How ISG15 Combats Viral Infection

Abstract

Interferon (IFN)-stimulated gene product 15 (ISG15) is a ubiquitin-like protein critical for the control of microbial infections. ISG15 appears to serve a wide variety of functions, which regulate multiple cellular responses contributing to the development of an antiviral state. ISG15 is a versatile molecule directly modulating both host and virus protein function which regulate many signaling pathways, including its own synthesis. Here we review the various roles ISG15 plays in the antiviral immune response, and examine the mechanisms by which viruses attempt to mitigate or exploit ISG15 activity.

INTRODUCTION

One of the first lines of defense against viral pathogens is the innate immune response restricting virus replication early after infection. Immune cells such as neutrophils and macrophages, as well as inflammatory cytokines such as interferons (IFNs) are critical elements of the innate immunity. One highly upregulated IFN-stimulated gene product (ISG) protein in the initial stages of the innate response to viral infection is ISG15. Human ISG15 is a 15kDa Ubiquitin (Ub)-like (Ubl) protein that structurally resembles two β-grasp Ub domains connected by a linker sequence(Narasimhan et al., 2005). ISG15 can act as both an effector and a signaling molecule in various phases of the innate immune response. ISG15 was first discovered in 1979 with references to a 15-kDa protein induced by IFN treatment(Farrell et al., 1979). ISG15’s Ubl structure was first discovered in 1987 when it cross-reacted with anti-Ub antibodies(Haas et al., 1987). It was quickly suspected of serving an immunological function due to ISG15 being one of the most highly upregulated genes during viral infections(Der et al., 1998; González-Sanz et al., 2016; Meraro et al., 2002).

Similar to Ub and other Ubls, ISG15 is able to regulate a wide range of cellular pathways tied to host immune responses. ISG15 can be post-translationally linked to a wide array of target proteins, both cellular and viral(Huang et al., 2014; Loeb and Haas, 1992; Okumura et al., 2007; Zhang et al., 2019; Zhao et al., 2016). The ISG15 conjugation mechanism mirrors that of ubiquitination as it requires three enzymes; the E1 activating enzyme (Ube1L), E2 conjugating enzyme (UbcH8), and an E3 ligase enzyme (HERC5, EFP, or TRIM25)(Zhang and Zhang, 2011). These enzymes work together to covalently bond the C-terminal glycine of the ISG15 LRLRGG motif to a lysine of the target protein (Berndsen and Wolberger, 2014; Wong et al., 2006). This process is initiated when ISG15 is converted from its 17kDa precursor form by currently unidentified cellular proteases into its 15kDa mature form. ISGylation is principally reversed by one host protease, ubiquitin-specific protease 18 (USP18)(Potter et al., 1999). Beyond acting through conjunction with other proteins, ISG15 has also been found to have numerous immunological roles in its free form, both intra-and extracellularly(Baldanta et al., 2017; Campbell and Lenschow, 2013; Napolitano et al., 2018; Recht et al., 1991; Swaim et al., 2017; Yeung et al., 2018).

In this review we describe the specific roles ISG15 plays in the antiviral immune response. ISG15 can vary considerably between species in sequence, structure and function(Deaton et al., 2016). ISG15 has been shown to be a vital part of the immune response in mice. The absence of ISG15 in knockout mice and mouse cell lines results in a significant reduction in the ability to mount a viable defense against a variety of viral infections(Lenschow et al., 2007; Speer et al., 2016). In contrast, human patients lacking ISG15 do not appear to be more susceptible to viral infection, but are more susceptible to some bacterial infections(Bogunovic et al., 2012). Various viruses have developed methods to counteract the antiviral effects of ISG15, including the reversal of ISGylation, sequestering of ISGylated proteins, and interfering with ISG15 synthesis(Daczkowski et al., 2017b; Gargan et al., 2018; Zhao et al., 2016).

