Progress on the research of anti-infection role of ubiquitin-like protein ISG15 against coronavirus

LIU Xinran, CHEN Tong, ZHANG Yu, CHEN Hong

(School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China)

Abstract：The ISG15 molecule is a ubiquitin-like protein that plays an important role in regulating innate immunity against viral infection. Previous studies have reported the antiviral mechanisms of ISG15 against viral infection, such as influenza virus, hepatitis B virus, HIV virus, Ebola virus, SARS-CoV, MERS-CoV and cytomegalovirus. Recently, a number of studies reported the function of ISG15 in SARS-CoV-2 infection, that ISG15 can be inactivated by the papain-like protease of SARS-CoV-2, thereby inhibiting its antiviral function. In this review, we presented the antiviral mechanism of ISG15, emphasizing the specificity of the antiviral effects of ISG15 against coronaviruses. Moreover, the immune evasion ability of coronavirus via papain-like protease (PLpro) by blocking ISG15-dominated innate immunity, as well as the potential therapeutic inhibitors of PLpro were also reviewed. From the perspective of the molecular mechanism of ISG15's antiviral innate immunity, the latest progress of anti-SARS-CoV-2 drugs was summarized, as well as the prospect of the related drug research and development.

Key words：ubiquitin-like protein ISG15; ISGylation; papain-like protease; coronovirus

ISG15 (interferon-stimulated gene 15) is an important molecule that plays a critical role in regulating the innate immunity system[1]. As a key molecule for anti-viral infection, ISG15 could be induced by interferon (IFN) and viral infection. As the first discovered ubiquitin-like protein (UBL), ISG15 can covalently conjugate with substrate proteins through ubiquitin-like modification (ISGylation), affecting the biological activity of target proteins[1]. In addition to ISGylation, ISG15 can also function as a secretory protein, binding to integrin receptors on membranes of immune cells such as NK (natural killer) cell and macrophages, regulating the production and secretion of interleukin 6 and IFN-γ[1].

The primary sequence of ISG15, which has small molecular weight, is conserved in mammalian species. The mature ISG15 molecule in human contains 159 amino acid residues with the molecular weight of about 15 KD. