# ISG15: Ubiquitin-Like Interferon-Stimulated Gene 15, ISGylation Cascade, and Viral Evasion


## Key Takeaways

- ISG15 is a ubiquitin-like protein rapidly induced by type I interferons, functioning through covalent conjugation (ISGylation), non-covalent signaling, and extracellular cytokine activity to mediate innate immunity against viral pathogens.
- The ISGylation cascade involves E1 (UBA7), E2 (UBE2L6), and E3 (HERC5/HERC6/TRIM25) enzymes, with deconjugation primarily mediated by USP18, which also negatively regulates type I interferon signaling.
- Viral evasion strategies include encoding deISGylases, such as the papain-like protease (PLpro) of coronaviruses and OTU domain proteases of nairoviruses, which cleave ISG15 from target proteins to suppress host antiviral responses.
- Autosomal recessive ISG15 deficiency presents as a type I interferonopathy characterized by susceptibility to mycobacterial infections, intracranial calcifications, and cutaneous manifestations, highlighting ISG15's critical role in both innate immunity and immune homeostasis.
- ISG15 exhibits pleiotropic roles beyond antiviral defense, influencing cancer biology, DNA damage responses, metabolism, and inflammatory diseases, making it a complex but promising therapeutic target for various pathologies.

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## Executive Summary & Key Metadata

ISG15 (Interferon-Stimulated Gene 15) encodes a 15–17 kDa ubiquitin-like (Ubl) protein that constitutes one of the most rapidly and robustly induced effectors of the type I interferon (IFN-I) response. First identified in 1979 as a 15 kDa protein induced by IFN-β, ISG15 has since been recognized as a pleiotropic molecule with three distinct functional modalities: (1) covalent conjugation to target proteins via an enzymatic cascade termed ISGylation; (2) non-covalent intracellular signaling functions; and (3) extracellular cytokine-like activity following secretion. The ISG15 system is evolutionarily conserved across vertebrates, from teleost fish to mammals, underscoring its fundamental role in innate immunity. Beyond its canonical antiviral functions, ISG15 has emerged as a critical regulator of cancer biology, DNA damage responses, metabolism, and autoinflammatory disease.

| Attribute | Value |
|---|---|
| **HGNC Symbol** | ISG15 |
| **UniProt Accession** | P05161 |
| **Representative PDB ID** | 1R4N |
| **Chromosomal Locus** | 1p36.33 (human) |
| **Gene Size** | ~2.5 kb (two exons, one intron in 5' UTR) |
| **Primary Molecular Function** | Ubiquitin-like protein modifier (ISGylation); cytokine; antiviral effector |
| **Key Interactors** | UBA7 (E1), UBE2L6/UBCH8 (E2), HERC5/HERC6 (E3), USP18 (deconjugase), MDA5, STING, STAT1 |
| **Disease & Pathology Associations** | ISG15 deficiency (autosomal recessive type I interferonopathy), susceptibility to mycobacterial disease, multiple cancers, viral infections (SARS-CoV-2, HIV-1, influenza), diabetic kidney disease, heart failure, preeclampsia |

---

## 1. Genomic Locus, Chromosomal Organization & Isoforms

### 1.1 Chromosomal Localization and Gene Structure

The human *ISG15* gene is located on the short arm of chromosome 1 at cytogenetic band 1p36.33, a gene-dense region frequently subject to copy number alterations in cancer. The gene spans approximately 2.5 kilobases of genomic DNA and exhibits a remarkably compact architecture: two exons separated by a single intron located within the 5' untranslated region (UTR). The coding sequence resides entirely within exon 2, a feature shared across vertebrate orthologs including teleost fish such as the large yellow croaker (*Larimichthys crocea*), gilthead seabream (*Sparus aurata*), Atlantic cod (*Gadus morhua*), and turbot (*Scophthalmus maximus*). This conserved intron-in-5'-UTR structure suggests strong selective pressure maintaining the genomic organization, potentially related to post-transcriptional regulatory mechanisms.