INDUCTION AND REGULATION OF ISG15 SIGNALING

As the name implies, ISG15 is an IFN-stimulated gene, and its expression is therefore upregulated upon IFN stimulation (FIG. 1). Type I IFN is induced when Pathogens-Associated Molecular Patterns (PAMPs), including double-stranded viral RNA (dsRNA), are detected in the cytoplasm by retinoic acid-inducible gene 1 protein (RIG-I) or other RIG-I-like receptors (RLRs) such as melanoma differentiation-associated protein 5 (MDA5). Upon binding to dsRNA RLRs undergo a conformational change that allows ubiquitination of their caspase recruitment domains (CARDs). The ubiquitinated CARDs are recruited to the mitochondria by mitochondrial antiviral-signaling protein (MAVS) where they initiate a signaling cascade leading to the downstream nuclear translocation of interferon regulatory factor 3 (IRF3) and NFkB. These transcription factors are important activators of the type I IFN promoters, resulting in increased production of IFN-αand IFN-β(Kalvakolanu and Borden, 1996; Meraro et al., 2002). IRF3 can directly activate the transcription of several ISGs including ISG15(Grandvaux et al., 2002). Type I IFNs are secreted and subsequently bind the IFN-α/βreceptor (IFNAR) on the cell membranes of the infected cell and neighboring cells. Activated IFNAR signals for Janus kinase 1 (JAK1) to phosphorylate signal transducer and activator of transcription (STAT) proteins, which form a complex with IRF9 called IFN-stimulated gene factor 3 (ISGF3). ISGF3 binds the IFN-sensitive response element (ISRE) within ISG promoters, increasing transcription and expression of hundreds of ISGs, including ISG15 and its conjugating enzymes Ube1L, UbcH8, EFP, TRIM25, and HERC5, as well as USP18 (FIG. 1)(Zhang and Zhang, 2011). IFN-mediated upregulation of ISGs results in an antiviral state, thus reducing viral spread.

(Figure 1).

(1) Upon infection viral RNA or DNA is released into the cytoplasm where it is detected by RIG-I or other RLRs. After binding to the nucleic acid the RLR will undergo a conformational change, exposing the CARDs. (2) The exposed CARDs can be ubiquitinated or ISGylated. Ubiquitinated CARDs activate MAVS, which leads to downstream activation of IRF3. Activated IRF3 increases transcription at the ISREs for IFNαand IFNβ. ISGyaltion of RIG-I inhibits this pathway by marking RIG-I for proteasomal degradation. (3) Type 1 IFNs bind extracellularly to IFNAR, activating JAK1. (4) Activated JAK1 phosphorylates STAT1 and STAT2, which bind to IRF9 to form ISGF3. ISGF3 binds to the ISREs of many ISGs including ISG15, Ube1L, UbcH8, and HERC5. (5) proISG15 is processed by an unknown protease into functional free ISG15. (6)USP18 can inhibit activation of the JAK/STAT pathway by binding competitively with IFNAR2 preventing JAK1 activation. (7) ISG15 stabilizes the interaction between IFNAR2 and USP18 by inhibiting SKP2 mediated Ub conjugation. This prevents proteasomal degradation of USP18, effectively down regulating production of ISG15. ISGylation of RIG-I CARDs downregulates MAVS activation and IFN production.

ISGylation of cellular proteins interferes with viral replication

ISG15 can hinder viral replication by interfering with the endogenous translation and exocytosis machinery that viruses hijack in order to replicate. Eukaryotic initiation factor 4E (eIF4E) facilitates translation initiation. eIF4E homologous protein (4EHP) binds to the cap of both cellular and viral mRNAs preventing translation by competing with eIF4E. ISGylation of 4EHP enhances this inhibitory effect on translation, likely due to a stabilization of the 4EHP-cap interaction. It has been proposed that ISG15 may selectively conjugate 4EHP-capped viral mRNAs as a mechanism of selective inhibition of viral RNA translation(Okumura et al., 2007). Potential targets include flaviviruses such as Dengue, West Nile, yellow fever, Kunjin, and Japanese encephalitis viruses, which contain capped positive sense RNA.

