ISG15, making it a challenge to understand how ISGylation can impact the overall function of a protein. One possibility is that modification of a protein alters its cellular localization and function, as was seen with the ISGylation of filamin B23. In addition, for proteins that multimerize or polymerize, the modification of only a small fraction of the protein could disrupt the assembly of protein complexes, as has been observed with influenza B virus (IBV) and human papilloma virus (HPV) (see discussion below).
ISGylation can be reversed by the deconjugating enzyme Ubl carboxy-terminal hydrolase 18 (USP18)24,25. The specificity of USP18 for ISG15 is achieved through the hydrophobic interaction between a hydrophobic patch in USP18 and a hydrophobic region unique to ISG15 (ref.25). Of note, independent of its deconjugating activity, USP18 also binds to the second chain of the interferon α/β receptor 2 (IFNAR2) complex, competing with Janus kinase 1 (JAK1) for binding and, therefore, functions as a negative regulator of interferon signalling26. Thus, studies involving USP18 need to be interpreted with caution to differentiate between effects on ISG15 conjugation or dysregulated interferon signalling27.
ISG15 also exists as an unconjugated protein. In vivo, ISG15 has been detected in the serum of patients treated with interferon and in virally infected mice28,29,30,31. Extracellular ISG15 has been proposed to function as a cytokine with several immunomodulatory activities, including the induction of natural killer (NK) cell proliferation32, the stimulation of interferon-γ (IFNγ) production5 and the induction of dendritic cell maturation33, and to function as a chemotactic factor for neutrophils34. Its mechanism of release is unclear as ISG15 does not contain a leader signal; however, recent studies have shown that it could be released via non-conventional secretion, including in exosomes35, through the release of neutrophil granules from secretory lysosomes or via apoptosis36. Recently, lymphocyte function-associated antigen 1 (LFA1) was identified as a cell surface receptor for extracellular ISG15. Binding of ISG15 to LFA1 stimulated the release of IFNγ and interleukin-10 (IL-10) from IL-12 primed cells37. The impact that extracellular ISG15 exerts during viral infection remains to be determined.
It has recently been shown that, similar to ubiquitin, free, intracellular ISG15 can non-covalently bind to intracellular proteins and modulate their functions3,38 (Fig. 2). Free intracellular ISG15 has been shown to bind to and block the enzymatic activities of enzymes39,40. ISG15 has also been shown to regulate type I interferon signalling by stabilizing USP18 (ref.4) and by interacting with leucine-rich repeat-containing protein 25 (LRRC25) to mediate the autophagic degradation of retinoic acid-inducible gene I protein (RIG-I; also known as DDX58)41. In the following sections, we discuss recent insights into ISGylation and the role of free intracellular and extracellular ISG15 during viral infections.
ISG15 and viral infection
Early on, it was hypothesized that ISG15 would regulate the host antiviral response42. This stemmed from the rapid upregulation of ISG15 and members of the conjugation cascade by both type I interferons and several viruses43.
Early in vitro experiments used either the overexpression or knockdown of mouse or human ISG15 in cultured cells and found that ISG15 inhibited the growth of many viruses (reviewed in refs44,45, Fig. 3). Additional evidence that ISG15 functions as an antiviral molecule came from the study of infected mice in which ISG15 or genes of the conjugation cascade were knocked out (Fig. 3). Mice lacking ISG15 or the ISG15 E1 enzyme, ubiquitin-activating enzyme E1 homologue (UBE1L; also known as UBA7), were more susceptible to Sindbis virus46, influenza A virus (IAV) and IBV30,46,47 than wild-type mice. During infection with IBV, mice lacking ISG15 or UBE1L displayed a 3–4 log increase in virus in their lungs compared with wild-type mice, and cells derived from these mice supported increased viral replication, supporting the hypothesis that protein ISGylation restricts viral replication30. Studies with coxsackievirus B3 virus (CVB3) also confirmed an antiviral role for ISG15 that is mediated through its conjugation activity48. Both Isg15−/− and Ube1l−/− mice infected with CVB3 displayed more severe myocarditis, increased viral loads and increased lethality following infection48. The development of Usp18-knock-in mice (in which the deconjugating activity of USP18 was disrupted while leaving its ability to regulate interferon signalling intact) revealed that an accumulation of ISG15 conjugates resulted in increased resistance to infection during IBV and vaccinia virus infection49. In these examples, ISG15 protected the host by functioning as a bona fide antiviral protein, inhibiting viral replication in a conjugation-dependent manner. Whether these targets are viral or host proteins is still under investigation. Perhaps the strongest evidence that ISG15 has an important antiviral role is the increasing number of viral immune-evasion proteins that target the ISG15 pathway. Efforts are now focused on determining the mechanisms by which ISG15 regulates these responses (see discussion below and Fig. 4).