The promoter region of *ISG15* contains canonical interferon-stimulated response elements (ISREs) and gamma-activated sequences (GAS), which serve as docking sites for the transcription factors IRF3, IRF7, IRF1, and STAT1/STAT2 heterodimers. The ISRE motif (consensus: AGTTTCNNTTTCNC/T) is located approximately 100–120 bp upstream of the transcription start site (TSS), while GAS elements (consensus: TTCNNNGAA) are positioned further upstream. Detailed promoter dissection studies in the context of [classical swine fever virus](/knowledge/viruses/livestock-viruses/classical-swine-fever-virus) (CSFV) infection have identified a critical regulatory region spanning nucleotides −487 to −325 relative to the TSS, which contains functional IRF1 binding sites. IRF1 directly binds this region and transactivates *ISG15* expression in response to double-stranded RNA (dsRNA) stimulation or CSFV infection, demonstrating that ISG15 induction is not exclusively dependent on the canonical IRF3/IRF7 axis.

### 1.2 Transcriptional Regulation and Epigenetic Control

The transcriptional regulation of *ISG15* is governed by a multilayered network that integrates signals from multiple innate immune pathways. The primary inducer is type I IFN (IFN-α/β), which activates the JAK-STAT signaling cascade, leading to the formation of the ISGF3 complex (STAT1-STAT2-IRF9) and its binding to ISRE elements. Type II IFN (IFN-γ) also induces ISG15 through STAT1 homodimers binding to GAS elements. Beyond the classical IFN pathways, ISG15 expression is directly induced by IRF3 following activation of the RIG-I-like receptor (RLR) and cGAS-STING pathways, providing a rapid, IFN-independent induction mechanism. IRF1 serves as an additional transcriptional activator, particularly in the context of viral infection.

Epigenetic regulation of the *ISG15* locus involves histone modifications and DNA methylation. Upon IFN stimulation, histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 acetylation (H3K27ac) are deposited at the promoter, correlating with transcriptional activation. Conversely, the polycomb repressive complex 2 (PRC2)-mediated H3K27me3 mark maintains the gene in a repressed state in unstimulated cells. Recent evidence indicates that the RNA N6-methyladenosine (m6A) modification pathway regulates ISG15 expression at the post-transcriptional level. Specifically, METTL3-mediated m6A modification of ISG15 mRNA modulates its stability and translation efficiency, with implications for doxorubicin-induced endothelial cell apoptosis. The m6A reader protein IGF2BP2 binds methylated ISG15 mRNA and enhances its stability, contributing to granulosa cell proliferation in polycystic ovary syndrome.

### 1.3 Isoforms and Splice Variants

The human *ISG15* gene produces a single predominant protein isoform of 165 amino acids. However, alternative splicing events generate minor transcript variants with potential functional significance. A naturally occurring short isoform lacking the C-terminal LRLRGG motif (the site of conjugation to target proteins) has been described, which cannot participate in ISGylation but retains cytokine-like activity. In teleost fish, two distinct ISG15 homologues have been characterized in several species, including the large yellow croaker (LcISG15-1 and LcISG15-2), crucian carp, and Asian seabass. These paralogs exhibit differential expression patterns and may have subfunctionalized to respond to distinct pathogen classes. For example, LcISG15-1 (159 amino acids) and LcISG15-2 (155 amino acids) in the large yellow croaker both contain two ubiquitin-like domains but differ in their C-terminal processing and conjugation efficiency. The presence of multiple ISG15 paralogs in fish but a single gene in mammals suggests that gene duplication events during teleost evolution contributed to functional diversification of the ISG15 system.

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## 2. 3D Protein Domain Architecture & Structural Biology

### 2.1 Overall Fold and Domain Organization

ISG15 is a two-domain protein composed of two tandem ubiquitin-like (Ubl) domains connected by a flexible linker. The N-terminal domain (residues 1–78 in human ISG15) shares ~30% sequence identity with ubiquitin, while the C-terminal domain (residues 79–165) shares ~35% identity. Each domain adopts the canonical β-grasp fold characteristic of ubiquitin and ubiquitin-like modifiers: a five-stranded β-sheet (β1-β5) packed against a single α-helix. The two domains are connected by a short, flexible linker region (residues 79–85) that permits interdomain mobility, a feature critical for the recognition of ISG15 by its cognate enzymes and viral proteases.

The C-terminal domain contains the essential LRLRGG motif (residues 151–155 in human ISG15), which is the site of activation by the E1 enzyme UBA7. This motif is structurally analogous to the C-terminal diglycine of ubiquitin and is exposed on the protein surface, facilitating recognition by the conjugation machinery. The C-terminal domain also harbors the major epitopes recognized by viral deISGylases, including the papain-like proteases (PLpro) of coronaviruses and the ovarian tumor (OTU) domain proteases of nairoviruses.