Furthermore, ISG15 has been described to interfere with the budding of virus-like particles (VLPs) by inhibiting endogenous enzymes required for this process. Ebola relies on ubiquitination of its VP40 matrix protein by E3 ligase Nedd4 to facilitate virion egress. ISGylation of Nedd4 prevents VP40 ubiquitination and inhibits budding(Malakhova and Zhang, 2008; Okumura et al., 2008; Pincetic et al., 2010; Seo and Leis, 2012). Budding of Avian Sarcoma Leukosis virus (ASLV) and human immunodeficiency virus (HIV)-1 requires recruitment of ESCRT-III complex proteins such as Vps4 and LIP5. ISGylation of the ESCTRT-III protein CHMP5, which regulates LIP5, prevents oligomerization of Vps4 and LIP5, halting the budding process(Pincetic et al., 2010).

ISGYLATION OF VIRAL PROTEINS

One of the best-studied ways ISG15 affects viral replication is through conjugation to viral proteins. ISGylation can hinder protein function, mark proteins for degradation, affect protein localization, and prevent the formation of protein complexes(Nakashima et al., 2015; Tang et al., 2010; Villarroya-Beltri et al., 2016; Zhang et al., 2019; Zhao et al., 2016). Unlike Ub and other Ubl proteins, ISG15 has a conjugation preference towards newly synthesized proteins(Durfee et al., 2010). The proposed model suggests that HERC5 associates with the 60S ribosomal subunit, which contains the exit tunnel, and allows for preferential ISGylation of newly synthesized proteins. However, only a small percentage of newly synthesized proteins is ISGylated. What drives certain proteins to become ISGylated over others during translation is currently unknown. However, many proteins produced during viral infection are viral proteins and ISGs, potentially allowing ISG15 to maximize its impact on viral replication.

ISGylation interferes with viral protein function

One of the first identified viral targets of ISG15 is the NS1 protein of Influenza A virus (IAV) (Tang et al., 2010). IAV NS1 can inhibit the production of type I IFNs, thereby preventing activation of ISGs including protein kinase R (PKR) and oligoadenylate synthetase (OAS), which results in increased translation of viral RNA (Bergmann et al., 2000; de la Luna et al., 1995; Min and Krug, 2006; Zhang et al., 2015). In addition, ISGylation inhibits the ability of NS1 to bind, among other things, dsRNA and PKR, reducing viral suppression of the pathways in which these factors are involved(Tang et al., 2010). ISGylated NS1 is also prevented from associating with importin-α, which mediates the translocation of NS1 into the nucleus(Zhao et al., 2010). ISGylation of NS1 also impacts its ability to form homodimers, which is required for many NS1 functions. The Coxsackie B3 virus 2A protease inhibits host translation by inhibiting the same eIF4E pathway as 4EHP. By cleaving eIF4G -the binding partner of eIF4E -Coxsackie B3 virus 2A shuts off host translation. ISGylation of the 2A protease restores cellular translation by inhibiting cleavage of eIF4G (Rahnefeld et al., 2014). Another viral ISGylation target is the pUL26 protein from Human cytomegalovirus (HCMV). Active pUL26 suppresses NF-κB signaling, but ISG15 conjugation inhibits this activity and results in decreased HCMV replication(Kim et al., 2016).

ISG15 interferes with oligomerization of viral proteins

Many viral proteins rely on forming oligomers or complexes to perform their functions. ISGylation of these viral proteins as an antiviral strategy is particularly effective. Conjugated ISG15 causes a steric hindrance, preventing further oligomerization once an ISGylated protein is incorporated. Therefore, ISGylation of a relatively small percentage of viral proteins can achieve a dominant inhibitory effect(Durfee et al., 2010; Zhao et al., 2016). A well-studied example of this is the nucleoprotein (NP) of Influenza B virus (IBV). NP is a component of the IBV ribonucleoprotein (RNP), and is critical for synthesizing viral RNA(Zhao et al., 2016). Because the incorporation of a single ISGylated NP into an RNP oligomer results in the entire oligomer being non-functional, ISGylated NPs have been observed to have a dominant effect over unmodified NPs. ISGylation of IBV NP results in reduced viral RNA synthesis and ultimately reduced viral replication(Zhao et al., 2016). Similarly, ISGylation of 10–30 percent of human papillomavirus (HPV) L1 capsid proteins results in a 70 percent decrease in infectivity(Durfee et al., 2010).