However, recent findings have challenged the notion that the dominant function of ISG15 is as an antiviral protein that directly inhibits viral replication. First, Isg15−/− mice are not susceptible to all viruses, and even for virus challenge experiments in which Isg15−/− mice have increased lethality, it is not always due to increased viral replication31,50. As discussed below, ISG15 can regulate cytokine responses31 and the host damage and repair response50. In addition, ISG15 has been shown to regulate cellular processes that include autophagy40,41,51 and metabolism52. Regulation of these processes could indirectly affect the outcome during viral infection. Second, the identification of ISG15-null patients who have a type I interferon autoinflammatory condition and, to date, no increased susceptibility to viral infection raises questions as to whether ISG15 is an essential antiviral molecule (reviewed in ref.45) (Box 1). This phenotype is in contrast to Isg15−/− mice that do not display type I interferon autoinflammation53, indicating that ISG15 may have divergent functions between different species. These observations increase our understanding of the ISG15 pathway but must also be taken into consideration when interpreting future studies on ISG15. For the remainder of this Review, we explore our understanding of the mechanism by which ISG15 regulates viral replication and the host response in both mice and humans.
Direct effects of ISG15 on viral replication
Recent studies have shown that the ISGylation of both host and viral proteins and the non-covalent binding of ISG15 to host proteins can disrupt viral replication. Several of these examples are discussed below.
ISGylation of viral proteins
ISGylation, through the localization of E3 ISG15–protein ligase HERC5 to polyribosomes, can target nascent proteins, making viral proteins, which are the dominant proteins within an infected cell, likely targets54. Although an extensive characterization of protein ISGylation during different viral infections has not been reported, several viral proteins have been identified as substrates for ISG15 conjugation (Fig. 3). In these studied examples, ISGylation of viral proteins can disrupt their interaction with host pathways that are required for replication, disrupt the oligomerization of viral proteins and/or the geometry of the virus, or disrupt viral protein function, resulting in reduced viral replication or the alteration of the host immune response.
The first viral protein that was found to be modified was the non-structural protein 1 of IAV (NS1/A)55,56. NS1/A is crucial to viral replication as it inhibits the induction of type I interferons57, blocks the activation of protein kinase R (PKR)58, selectively enhances viral mRNA translation59 and interferes with cellular mRNA processing60,61. Modification of the lysine at position 41 (K41) of NS1/A inhibited the nuclear translocation of NS1/A by disrupting its interaction with importin-α55, leaving the virus susceptible to inhibition by interferon. In a second study, ISGylation of NS1/A at distinct sites disrupted its interaction with several binding partners, including the amino terminus of PKR, the RNA-binding domain of NS1/A, U6 small nuclear RNA (snRNA) and double-stranded RNA (dsRNA), limiting its ability to disrupt the host antiviral response56. Together, these findings provided the initial evidence that ISG15 can modify viral proteins and directly antagonize virus replication.
The CVB3 protease 2 A (2APro) is also targeted for ISGylation48. The 2APro protein mediates the cleavage of the mammalian eukaryotic translation initiation factor 4γ1 (eIF4G1), resulting in shut-off of host cell protein synthesis, which in turn promotes viral replication. ISGylation of 2APro hinders the cleavage of eIF4G during CVB3 infection, diminishing host cell shut-off and reducing CVB3 replication48.
Studies of IBV and HPV have suggested that ISGylation of viral proteins blocks oligomerization, disrupting the function and geometry of viral complexes. Oligomerization of IBV nucleoprotein (NP) forms the viral ribonucleoprotein (vRNP), which is required for viral RNA synthesis. ISGylated IBV NP acts as a dominant-negative inhibitor of the oligomerization of unmodified NP, which restricts viral RNA synthesis and reduces IBV replication62. The HPV capsid protein L1 can also be ISGylated and then incorporated into viral particles. Both the number and infectivity of particles that have incorporated ISGylated L1 protein were found to be decreased, possibly owing to alterations in the geometry of the viral capsid54.