### 2.2 Structural Dynamics and Interdomain Communication

The interdomain interface of ISG15 is characterized by a hydrophobic patch that mediates intramolecular contacts between the N- and C-terminal domains. Nuclear magnetic resonance (NMR) and X-ray crystallographic studies have revealed that the two domains adopt a compact, closed conformation in solution, with the interdomain interface burying approximately 800 Ų of solvent-accessible surface area. However, the linker region exhibits significant conformational flexibility, allowing the protein to sample open conformations in which the two domains are separated. This conformational plasticity is functionally important: the closed conformation is recognized by the E1 and E2 enzymes, while the open conformation is required for engagement with certain viral proteases and cellular substrates.

Structural studies of ISG15 from the bat species *Myotis davidii* have provided insights into the evolutionary conservation of the interdomain interface and its impact on viral protein engagement. The bat ISG15 structure reveals that while the overall fold is conserved, species-specific variations in the interdomain interface residues alter the conformational equilibrium and affect binding to viral OTU domain proteases. These findings suggest that the ISG15 interdomain interface has been a target of evolutionary selection, potentially driven by host-pathogen arms races.

### 2.3 Post-Translational Modifications of ISG15 Itself

ISG15 is itself subject to post-translational modifications that modulate its function. The protein can be ISGylated on internal lysine residues, leading to the formation of poly-ISG15 chains. Mass spectrometry analyses have identified K29, K46, and K158 as major ISGylation sites within human ISG15. The functional significance of poly-ISG15 chains remains incompletely understood, but they may enhance the avidity of ISG15-substrate interactions or serve as degradation signals. Additionally, ISG15 can be phosphorylated on serine and threonine residues within the linker region, potentially modulating its conformational dynamics and interactions with binding partners.

### 2.4 Interactive 3D Visualization

The three-dimensional structure of human ISG15 has been determined by NMR spectroscopy (PDB: 1R4N), revealing the two-domain architecture in atomic detail. The structure provides a framework for understanding how ISG15 engages its enzymatic partners and how viral proteases recognize and cleave this host defense protein.

> **[Interactive 3D Protein Visualizer: Load ISG15 (PDB: 1R4N)](/tools/protein-structure-viewer?source=direct&pdbId=1R4N)**

The visualizer allows exploration of the N-terminal and C-terminal Ubl domains, the flexible linker, the C-terminal LRLRGG conjugation motif, and surface residues involved in protein-protein interactions. Users can toggle between cartoon, surface, and electrostatic potential representations to examine the structural basis of ISG15 function.

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## 3. Cellular Signaling Pathways & Molecular Function

### 3.1 The ISGylation Cascade

The covalent conjugation of ISG15 to target proteins, termed ISGylation, proceeds through a three-step enzymatic cascade analogous to ubiquitination. The pathway is activated by type I IFN signaling and involves the following components:

**E1 Activating Enzyme (UBA7/UBE1L):** UBA7 is the sole E1 enzyme for ISG15 and catalyzes the ATP-dependent activation of the C-terminal glycine of ISG15, forming a high-energy thioester intermediate. UBA7 is itself an IFN-stimulated gene, ensuring coordinated upregulation of the entire ISGylation machinery upon IFN stimulation. The evolutionary origin of UBA7 has been traced to a gene duplication event of the ubiquitin-activating enzyme UBA1, which occurred early in vertebrate evolution.

**E2 Conjugating Enzyme (UBE2L6/UBCH8):** UBE2L6 is the primary E2 enzyme for ISG15 conjugation, receiving the activated ISG15 from UBA7 via a transthioesterification reaction. UBE2L6 exhibits specificity for ISG15 over ubiquitin, determined by distinct surface residues that recognize the unique features of ISG15's C-terminal domain.

**E3 Ligases (HERC5, HERC6, TRIM25):** The HECT domain and RCC1-like domain-containing protein 5 (HERC5) is the predominant E3 ligase for ISG15 in human cells. HERC5 is a large (~320 kDa) protein that contains a C-terminal HECT domain responsible for catalyzing the final transfer of ISG15 to substrate lysine residues. Unlike ubiquitin E3 ligases, HERC5 exhibits broad substrate specificity, ISGylating hundreds of cellular proteins involved in translation, RNA processing, and cytoskeletal organization. HERC6, a paralog of HERC5, serves a similar function in mice. TRIM25, a RING finger E3 ligase, also possesses ISG15 E3 activity and contributes to ISGylation of specific substrates, including the RIG-I-like receptor MDA5.

**Deconjugating Enzymes (USP18, USP43, USP24):** The ubiquitin-specific protease 18 (USP18) is the principal ISG15 deconjugase, removing ISG15 from target proteins and thereby reversing ISGylation. USP18 is itself an IFN-stimulated gene and plays a dual role in IFN signaling: (1) it deISGylates substrates, and (2) it negatively regulates type I IFN signaling by binding to the IFN-α/β receptor subunit 2 (IFNAR2) and preventing JAK1 association, thereby dampening the IFN response. USP18 stability is dependent on ISG15 binding, creating a negative feedback loop in which ISG15 stabilizes its own deconjugase. USP24 has recently been identified as a cross-reactive deubiquitinase that can remove both ubiquitin and ISG15 from substrates, with a specific role in deISGylating the RNA helicase MOV10 to regulate IFN-I production.

### 3.2 Substrate Selection and Functional Consequences of ISGylation

Proteomic studies have identified over 300 cellular proteins that undergo ISGylation in response to IFN stimulation. These substrates span diverse functional categories, including:

- **Innate immune signaling proteins:** RIG-I, MDA5, STING, IRF3, IRF7, and STAT1 are all ISGylated, with consequences ranging from enhanced signaling to altered stability.
- **Translation machinery:** Multiple ribosomal proteins and translation initiation factors are ISGylated, leading to inhibition of host protein synthesis during viral infection.
- **Cytoskeletal proteins:** Actin, tubulin, and intermediate filament proteins are ISGylation substrates, potentially affecting viral entry and egress.
- **DNA damage response proteins:** ISG15 and ISGylation play critical roles in maintaining genome stability by regulating replication fork stability and DNA repair pathways.

The functional consequences of ISGylation are substrate-specific. For example, ISGylation of MDA5 at specific lysine residues enhances its ability to activate downstream signaling, promoting IFN production and antiviral immunity. Conversely, ISGylation of STING modulates its activity in DNA sensing pathways, with both positive and negative regulatory effects depending on the cellular context. ISGylation of the TGF-β receptor type 1 (TGFβR1) promotes its stability and signaling, contributing to acute kidney injury and the AKI-to-CKD transition. In the context of liver fibrosis, ISG15 deficiency in hepatic stellate cells promotes TGFβ2-induced fibrosis by counteracting CREB1 ISGylation, demonstrating the context-dependent nature of ISG15 function.

### 3.3 Non-Covalent and Extracellular Functions of Free ISG15

In addition to its role as a covalent modifier, free (unconjugated) ISG15 exerts multiple biological functions. Intracellularly, free ISG15 can bind to and regulate the activity of specific proteins. For example, free ISG15 binds to the E3 ligase GRAIL1 (RNF128), promoting its degradation and thereby modulating CD3 expression on T cells, which has implications for esophageal adenocarcinoma survival. Free ISG15 also positively regulates type I IFN signaling by stabilizing USP18, as described above.

Extracellularly, ISG15 functions as a cytokine with immunomodulatory activity. Secreted ISG15 is produced by various cell types, including neutrophils, monocytes, and epithelial cells, and acts on neighboring cells through a yet-to-be-fully-characterized receptor mechanism. Extracellular ISG15 has been shown to:

- **Induce IFN-γ production** from T cells and natural killer (NK) cells, promoting Th1-type immune responses.
- **Stimulate dendritic cell maturation** and enhance their ability to activate CD8+ T cells.
- **Promote macrophage M2-like polarization**, contributing to immune suppression in the tumor microenvironment.
- **Trigger ISGylation in recipient cells** through a non-canonical, type I IFN-independent mechanism, representing a novel mode of intercellular communication.

The cytokine-like functions of ISG15 are particularly relevant in the context of bacterial infections. During *Chlamydia trachomatis* infection, secreted ISG15 exerts immunomodulatory effects on IFN-γ defense and inflammation, dampening the host immune response. Similarly, *Chlamydia*-driven ISG15 expression in epithelial cells dampens the immune response independently of ISGylation, highlighting the importance of free ISG15 in bacterial pathogenesis.

### 3.4 ISG15 in Metabolism and Cellular Stress Responses

Recent studies have revealed unexpected roles for ISG15 in cellular metabolism and stress responses. ISG15 regulates lipid metabolism during vaccinia virus infection, with ISG15 deficiency leading to altered macrophage lipid profiles. In the heart, ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection, protecting cardiomyocytes from virus-induced metabolic stress. ISG15 also modulates adipose thermogenesis through IRF3-mediated reprogramming of glycolysis, linking innate immune signaling to metabolic regulation.