ISGylation can potentially target proteins for degradation

It has been suggested that ISG15 may serve a similar or redundant role to Ub in the autophagosomal and proteasomal degradation pathways. When stimulated by type I IFN, both free ISG15 and ISGylated proteins localize with histone deacetylase 6 (HDAC6) and Ub-binding protein p62(Nakashima et al., 2015). HDAC6 controls autophagosome maturation in the process of clearing ubiquitinated protein aggregates, and p62 acts as a major chaperone protein(Lee et al., 2010; Nakashima et al., 2015). p62 is also critical to the proteasomal degradation pathway(Liu et al., 2016). In the presence of the proteasome inhibitor MG132 p62-linked autophagy increases, as does co-localization between p62, HDAC6, and ISG15(Nakashima et al., 2015). The autophagy and proteasomal degradation pathways are utilized to degrade ISGylated and ubiquitinated proteins. They are effective at lowering levels of viral proteins as well as misfolded endogenous proteins. Infection by HIV-1 induces accumulation of misfolded tumor suppressor p53, an antiviral factor that is suspected to inhibit HIV-1 long terminal repeat promoter activity(Cooper et al., 2013; GENINI et al., 2001; Osei Kuffour et al., 2019). ISG15 conjugation to the misfolded p53 by HERC5 and TRIM25 leads to p53 degradation, and in the absence of ISG15 misfolded p53 accumulates and enhances HIV-1 replication(Huang et al., 2014; Osei Kuffour et al., 2019; Park et al., 2016). One example of ISGylation leading to p62-mediated degradation is found in the negative regulation of RIG-I signaling. LRRC25 binds to ISGylated RIG-I and promotes association with p62(Du et al., 2018). ISG15 has also been linked to increased basal and infection-induced autophagy during Listeria monocytogenes infection by modifying mTOR, WIPI2, AMBRA1, and RAB7(Zhang et al., 2019). Despite both Ub and ISG15 being associated with protein degradation, ISG15-Ub mixed chains are not degradation signals. Furthermore ISG15 can conjugate to Lys-29 of Ub, and ISGylation of cellular ubiquitinated proteins appears to slow turnover(Fan et al., 2015).

Free ISG15 negatively regulates type I IFN signaling in humans

Despite the fact that ISG15 is generally considered an antiviral protein, ISG15 has also been described to negatively regulate type I IFN signaling, at various places in this pathway. Type I IFN signaling is critical for antiviral innate immune responses, but excessive IFN signaling can result in autoinflammatory pathogenesis(Tokarz et al., 2004; Zhang et al., 2015). To mitigate this the human JAK/STAT pathway is regulated through a negative feedback loop, in which ISG15 and USP18 play an important role. STAT2 recruits USP18 to IFNAR2. USP18 binds competitively at the IFNAR2-JAK1 binding site, displacing JAK1. In humans ISG15 binds to USP18, inhibiting SKP2-mediated ubiquitination, which would result in proteasomal degradation of USP18(Vuillier et al., 2019). By preventing USP18 degradation ISG15 stabilizes the interaction between IFNAR2 and USP18 (FIG. 1). Bound USP18 interferes with receptor dimerization and JAK activation, preventing the formation of ISGF3(Arimoto et al., 2017). This ultimately results in decreased expression of ISGs and moderating the IFN response. The role of ISG15 in this negative feedback regulation system appears to be specific to humans. In mice USP18 is a negative regulator of the JAK/STAT pathway but is not ISGylated to prevent degradation(Speer et al., 2016). This may account for part of the difference in ISG15 function between species(Speer et al., 2016). In addition, ISG15 acts as a negative regulator of RIG-I activation. As opposed to increasing MAVS and IRF3 activation as seen with ubiquitination, ISGylation of RIG-I CARDs results in decreased activation of these proteins and decreased production of type I IFNs(Kim et al., 2008).