In addition to disrupting viral replication, ISGylation of viral proteins can dampen the host innate immune response. The ISGylation of NS1/A disrupted its ability to interact with components of the interferon response, such as PKR, U6 snRNA and dsRNA, limiting the ability of NS1/A to disrupt the innate immune response56. ISGylation also inhibited human cytomegalovirus (HCMV) gene expression and virion release. HCMV pUL26 is known to inhibit tumour necrosis factor-α (TNFα)-induced nuclear factor-κB (NF-κB) activation63. It was shown that the HCMV pUL26 protein forms both covalent and non-covalent interactions with ISG15. ISGylation of pUL26 altered pUL26 stability and inhibited its ability to suppress NF-κB signalling64. Therefore, the ISGylation of viral proteins can also interfere with the viral modulation of the host immune response.
Altogether, these examples illustrate the ability of ISGylation of viral proteins to reduce the efficiency and quality of viral progeny production and to limit the ability of viral proteins to regulate the host immune response.
Inhibition of virus egress
Several studies have found that ISG15 can impact virus egress. In these examples, it is not the ISGylation of viral proteins but rather the modification of host proteins that are required for viral release that are impacted by ISG15 (Fig. 3).
The first evidence of ISG15 inhibiting virus release came from studies on HIV-1 replication. Co-transfection of a plasmid expressing ISG15 with HIV-1 proviral DNA inhibited the release of HIV-1 but had no impact on HIV-1 protein production65. Expression of ISG15 inhibited the mono-ubiquitylation of the HIV-1 Gag polyprotein and disrupted the interaction between Gag and the host tumour susceptibility gene 101 protein (TSG101), both of which are required for HIV-1 budding and release. Recent studies also found that the transport of IAV haemagglutinin (HA) to the cell surface in a semi-intact cell system was inhibited by the ISGylation of TSG101. In this study, HA transport was restored when samples were treated with deISGylases such as USP18 or the ovarian tumour domain (OTU)-containing L protein of Crimean–Congo haemorrhagic fever virus (CCHFV)66. A recent study further indicated that ISG15 conjugation decreases the number of multivesicular bodies (MVBs) and impairs exosome secretion by triggering the aggregation and degradation of MVB proteins by lysosomes, including TSG101 (ref.67).
ISG15 was also shown to inhibit budding and release of Ebola virus-like particles (VLPs) and avian sarcoma leukosis virus (ASLV). Release of Ebola VLPs requires the ubiquitylation of the viral matrix protein VP40, which is mediated by host ubiquitin protein ligase NEDD4 (ref.68). ISG15, when co-expressed with VP40, inhibited Ebola VLP release. ISG15 inhibited the activity of NEDD4 and blocked the ubiquitylation of VP40 and the interaction between NEDD4 and ubiquitin E2 conjugating enzymes, preventing ubiquitin from being transferred to NEDD4 (refs39,69). Interestingly, a recent study found that ITCH, another E3 ubiquitin ligase, also interacts with VP40 to regulate viral budding via an identical protein domain that is used by NEDD4. Whether the anti-budding function of ISG15 extends to ITCH ligases as well remains unknown70. ISG15 also inhibited ASLV VLP release by inhibiting the recruitment of the host ATPase vacuolar protein sorting-associated protein 4A (VPS4A) to the endosomal sorting complex required for transport (ESCRT), which is required for ASLV budding71,72,73. This inhibition correlated with ISGylation of the host protein charged multivesicular body protein 5 (CHMP5), which is required for VPS4A recruitment. Therefore, ISG15 is able to limit the replication of some viruses through covalent and non-covalent modifications of host proteins that are involved in protein sorting and transport pathways.