In the context of cancer metabolism, ISG15 and ISGylation are required for pancreatic cancer stem cell mitophagy and metabolic plasticity, enabling these cells to adapt to nutrient-limiting conditions. ISG15 enhances the activity of γ-glutamate cysteine ligase to suppress apoptosis in high-fat diet-promoted hepatocellular carcinoma, protecting cancer cells from oxidative stress. These findings establish ISG15 as a metabolic regulator with broad implications for disease pathogenesis.

### 3.5 Protein-Protein Interaction Networks

The ISG15 interaction network is extensive and context-dependent. Key protein-protein interactions include:

| Interactor | Type | Functional Consequence | Reference |
|---|---|---|---|
| UBA7 | Enzymatic (E1) | ISG15 activation | |
| UBE2L6 | Enzymatic (E2) | ISG15 conjugation | |
| HERC5 | Enzymatic (E3) | Substrate ISGylation | |
| USP18 | Enzymatic (deconjugase) | DeISGylation; IFN signaling regulation | |
| MDA5 | Substrate | Enhanced antiviral signaling | |
| STING | Substrate | Regulation of DNA sensing | |
| MOV10 | Substrate (via USP24) | Regulation of IFN-I production | |
| GRAIL1 (RNF128) | Binding partner | CD3 regulation; T cell function | |
| HDAC6 | Binding partner | Selective autophagy of ISG15 conjugates | |
| SQSTM1/p62 | Binding partner | Selective autophagy | |
| SERBP1 | Substrate | Glioblastoma stemness | |
| TGFβR1 | Substrate | Kidney injury and fibrosis | |
| CREB1 | Substrate | Liver fibrosis | |
| c-Myc | Transcriptional target | Granulosa cell proliferation | |

STRING and BioGRID databases list over 100 experimentally validated ISG15 interactors, reflecting the pleiotropic nature of this protein. The interaction network is dynamically remodeled upon IFN stimulation, with the ISGylation machinery and its substrates being coordinately upregulated.

### 3.6 ISG15 in DNA Damage Response and Genome Stability

ISG15 plays a critical role in maintaining genome stability through multiple mechanisms. ISG15 and ISGylation are induced upon DNA damage and replication stress, linking innate immune signaling to the DNA damage response. In BRCA-defective cells, IFN restores replication fork stability and cell viability via ISG15, suggesting that ISG15 may protect cells from replication-associated DNA damage. ISG15 also regulates the DNA damage response by modulating the stability and activity of key repair proteins, including BRCA1 and 53BP1.

In ataxia telangiectasia (A-T) cells, ISG15 attenuates post-translational modifications of mitofusins and impairs the congression of damaged mitochondria, linking ISG15 to mitophagy and mitochondrial quality control. These findings suggest that ISG15 dysfunction may contribute to the neurodegenerative phenotypes observed in A-T and related disorders.

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## 4. Pathogenic Hotspot Mutations & Clinical Differentials

### 4.1 ISG15 Deficiency: A Type I Interferonopathy

Autosomal recessive ISG15 deficiency is a rare inborn error of immunity caused by loss-of-function mutations in the *ISG15* gene. The condition was first described in patients with susceptibility to mycobacterial disease, particularly disseminated Bacillus Calmette-Guérin (BCG) infection. The molecular basis of this susceptibility lies in the extracellular cytokine function of ISG15: free ISG15 is required for IFN-γ production by T cells and NK cells, and its absence impairs the IFN-γ-dependent antimycobacterial response.

Beyond mycobacterial susceptibility, ISG15 deficiency manifests as a type I interferonopathy characterized by:

- **Intracranial calcifications:** Present in the majority of affected individuals, likely resulting from enhanced type I IFN signaling due to USP18 instability in the absence of ISG15.
- **Cutaneous manifestations:** Ulcerating skin lesions that heal with scar formation, reflecting the role of ISG15 in skin homeostasis.
- **Neurological features:** Seizures and neurodevelopmental delay in some patients.
- **Recurrent infections:** Including viral respiratory infections and parenchymal pneumonia.