ISG15 AS AN EXTRACELLULAR SIGNALING MOLECULE

In addition to contributing to intracellular immune responses, ISG15 also plays an important role in the extracellular immune response. ISG15 can initiate the production and secretion of a wide variety of antiviral and anti-bacterial factors such as type III IFNs, reactive oxygen species (ROS), and nitric oxide (NO), as well as act as an extracellular cytokine itself (FIG. 2). Type III IFNs are proinflammatory, but also upregulate ISG15 production in a similar manner to type I IFNs. The mechanism of ISG15 secretion or release is still uncertain, but it does not involve a hydrophobic leader protein and is not inhibited by blocking classical secretion mediated by the Endoplasmic Reticulum-Golgi pathway (D’Cunha et al., 1996). There is speculation that it could involve exosomal trafficking, or apoptosis(Campbell and Lenschow, 2013; Dos Santos and Mansur, 2017; Perng and Lenschow, 2018; Sun et al., 2016). Extracellular free ISG15 binds to lymphocyte function-associated antigen 1 (LFA-1), a surface receptor on dendritic cells (DCs), natural killer (NK) cells, T cells, and macrophages(Baldanta et al., 2017; Napolitano et al., 2018; Recht et al., 1991; Swaim et al., 2017; Yeung et al., 2018). Depending on the cell type this can trigger proliferation and maturation. Upon co-stimulation with IL-12, ISG15 binding to LFA-1 can initiate production of IFN-γ and IL-10 (FIG. 2)(Baldanta et al., 2017; Napolitano et al., 2018; Padovan et al., 2002; Recht et al., 1991; Swaim et al., 2017; Yeung et al., 2018).

(Figure 2).

Extracellular ISG15 directly affects pathogenesis or activates various immune cell types. (1) It inhibits the virus replication at the earliest stages, potentially by preventing entry into the cell. (2) It is an effective chemoattractant for neutrophils. It binds to the LFA-1 receptor on dendritic cells, NK cells, and T cells. This receptor is present in macrophages but has yet to be confirmed as the mechanism by which ISG15 activates macrophages. (3) In dendritic cells it initiates maturation and IFN-γ production. (4) In macrophages it causes polarization to the M1 phenotype, resulting in production of reactive oxygen species and nitric oxide. It also increased autophagy and mitophagy of infected cells and organelles. (5) In T cells and NK cells upon co-stimulation with IL-12 it stimulates production of IFN-γ. (6) In NK cells it also stimulates production of IL-10, which inhibits IFN-γ production in T cells. (7) Exosomal trafficking is one of the proposed methods by which ISG15 may be secreted from a cell but that mechanism is still unclear.

ISG15 induces an antiviral state in immune cells

ISG15 is a potent activator and recruiter of immune cells. In the presence of ISG15 macrophages increase in polarization towards the proinflammatory and antiviral M1 phenotype. M1 macrophages display higher production of antiviral factors such as ROS and NO(FIG. 2). In addition, ISG15-stimulated macrophages display increased autophagy and mitophagy of infected cells and organelles (Baldanta et al., 2017). The mechanism of by which ISG15 induces M1 polarization in macrophages is currently unknown, but the expression of LFA-1 receptors by macrophages suggest it may be through a similar mechanism to NK cells.

NK cells respond to ISG15 binding LFA-1 by proliferating and increasing production of IFN-γ and IL-10 (Swaim et al., 2017). Increased production of the pro-inflammatory cytokine IFN-γ is a common effect of activation by ISG15, occurring in NK cells, DCs, and T cells(Napolitano et al., 2018; Recht et al., 1991; Swaim et al., 2017). Increased production of IL-10 in response to ISG15 signaling appears to be negative feedback inhibition to prevent damage from an overactive inflammation response. IL-10 inhibits T cell activation and downregulates proinflammatory cytokine production (Couper et al., 2008). In CD8+ T cells ISG15 binding increases activation (Yeung et al., 2018). Both CD4+ and CD8+ T cells increase IFN-γ production in the presence of free ISG15 but only after being primed with IL-2 (Recht et al., 1991).

ISG15 causes CD8α+ DCs to increase production of IL-1β and IFN-γ as well(Napolitano et al., 2018). Additionally, the presence of free ISG15 induces dendritic cell phenotypic maturation. Incubation with ISG15 in growth media induced production of E-cadherin, CD15, and CD86 by dendritic cells. Expression of these genes is typically associated with a mature phenotype, and CD86 acts as a co-stimulatory signal to activate T cells. The presence of anti-ISG15 antibodies in the media nullified these effects (Padovan et al., 2002).