Modulation of viral latency
Recent studies have implicated ISG15 in the regulation of viral latency. Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma, the most common HIV/AIDS-associated cancer worldwide. Transcriptional analysis of KSHV-infected primary human oral fibroblasts identified a series of strongly induced ISGs, including ISG15. Knockdown of ISG15 expression with small interfering RNA (siRNA) in these latently infected cells increased the expression of viral lytic genes and increased virion release, implicating ISG15 in the maintenance of KSHV latency74. One proposed mechanism for this was through the regulation of specific KSHV microRNAs that are known to modulate KSHV latency74. In a second study, KSHV infection was reactivated in primary effusion lymphoma cells by various means, and both ISG15 and ISG15 conjugate levels were found to be increased, along with activation of the type I interferon system75. Knockdown of either ISG15 or the E3 ligase HERC5 resulted in an increase in KSHV reactivation and an increase in the production of infectious virus75. These results suggest that ISG15 conjugation modulates both KSHV replication and reactivation from viral latency.
Indirect effects of ISG15 on viral infection
In addition to direct effects on viral replication, recent studies have also found that ISG15 influences the host response by functioning as an immunomodulatory protein, regulating the host damage and repair response during viral infection and modulating host signalling pathways that can indirectly limit or alter viral pathogenesis. In some cases, these actions are mediated by ISGylation of target proteins, and more recently, it has been found that unconjugated ISG15 can mediate these effects.
Immunomodulatory functions of ISG15
Early reports noted that ISG15 could be released from cells and that recombinant ISG15 could stimulate IFNγ production. This was recently confirmed in a study of individuals with ISG15 deficiency that presented with increased susceptibility to mycobacterial infection5 (Box 1). In this study, the production of IFNγ by NK cells and lymphocytes that was induced by ISG15 was increased when the cells were co-stimulated with IL-12. Earlier studies also implicated that the cytokine activity of ISG15 drives NK cell proliferation, dendritic cell maturation and neutrophil recruitment. Recently, LFA1 was identified as a cell surface receptor for ISG15, and its binding to this receptor mediated the release of IFNγ and IL-10 from cells pretreated with IL-12 (ref.37). Whether LFA1 also contributes to other activities that have been attributed to ISG15 and whether the cytokine-like activity of ISG15 has a role during viral infection remain to be elucidated.
The unconjugated form of ISG15 has also been shown to counteract the inflammatory response during viral infection. During chikungunya infection, neonatal mice lacking ISG15 were found to be more susceptible to viral infection; however, this protection was independent of UBE1L-mediated ISGylation, and the increased lethality observed in the Isg15−/− mice was not due to an increase in viral titres31. Instead, the infected Isg15−/− mice developed an exaggerated immune response, displaying a significant increase in pro-inflammatory cytokines and chemokines compared with wild-type or Ube1−/−mice31. Preliminary data indicate that death of the animals occurred in a manner that is consistent with a cytokine storm, and the survival of Isg15−/− mice could be prolonged when they were treated with TNFα-blocking antibody before infection76. In a separate study, ISG15-deficient mice were more susceptible to vaccinia virus infection and produced elevated levels of the pro-inflammatory cytokine IL-6 (ref.77). Taken together, these findings suggest that unconjugated ISG15 negatively regulates the production of pro-inflammatory cytokines and chemokines during certain viral infections. It is still unclear whether these effects are mediated by intracellular or extracellular unconjugated ISG15 and which cell types are responsible for the increased cytokine production.
Regulation of host damage and repair pathways
The host response to a pathogen includes the upregulation of genes that limit pathogen replication (disease resistance) and protect the host from tissue damage, independent of controlling pathogen burden (disease tolerance)78. Recent findings support a role for ISG15 in the regulation of disease tolerance. In a mouse model of IAV and Sendai virus infection (SeV), ISG15 protected mice from virus-induced lethality47,79. Both in vitro and in vivo analyses revealed that the ISG15-mediated protection neither restricted viral replication nor modulated cytokine or chemokine production within the lung tissue. Instead, it was determined that ISG15 regulated the damage and/or repair of the respiratory epithelium following infection50. To date, this is the only evidence that the ISG15 pathway regulates disease tolerance during viral infection. However, recent studies identified pathways that are targeted by ISG15 and are involved in homeostasis, including the regulation of apoptosis and autophagy23,40,51,80,81. It will be important to determine if ISG15 facilitates disease tolerance by coordinating these pathways.