### 4.2 Specific Pathogenic Variants

Several pathogenic ISG15 mutations have been characterized at the molecular level:

| Variant | Mutation Type | Protein Consequence | Clinical Phenotype | Reference |
|---|---|---|---|---|
| c.335G>A (p.Cys112Tyr) | Missense | Disruption of C-terminal domain fold | ISG15 deficiency; BCG disease | |
| c.420C>A (p.Tyr140*) | Nonsense | Truncated protein lacking LRLRGG motif | Type I interferonopathy; cutaneous lesions; pneumonia | |
| c.337C>T (p.Arg113*) | Nonsense | Truncated protein | ISG15 deficiency; skin ulcers | |
| c.146G>A (p.Trp49*) | Nonsense | Truncated N-terminal domain | ISG15 deficiency; mycobacterial disease | |
| c.331C>T (p.Arg111*) | Nonsense | Truncated protein | ISG15 deficiency; recurrent infections | |

The p.Tyr140* mutation is particularly instructive: it produces a truncated protein lacking the C-terminal LRLRGG conjugation motif, abolishing ISGylation while potentially retaining some cytokine-like activity. Sibling patients with this mutation exhibit diverse phenotypes, including cutaneous lesions and recurrent parenchymal pneumonia, highlighting the variable expressivity of ISG15 deficiency.

### 4.3 ISG15 in Cancer: Mutations and Expression Alterations

While germline ISG15 mutations are rare, somatic alterations in ISG15 expression are common in cancer. ISG15 is overexpressed in numerous tumor types, including breast, colorectal, ovarian, pancreatic, prostate, renal, and oral squamous cell carcinomas. The prognostic significance of ISG15 expression is context-dependent:

- **Poor prognosis:** In invasive breast cancer, high ISG15 expression correlates with lymphovascular invasion and worse survival. In solitary fibrous tumors, ISG15 serves as a prognostic biomarker. In clear cell renal cell carcinoma, ISG15 promotes tumor progression via the IL6/JAK2/STAT3 signaling pathway.
- **Favorable prognosis:** In some cancer types, ISG15 expression is associated with improved outcomes, reflecting its role in promoting antitumor immunity.

Somatic mutations in ISG15 are uncommon in cancer, but copy number alterations at the 1p36.33 locus are frequent. The 1p36 region is subject to both amplifications and deletions in various malignancies, with the net effect on ISG15 expression depending on the specific alteration and tumor context.

### 4.4 ISG15 in Inflammatory and Autoimmune Diseases

ISG15 dysregulation contributes to the pathogenesis of multiple inflammatory and autoimmune conditions:

- **Psoriasis:** ISG15 is increased in psoriatic lesions and promotes keratinocyte proliferation via the HIF-1α signaling pathway.
- **Vitiligo:** ISG15-USP18 dysregulation by oxidative stress promotes IFN-γ secretion from CD8+ T cells, contributing to autoimmune melanocyte destruction.
- **Inflammatory bowel disease:** Intestinal epithelial cells express immunomodulatory ISG15 during active ulcerative colitis and Crohn's disease.
- **Experimental autoimmune uveitis:** Chronic sleep deprivation promotes disease through STAT1 phosphorylation and ISG15 expression.
- **Diabetic kidney disease:** Elevated ISG15 promotes disease by modulating renal tubular epithelial cell pyroptosis.
- **Acute kidney injury:** ISG15 accelerates AKI and the AKI-to-CKD transition by promoting TGFβR1 ISGylation.
- **Heart failure:** Pressure overload induces ISG15, which facilitates adverse ventricular remodeling.
- **Preeclampsia:** Reduced ISG15 levels impair extravillous trophoblast invasion.

### 4.5 Clinical Differential Diagnosis

The differential diagnosis of ISG15 deficiency includes other type I interferonopathies, such as Aicardi-Goutières syndrome (caused by mutations in TREX1, RNASEH2A/B/C, SAMHD1, ADAR, IFIH1), STING-associated vasculopathy with onset in infancy (SAVI), and USP18 deficiency. Key distinguishing features of ISG15 deficiency include the combination of mycobacterial susceptibility, intracranial calcifications, and skin ulcers, which are not typical of other interferonopathies. Genetic testing for ISG15 mutations is essential for definitive diagnosis.

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## 5. Host-Pathogen & Viral Interactions

### 5.1 Antiviral Mechanisms of ISG15

ISG15 exerts antiviral activity through multiple mechanisms, targeting various stages of the viral life cycle:

**Inhibition of viral entry and uncoating:** ISG15 modification of viral envelope proteins can interfere with receptor binding and membrane fusion. For example, ISGylation of the influenza A virus NS1 protein inhibits its function, reducing viral replication.