Aside from binding to the LFA-1 receptor, free ISG15 can act as a chemoattractant for neutrophils (Owhashi et al., 2003). Because ISG15 is highly concentrated at sites of infection and apoptotic cells it is effective in recruiting neutrophils to areas of need. When left unchecked this elevated proinflammatory state can cause damage. Therefore, the IFN regulatory activity of USP18 is critically important to maintain signaling levels under normal conditions and restoring homeostasis after an infection. By inhibiting the JAK/STAT pathway and suppressing the downstream effects of type I IFN signaling USP18 effectively reduces auto-inflammatory pathogenesis. The absence of USP18 in humans has been observed to result in severe interferonopathy(Gruber et al., 2020; Zhang et al., 2015).

Interference with viral cell entry

In addition to activating immune cells and extracellular pathways, ISG15 can interfere with viral infection at its earliest stages. ISG15 interferes with norovirus entry or uncoating. ISG15 is a known inhibitor of norovirus replication and this inhibition occurs upstream of virus transcription(Rodriguez et al., 2014). It has been proposed that ISG15 reduces Zika virus (ZIKV) infection by preventing viral entry as well. The presence of free extracellular ISG15 reduces intracellular viral titers during ZIKV infection (Singh et al., 2019).

VIRAL COUNTERMEASURES TO ISG15 SIGNALING

Various viruses have developed strategies to circumvent ISG15 interference. Most strategies to suppress ISG15 signaling principally appear to fall along two lines, viral protease mediated deISGylating and sequestering ISGylated proteins. However, some viruses have additional mechanisms that allow them to suppress ISG15 antiviral activities.

Viral deISGylases

The initial suggestion that viruses could be encoding proteases with deubiquitinase function (DUBs) to counter innate immune responses came from structural similarities between papain-like proteases (PLP) from coronaviruses to host USPs such as HAUSP(Barretto et al., 2005; Sulea et al., 2005). Shortly thereafter, these proteases were among the first to be found to be multifunctional in their ability to reverse ubiquitination, as well as ISGylation(Lindner et al., 2005). Prominent examples of coronaviruses that encode PLPs possessing deISGylase activity include Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle East Respiratory Syndrome coronavirus (MERS-CoV), as well as the lesser known mouse hepatitis virus (MHV)(Mielech et al., 2015; Mielech et al., 2014; Ratia et al., 2014). However, the full breath of PLPs from coronaviruses that can robustly process ISG15 is currently unknown and has been somewhat impeded by the revelation that viral PLPs are specific for certain host’s ISG15(Daczkowski et al., 2017a). In contrast to the highly conserved Ub, ISG15 displays significantly more sequence variation between host species (Dzimianski et al., 2019a). Sequence variation within the C-terminal β-grasp Ubl domain, or differences that impact the domain-domain interactions within ISG15 have been observed to impact the ability of coronavirus PLPs to engage with ISG15(Daczkowski et al., 2017a; Langley et al., 2019). This phenomenon has been suggested to occur because PLPs are likely specifically adapted to cleave the ISG15 of their predominate host reservoirs, but lack deISGylase activity in other species(Langley et al., 2019). As unclear about how many coronaviruses encode for PLPs that have deISGylase activity, so is the exact role and impact viral deISGylase activity plays in modulating host immune function. The combination of both deISGylase and DUB activity in coronaviruses have been observed to contribute to the suppression of the innate immune response by acting in part on IFN-βand NF-κB signaling pathways(Bailey-Elkin et al., 2014; Clementz et al., 2010; Mielech et al., 2014; Ratia et al., 2014). Yet the individual contribution and role of viral deISGylase activity remains unclear. However, the recent development of several new molecular tools in the form of altered MERS-CoV PLPs that lack DUB, deISGylase, or both activities may help elucidate the exact role of ISG15 during CoV infection (Clasman et al., 2019; Daczkowski et al., 2017b).

While the exact contribution of PLP deISGylase activity on immune suppression remains unknown, this is not entirely the case with the other well-studied family of viral deISGylases, viral ovarian tumor (OTU) domain proteases. Viral deISGylases belonging to the OTU superfamily are encoded by multiple viral families, including nairoviruses, arteriviruses and tenuiviruses(Bester et al., 2018; Frias-Staheli et al., 2007; Zhang et al., 2007). Like PLPs, OTUs in general have shown potential to possess the ability to reverse ISGylation in addition to a DUB role(Frias-Staheli et al., 2007). The Erve nairovirus even possesses an OTU domain containing deISGylase activity, but lacking DUB activity, underscoring the importance of viral deISGylase function for particular viruses(Deaton et al., 2016). Similar to viral PLPs, viral OTUs have shown to be specific for ISG15 from certain species, including those they productively infect(Dzimianski et al., 2019b).

Specific mutations within the OTU domain from the Crimean-Congo hemorrhagic fever (CCHFV) nairovirus that silenced DUB activity, or both DUB and deISGylase activities allowed narrowing down the mechanism by which viral OTU deISGylase function manipulates host immunity. This has suggested a distinct function for viral deISGylase activity for nairoviruses. Whereas DUB activity was shown to downregulate type I IFN signaling, OTU deISGylase activity appeared to stabilize CCHFV L-protein levels at a later stage in infection (Scholte et al., 2017).

Sequestering proteins

Another countermeasure viruses employ against ISG15 signaling is the production of proteins that sequester ISG15 and ISGylated proteins. The Vaccinia virus E3 protein sequesters free ISG15(Guerra et al., 2008; Smith et al., 2013). By reducing unconjugated ISG15 Vaccinia E3 prevents interference with viral proteins or propagation of antiviral signals. NS1B from IBV on the other hand sequesters ISGylated proteins in a species dependent manner, particularly ISGylated viral proteins(Zhao et al., 2016). This species dependent behavior has been observed to influence the replication of IBV in humans over their mouse and other animal counterparts(Jiang and Wang, 2019; Sridharan et al., 2010). As previously stated, ISGylation of viral proteins that need to form complexes to function is a particularly effective method of viral suppression due to the relatively small percentage of proteins that need to be ISGylated to elicit an antiviral effect. By sequestering ISGylated components of these complexes IBV drastically reduces the efficacy of that method of viral suppression (FIG. 3). For both of these viruses expression of their respective sequestering proteins appears to be critical to preventing viral clearance(Guerra et al., 2008; Zhao et al., 2016).

(Figure 3).

Viral countermeasures to ISG15 production and activity. (1) The Vif protein from HIV-1 degrades STAT1 and IE1 from HCMV sequesters STAT2, both of which are critical to upregulating ISG15 synthesis. (2) Vaccinia virus E3 protein sequesters free ISG15, preventing it from conjugating to targets or acting as a signaling protein. (3) pUL50 of HCMV binds to and causes proteasomal degradation of Ube1L, an essential E1 activator protein that facilitates ISGylation. (4) KSHV and HCMV both produce proteins that interfere with the HERC5 E3 ligase. Both reduce ISG15 conjugation to target proteins. (5) Viral DUBs and deISGylases such as OTUs and PLPs cleave the conjugation between ISG15 and target proteins, returning ISGylated proteins to their normal state. (6) NS1B from IBV sequesters ISGylated proteins, preventing them from being incorporated into oligomers.

Inhibition of ISG15 production and conjugation

Some other less common methods viruses employ to counteract ISG15 activity include inhibition of ISG15 conjugation to targets and interfering with the ISG15 synthesis and conjugation pathways (FIG. 3). Kaposi’s sarcoma-associated herpesvirus (KSHV) and HCMV both encode proteins that interfere with the HERC5 E3 ligase(Jacobs et al., 2015; Kim et al., 2016). HCMV also encodes pUL50, a protein that binds to Ube1L leading to proteasomal degradation, resulting in reduced ISG15 activation(Lee et al., 2018). In addition HCMV also produces an immediate-early protein 1(IE1) that reduces overall ISG15 production(Bianco and Mohr, 2017; Kim et al., 2016). It does so by sequestering STAT2, preventing formation of the ISGF3 complex and inhibiting ISRE activation(Huh et al., 2008; Krauss et al., 2009; Paulus et al., 2006). This reduces not only the amount of ISG15 available, but also the amount of ISG15 conjugating enzymes (Ube1L, UbcH8, HERC5). HIV-1 is capable of degrading components of the JAK/STAT pathway to reduce the effectiveness of type 1 IFN signaling (Gargan et al., 2018). The HIV-1 protein Vif inhibits IFN-αsignaling by degrading STAT1 and STAT3. By reducing STAT1 HIV-1 effectively reduces the amount of ISG15 produced to fight infection.