Modulation of host signalling pathways that impact viral infection
Proteomic studies have identified hundreds of host proteins that are ISGylated upon interferon stimulation18,19. Many of these are ISGs that are involved in the regulation of the innate immune response, including signal transducer and activator of transcription 1 (STAT1), JAK1 (ref.82), RIG-I, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1)19, PKR and interferon-regulated resistance GTP-binding protein MxA (also known as Mx1)19. For a subset of these potential targets, modification has been validated, and the impact of ISGylation on their function has been investigated in detail (Table 1).
PKR is an interferon-induced protein that binds to dsRNA and, once activated, can phosphorylate the eukaryotic translation initiation factor eIF2α and inhibit cellular mRNA translation. PKR is ISGylated on lysines 69 and 159 after interferon or LPS stimulation83. ISGylated PKR exhibited an RNA-independent, constitutive activation that resulted in decreased protein synthesis83. However, it remains unclear whether ISGylation of PKR results in direct antagonism of virus replication.
The ISGylation of interferon regulatory factor 3 (IRF3) and STAT1 interferes with ubiquitylation and subsequent degradation of these proteins79,84. IRF3 is a transcription factor that, once phosphorylated, moves from the cytoplasm to the nucleus to form a complex with CREB-binding protein (CREBBP) and activates the transcription of IFNα, IFNβ and additional ISGs. STAT1 is a member of the STAT protein family and functions as a transcription factor involved in the upregulation of genes induced by type I, II and III interferons. During SeV infection, IRF3 is ISGylated, and this inhibits the interaction between IRF3 and peptidyl-prolyl cis–trans isomerase NIMA-interacting 1 (PIN1), preventing the ubiquitylation and subsequent degradation of IRF3 (ref.79). Similarly, STAT1 is ISGylated in human cells82, which was found to maintain the levels of phosphorylated, activated STAT1 and downstream signalling. In both cases, the result of ISGylation is a more robust interferon response that limits viral replication. Recent studies of individuals with inherited ISG15 deficiency who displayed signs of type I interferon autoinflammation4,5 (Box 1) revealed that intracellular unconjugated ISG15 has immunomodulatory functions. In vitro characterization of cells isolated from these patients showed elevated type I interferon secretion and a hyperresponsiveness to type I interferon stimulation, including prolonged STAT1 and STAT2 phosphorylation. This was attributed to the low level of USP18 protein expression in these cells4. This defect could be restored by either wild-type or non-conjugatable ISG15, implicating non-covalent interactions between ISG15 and USP18. Mechanistic studies demonstrated that ISG15 binds to and stabilizes USP18 by preventing its ubiquitylation by S-phase kinase-associated protein 2 (SKP2)4 (Fig. 2). These results suggest that human intracellular unconjugated ISG15 is crucial for USP18-mediated downregulation of type 1 interferon signalling during viral infections. Consistent with this, human ISG15-deficient cells primed with interferons displayed prolonged ISG expression, which in some cases provided resistance to viral infection53. Interestingly, this enhanced type I interferon signalling has not been observed in mice (Box 1), raising interesting questions about the divergent function of ISG15 between species.
The regulation of interferon signalling by ISG15 and USP18 also contributes to the unexpected proviral activity for ISG15 that has been associated with chronic viral hepatitis. The expression of ISG15 and members of its conjugation cascade was found to be upregulated in individuals persistently infected with hepatitis C virus (HCV) who had failed treatment with IFNα85,86,87. Consistent with this, in vitro studies also showed that knockdown of ISG15 increased the sensitivity of HCV-infected cells to IFNα or IFNα–ribavirin therapies87,88. Mechanistic studies found that ISG15 expression was induced under these conditions by unphosphorylated interferon-stimulated gene factor 3 (U-ISGF3)89 (Fig. 1). U-ISGF3 is a tripartite transcription factor composed of interferon regulatory factor 9 (IRF9) and unphosphorylated STAT1 and STAT2. The level of U-ISGF3 is significantly increased in response to IFNλ and IFNβ during chronic HCV infection and sustains the expression of ISG15 and a subset of ISGs, which restricts HCV replication. The sustained ISG15 expression led to stabilization of USP18 levels, which decreased signalling through the type I interferon receptor84,89. Therefore, the regulation of interferon signalling that is mediated by ISG15 contributes to the maintenance of chronic HCV.