**Inhibition of viral replication:** ISG15 and ISGylation target viral polymerases and replication complexes. Free ISG15 inhibits [peste des petits ruminants virus](/knowledge/viruses/livestock-viruses/peste-des-petits-ruminants-virus) (PPRV) replication by disrupting the interaction between the nucleoprotein and phosphoprotein, thereby inhibiting viral RNA synthesis. ISG15 also restricts Zika virus replication in primary human corneal epithelial cells.

**Inhibition of viral assembly and budding:** ISG15 blocks retrovirus release from cells late in the budding process, interfering with the interaction between the viral Gag polyprotein and the host ESCRT machinery. This mechanism is particularly relevant for HIV-1 and other retroviruses.

**Modulation of innate immune signaling:** ISG15 enhances antiviral signaling by ISGylating and activating key sensors such as MDA5 and RIG-I. ISG15-dependent activation of MDA5 is critical for the detection of picornaviruses and coronaviruses, and is antagonized by the SARS-CoV-2 papain-like protease (PLpro).

**Regulation of viral gene expression:** ISG15 can modify viral transcription factors and regulatory proteins, altering viral gene expression programs.

### 5.2 Viral Evasion Strategies

Viruses have evolved sophisticated strategies to counteract the ISG15 system:

**Viral deISGylases:** Many viruses encode proteases that cleave ISG15 from target proteins. The most well-characterized are:

- **Coronavirus PLpro:** The papain-like protease of SARS-CoV-2 and other coronaviruses cleaves ISG15 conjugates, counteracting ISG15-dependent activation of MDA5 and evading host innate immunity. PLpro exhibits specificity for ISG15 over ubiquitin, and its deISGylase activity is essential for viral pathogenesis.
- **Nairovirus OTU domain proteases:** Crimean-Congo hemorrhagic fever virus and related nairoviruses encode OTU domain proteases that cleave ISG15, suppressing the antiviral response.
- **Arterivirus papain-like proteases:** [Equine arteritis virus](/knowledge/viruses/livestock-viruses/equine-arteritis-virus) and [porcine reproductive and respiratory syndrome virus](/knowledge/viruses/livestock-viruses/porcine-reproductive-and-respiratory-syndrome-virus) encode PLPs with deISGylase activity.

**Inhibition of ISG15 expression:** Some viruses suppress ISG15 transcription by targeting the IFN signaling pathway. For example, the influenza A virus NS1 protein inhibits IFN production, thereby reducing ISG15 expression. Hepatitis C virus (HCV) modulates ISG15 expression through the proteasome subunit alpha type-6 (PSMA6), which putatively regulates ISG15 as a proviral host gene.

**Sequestration of free ISG15:** Certain viral proteins bind free ISG15 and prevent its interaction with cellular targets. The vaccinia virus E3 protein binds ISG15 and inhibits its antiviral activity.

**Exploitation of ISG15 for viral benefit:** In some contexts, ISG15 can promote viral replication. ISG15 deficiency restricts HIV-1 infection, suggesting that ISG15 has proviral effects in the context of HIV-1. The mechanism involves ISG15-mediated regulation of the IFN response: in the absence of ISG15, the IFN response is enhanced, leading to greater restriction of HIV-1 replication.

### 5.3 ISG15 in Specific Viral Infections

**SARS-CoV-2:** ISG15 plays a complex role in COVID-19. The SARS-CoV-2 PLpro deISGylates MDA5, counteracting ISG15-dependent antiviral signaling. Free ISG15 and protein ISGylation are emerging as important regulators of SARS-CoV-2 infection. ISG15 also modulates the inflammatory response to SARS-CoV-2, with potential implications for disease severity.

**HIV-1:** ISG15 blocks HIV-1 release by interfering with Gag processing and budding. However, ISG15 deficiency restricts HIV-1 infection, indicating that ISG15 also has proviral effects. The balance between these opposing activities determines the net effect of ISG15 on HIV-1 replication.

**Influenza virus:** ISG15 ISGylates the NS1 protein of influenza A virus, inhibiting its function and reducing viral replication. The NS1 protein of some influenza strains counteracts this by binding ISG15 and preventing its conjugation.

**Zika virus:** ISG15 restricts Zika virus replication in primary human corneal epithelial cells, suggesting a role in ocular antiviral defense.

**Coxsackievirus B3:** ISG15 blocks cardiac glycolysis and ensures sufficient mitochondrial energy production during Coxsackievirus B3 infection, protecting the heart from virus-induced metabolic stress. ISG15 also plays a critical role in host defense against virus-induced cardiomyopathy.