CONCLUSIONS

ISG15 is a functionally versatile Ubl protein that affects many aspects of the antiviral immune response. It is important as both a signaling molecule and effector protein. Intracellularly, ISG15 interferes with many viral processes directly, and is involved in many antiviral-signaling pathways. Extracellularly, ISG15 can directly interact with viruses to prevent infection, as well as activate various immune cells to promote viral clearance. ISG15 can also promote the production of many extracellular antiviral cytokines. Aligning with ISG15 being one of the most highly upregulated proteins during viral pathogenesis, not surprising the information known to date on ISG15 highlights it as having central role in myriad of host immune mechanisms. Mechanisms that viruses from diverse families seek to stymie in different ways to favor virulence.

Surprisingly, what is known about ISG15 and its immunological role is likely still in its infancy. While some mechanisms such as the interplay between ISG15 and IBV are well understood, others such as how ISGylation targets are determined and which viruses have evolved to target ISG15 activity over Ub activity remain unclear. Despite ISG15’s importance as an extracellular signaling molecule the process by which ISG15 is secreted is still largely unknown. Additionally, how 4EHP and ISG15 work synergistically to downregulate viral mRNAs over host mRNAs translation is limited. This limited knowledge also extends to what degree species to species variation within ISG15s contribute to protection from viruses spilling over into another host. Already ISG15 species-species variation appears to impact the effectiveness of immune evasion mechanisms of IBV, nairoviruses, and coronaviruses. Then there are the species to species variations in how hosts utilize ISG15 as a regulator when it comes to USP18. How all of these species-species differences translate to disease outcomes is not well understood. So as much as many things are known about ISG15’s roles, there seems to be as much yet to be revealed. Taking into account that numerous host and viral pathways that have been already been identified that could lend themselves to therapeutic intervention, the benefit of filling in the knowledge gaps regarding ISG15 should not be underestimated.

Highlights:

• ISG15 is a versatile ubiquitin-like protein that regulates antiviral responses

• ISG15 can operate as a as both an effector and signal protein

• Viruses have developed several strategies to counteract ISG15 antiviral functions

• ISG15 structure and function varies between species

Abbreviations:

IFN

interferon

ISG15

interferon-stimulated gene product 15

Ub

ubiquitin

Ubl

ubiquitin-like

DUB

deubiquitinase

USP18

ubiquitin-specific peptidase 18

dsRNA

double-stranded RNA

RIG-I

retinoic acid-inducible gene 1 protein

RLR

RIG-I-like receptor

MDA5

melanoma differentiation-associated protein 5

CARD

caspase recruitment domain

MAVS

mitochondrial antiviral-signaling protein

IRF

interferon regulatory factor

ISRE

interferon sensitive response element

IFNAR

IFN-α/β receptor

JAK1

Janus kinase 1

STAT

signal transducer and activator of transcription

ISGF3

interferon-stimulated gene factor 3

eIF4E

eukaryotic initiation factor 4E

4EHP

eIF4E homologous protein

VLP

virus-like particle

ASLV

avian sarcoma leukosis virus

IAV

influenza A virus

PKR

protein kinase R

OAS

oligoadenylate synthetase

NP

nucleoprotein

IBV

influenza B virus

RNP

ribonucleoprotein

HPV

human papillomavirus

HDAC6

histone deacetylase 6

HIV-1

human immunodeficiency virus-1

ROS

reactive oxygen species

NO

nitric oxide

LFA-1

lymphocyte function-associated antigen 1

DC

dendritic cell

NK

natural killer

ZIKV

Zika virus

PLP

papain-like protease

SARS-CoV

Severe Acute Respiratory Syndrome coronavirus

MERS-CoV

Middle East Respiratory Syndrome coronavirus

MHV

mouse hepatitis virus

OTU

ovarian tumor domain protease

KSHV

Kaposi’s sarcoma-associated herpesvirus

HCMV

human cytomegalovirus

IE1

immediate-early protein 1