ISG15 can also modulate the metabolic activities and function of macrophages during the host antiviral response52. Cells lacking ISG15 were deficient in mitochondrial respiration, oxidative phosphorylation, mitophagy and reactive oxygen species production52. In the absence of ISG15, interferon-primed, bone marrow-derived macrophages failed to produce nitric oxide and arginase 1, molecules that limit vaccinia virus infection. Although the detailed molecular mechanism remains unclear, this finding demonstrates that, in this cell type, ISG15 can also modulate cellular metabolic activities.
The ISG15 pathway has also been implicated in non-viral infectious diseases (Box 2), non-infectious diseases, such as cancers, and other cellular functions, such as the regulation of interferon-induced apoptosis23. The identification of further host proteins that are modified by or interact with ISG15 may provide important insights into the function of the ISG15 pathway.
Viral evasion strategies
Viruses acquired different immune-evasion strategies to counteract the ISG15 pathway, highlighting the importance of ISG15 in the host antiviral response (Fig. 4).
Influenza B virus NS1
The NS1 protein of IBV (NS1/B) was the first viral protein identified to have immune-evasion activity against the ISG15 pathway11. Initially, NS1/B was found to only bind human and primate ISG15 and inhibit its interaction with the E1 enzyme UBE1L, thereby inhibiting IFNβ-induced ISGylation90,91. However, recent studies in which a recombinant IBV was engineered to encode an NS1/B protein that is defective in ISG15 binding revealed that NS1/B does not inhibit ISGylation in IBV-infected cells. Instead, NS1/B binds to and sequesters ISGylated viral proteins, particularly ISGylated viral NP62. This prevents the incorporation of ISGylated NPs into vRNPs, which was previously shown to inhibit viral RNA synthesis.
Vaccinia virus E3
The vaccinia virus E3L protein inhibits ISG15 conjugation to promote viral replication. In in vitro assays, the vaccinia virus E3L protein binds to both human and mouse ISG15 and antag-onizes ISGylation77. Infection of mouse embryonic fibroblasts with a ΔE3L mutant vaccinia virus resulted in ISG15 conjugate formation and reduced viral replication in wild-type cells compared with ISG15-deficient cells. Isg15−/− mice exhibited increased lethality compared with wild-type mice when infected with the ΔE3L mutant vaccinia virus. The mutant virus also exhibited a ~25-fold increase in virus replication in Isg15−/− cells compared with wild-type cells92. These results suggest that ISG15 conjugation restricts vaccinia virus replication and that the E3L protein functions as an immune-evasion effector.
Several viruses, especially members of the order Nidovirales, which includes the coronaviruses, encode enzymes capable of deconjugating ubiquitin and ISG15 from target proteins to antagonize host responses. CCHFV, porcine reproductive and respiratory syndrome virus (PPRSV) and equine arteritis virus (EAV) encode an L protein that contains OTU-containing proteases. These proteins have been shown to reduce the total pool of both ubiquitin and ISG15 conjugates in a cell93. Coronaviruses, including severe acute respiratory syndrome-coronavirus (SARS-CoV), Middle East respiratory syndrome-coronavirus (MERS-CoV), mouse hepatitis virus strain 3 (MHV-3) and human coronavirus-NL63 (HCoV-NL63), encode papain-like proteases (PLPs) that also deubiquitylate and deISGylate target proteins94,95,96,97. The pharmacological inhibition of the PLP2 enzyme in vitro led to an increase of protein ISGylation and decreased viral replication during MHV-3 replication98. A recombinant Sindbis virus system has been used with both CCHFV and SARS-CoV to evaluate the impact of the deISGylating activity of these proteases on viral pathogenesis93,99. For example, co-expression of either the CCHFV L protein OTU domain or the SARS-CoV PLpro with ISG15 by a recombinant Sindbis virus abolished the protection provided by the expression of ISG15 alone during infection of Ifnar−/− mice. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked de-ISGylation in infected cells, and the administration of a PLpro inhibitor protected these mice from lethal infection, demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. Although these examples highlight another potential mechanism of circumventing ISG15, direct evidence for ISG15 antagonism by these proteins during viral infection remains to be demonstrated. Recent biochemical and structural studies have revealed that viral deconjugating enzymes have different specificities for the various forms of polyubiquitin chains and bind to ISG15 in a species-specific manner100,101,102,103,104,105,106. Therefore, the biological consequences of these different viral proteases will vary depending upon the virus and host being studied107.