**[Viral hemorrhagic septicemia virus](/knowledge/viruses/aquatic-viruses/viral-hemorrhagic-septicemia-virus) (VHSV):** CRISPR/Cas9-mediated knockout of ISG15 in EPC cells increases susceptibility to VHSV infection, demonstrating the antiviral role of ISG15 in fish.

**[Pseudorabies virus](/knowledge/viruses/livestock-viruses/pseudorabies-virus) (PRV):** Free ISG15 inhibits PRV infection by positively regulating type I IFN signaling.

**Lymphocytic choriomeningitis virus (LCMV):** ISG15 drives immune pathology and respiratory failure during systemic LCMV infection, indicating that ISG15 can also contribute to immunopathology.

### 5.4 Bacterial and Parasitic Infections

ISG15 also plays roles in immunity against bacterial and parasitic pathogens:

**Mycobacterium tuberculosis:** ISG15 and ISGylation are important host defense mechanisms against M. tuberculosis infection. ISG15 deficiency predisposes to mycobacterial disease, particularly BCG infection.

**Chlamydia trachomatis:** Chlamydia-driven ISG15 expression dampens the immune response of epithelial cells, both dependently and independently of ISGylation. Secreted ISG15 exerts immunomodulatory effects on IFN-γ defense and inflammation.

**[Listeria monocytogenes](/knowledge/bacteria/livestock-bacteria/listeria-monocytogenes-circling-disease-ruminants-silage):** A Listeria-based vaccine targeting ISG15 exerts anti-tumor efficacy in renal cell carcinoma, demonstrating the potential of ISG15 as a tumor-associated antigen.

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## 6. [Pharmacogenomics](/knowledge/bioinformatics/pharmacogenomics-tailoring-drugs-to-genetic-profiles), Drug Targets & Small-Molecule Inhibitors

### 6.1 ISG15 as a Therapeutic Target

The multifaceted roles of ISG15 in cancer, viral infection, and inflammatory diseases have made it an attractive therapeutic target. Several strategies are being pursued:

**Inhibition of ISG15 conjugation:** Small-molecule inhibitors targeting the ISG15 E1 enzyme UBA7 or the E2 enzyme UBE2L6 could block ISGylation globally. However, the structural similarity between the ISG15 and ubiquitin conjugation cascades poses challenges for selectivity. Curcumin has been shown to partially prevent ISG15 activation via UBA7 and decrease ISGylation, representing a natural product-based approach.

**Inhibition of ISG15 deconjugases:** Inhibitors of USP18 could enhance ISGylation and promote antiviral immunity. However, USP18 also negatively regulates type I IFN signaling, and its inhibition could have complex effects.

**Targeting ISG15 in cancer:** Given the pro-tumorigenic roles of ISG15 in many cancer types, strategies to reduce ISG15 expression or activity are being explored. These include:

- **Antisense oligonucleotides (ASOs)** targeting ISG15 mRNA.
- **Small interfering RNAs (siRNAs)** for ISG15 knockdown.
- **CRISPR/Cas9-mediated gene editing** to disrupt ISG15 in tumor cells.

**ISG15-based vaccines:** ISG15 is emerging as a tumor-associated antigen in colorectal cancer and renal cell carcinoma. A Listeria-based vaccine targeting ISG15 has shown anti-tumor efficacy in renal cell carcinoma models. Modified vaccinia virus Ankara (MVA)-based vectors expressing cell-free ISG15 increase IFN-I production and improve HIV-1-specific CD8 T cell immune responses, suggesting potential applications in vaccine development.

**Janus kinase (JAK) inhibition:** In ISG15 deficiency, the enhanced type I IFN signaling can be therapeutically targeted with JAK inhibitors. Case reports demonstrate that JAK inhibition is effective in treating the inflammatory manifestations of ISG15 deficiency.

### 6.2 Investigational Compounds and Drug Development

The development of specific ISG15-targeted therapeutics is at an early stage. Key challenges include:

- **Selectivity:** Achieving selectivity for ISG15 over ubiquitin and other Ubls.
- **Delivery:** Efficient delivery of nucleic acid-based therapeutics to target tissues.
- **Context-dependence:** The dual pro- and anti-tumorigenic roles of ISG15 complicate therapeutic targeting.

Chemo-enzymatic synthesis approaches have been developed to produce ISG15 tools for drug discovery, including ISG15-fluoromethylketone probes and ISG15-vinyl methyl ester derivatives.

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