HCMV IE1 and puL26
ISG15 conjugation inhibits HCMV growth by reducing viral gene expression and inhibiting virion release64. To overcome this, HCMV has evolved multiple countermeasures. The major immediate-early protein IE1 reduces ISG transcription by sequestering STAT2 and preventing interferon-sensitive response element (ISRE) binding. Ectopic expression of HCMV IE1 limited ISG15 protein conjugation, presumably through the decreased expression of components of the ISG15 conjugation machinery, such as HERC5 (ref.108). An IE1 deletion virus robustly induced interferon signalling, including the expression of ISG15 and ISG15 conjugates64. In addition, p21 and p27, two tegument proteins encoded by the gene pUL26 that are involved in virion stability and downregulation of NF-κB signalling, non-covalently interact with ISG15, UBE1L and HERC5 (ref.64). The expression of UL26–p21 reduced the levels of ISG15 conjugates in cells co-transfected with ISG15 and the conjugating enzymes. Interestingly, as discussed earlier, pUL26 itself is a target of ISG15 conjugation. ISGylation of pUL26 alters its stability and inhibited its ability to suppress NF-κB signalling. In the absence of pUL26, HCMV growth is more sensitive to IFNβ treatment.
ISG15 conjugation limits KSHV replication and modulates viral latency74,75. KSHV vIRF1 protein, which is expressed upon Toll-like receptor 3 (TLR3) activation and interferon induction, interacts with ISG15 E3 ligase HERC5. Interaction between vIRF1 and HERC5 decreased the levels of TLR3-induced ISG15 conjugation and cellular IRF3, suggesting that vIRF1 affects ISG15 conjugation and the interferon response, which could contribute to effective KSHV replication75.
Taken together, these examples highlight the convergent evolution of viral proteins that antagonize the ISG15 pathway, providing further support for the importance of this pathway in viral pathogenesis.
ISG15 has been shown to have an important role during infection for a broad range of viruses. Recent studies have advanced our understanding by elucidating how ISG15 antagonizes viral replication during acute and latent infections; identifying immune-evasion strategies; beginning to characterize how ISG15 alters disease pathogenesis, including its ability to limit tissue damage and to modulate human type I interferon signalling; and identifying the first cell surface receptor for unconjugated ISG15, which can regulate cytokine release. The recent identification of ISG15-deficient patients5 and the subsequent characterization of ISG15 regulation of type I interferon signalling have shed light on the complexity of this pathway and have prompted a re-evaluation of the role of ISG15 (ref.4). There are still many important questions that will need to be answered for this pathway to be fully understood.
How does protein ISGylation reshape the global post-translational modification profile of infected or immune cells in response to pathogen invasion? How ISGylation modulates viral proteins, viral replication and host homeostasis is still poorly understood. The ISG15 conjugation system has been intimately tied to protein translation, but it also results in the targeted modification of proteins, as outlined above. How the modification of a small fraction of the total pool of a protein can affect its overall function within a cell is still unclear. Possibilities include the ability of ISGylated proteins to disrupt oligomerization of proteins or to alter the cellular localization of proteins. Another intriguing possibility is that ISGylation could serve as a warning sign to the cell that it is infected. Recent studies have shown that ISG15 can interact with the autophagy pathway, which is known to regulate a variety of processes, including protein degradation, antigen presentation, cytokine signalling and cell death40,41,51. ISGylated proteins, through their interaction with autophagy pathways or other cellular pathways yet to be determined, could function as a danger signal, activating host responses that could serve to limit the infection and protect the host. The application of novel proteome analyses12,109 will be crucial for determining the conjugation preference, scope and potential biological outcomes of protein ISGylation. Although initial proteomic studies to identify ISGylated proteins have been performed in interferon-stimulated cells, this analysis will need to be expanded to different cell types and viruses.
What role does extracellular ISG15 have in the host response to viruses? Utilizing tools that inhibit ISG15 conjugation has allowed researchers to begin to decipher whether phenotypes that are attributed to ISG15 deficiency are conjugation-dependent or independent. However, tools that differentiate between the functions of unconjugated extracellular and intracellular ISG15 are lacking. The recent identification of LFA1 as an ISG15 receptor may facilitate the biological characterization of extracellular ISG15.
How does intracellular, unconjugated ISG15 modulate cellular pathways to limit pathogen burden or damage during infection? In vivo studies have provided evidence that it functions during viral pathogenesis to limit tissue damage, independent of its ability to directly antagonize viral replication50. The ability of ISG15 to non-covalently bind to USP18 and to regulate type I interferon signalling in humans indicates that ISG15 may interact with other unidentified intracellular proteins, independent of conjugation, to regulate additional cellular processes. Uncovering the binding partners will be instrumental in understanding its molecular mechanism of action.
In recent years, ISG15 has been used as a marker of antiviral treatment110,111 and as an immune adjuvant to enhance T cell antitumour immunity112. Further characterization of the ISGylation pathway could help to identify druggable targets, offering new opportunities to intervene in the progression of many diseases.
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The authors thank the members of the Lenschow laboratory for their critical reading of the manuscript during its preparation. The authors gratefully acknowledge support from the US National Institutes of Health (NIH) R01 AI080672 and Pew Charitable Trusts. Y.P. is funded through a Children Discovery Institute postdoctoral fellowship and the NIH postdoctoral training grant T32 CA009547.
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
A small regulatory protein that can be added to a substrate protein by a process known as ubiquitylation and can alter the function of the substrate protein through degradation, localization and protein–protein interactions.
- Ubiquitin-like modifiers
(Ubls). Small regulatory proteins that possess ubiquitin folds and are often conjugated onto a target protein similar to ubiquitin to alter function.
- Genotoxic stressors
Agents that damage the genetic information within a cell, causing mutations or diseases.
- Proteasome-mediated degradation
A cellular process to regulate the concentration of proteins and to degrade misfolded proteins by proteolysis, a chemical reaction that breaks peptide bonds.
- Leader signal
A short peptide present at the amino terminus of newly synthesized proteins that are destined for the secretory pathway.
- Secretory lysosomes
Dual-function organelles that could be used as a lysosome for degradation and hydrolysis and for storage of secretory proteins within the cell.
A double-stranded RNA helicase enzyme that functions as a cytosolic pattern-recognition receptor that recognizes short double-stranded or single-stranded RNA from viruses and triggers an antiviral response.
- Usp18-knock-in mice
Mice in which the endogenous USP18 gene was replaced with a USP18 gene mutated so that it maintains its ability to bind to and inhibit signalling through the type I interferon receptor but its de-ISGylating capacity is lost, resulting in the accumulation of ISG15 conjugates.
- Protein kinase R
(PKR). An interferon-induced, dsRNA-activated protein kinase that phosphorylates the eukaryotic translation initiation factor (eIF2α) in response to dsRNA and cellular stress, including viral infections.
- Ovarian tumour domain
(OTU domain). A domain that is a shared protein region of a family of deubiquitylating proteolytic enzymes involved in processing of ubiquitin precursors.
- Exosome secretion
A cellular secretion pathway mediated by the release of small membrane vesicles from multivesicular endosomes.
- Viral latency
A type of persistent viral infection in which the pathogenic virus lies dormant without killing infected cells until it is reactivated by certain stimuli.
- Aicardi–Goutières syndrome
(AGS). A rare, early-onset childhood inflammatory disorder characterized by elevated levels of type I interferons that results in skin and central nervous system manifestations.
- Pathogen burden
The number of pathogens in an infected host that require the immune system for eradication.
An antiviral medication used to treat hepatitis C, respiratory syncytial virus and other viral infections.
A type of autophagy in which a defective and/or dysfunctional mitochondrion is selectively degraded by the lysosome.
- Interferon-sensitive response element
(IRSE). A specific nucleotide sequence located in the promoters of interferon-stimulated genes (ISGs) that can bind to interferon stimulated gene factor 3 (ISGF3) or other transcriptional complexes upon type I interferon stimulation to initiation transcription of ISGs.
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Perng, YC., Lenschow, D.J. ISG15 in antiviral immunity and beyond. Nat Rev Microbiol 16, 423–439 (2018). https://doi.org/10.1038/s41579-018-0020-5
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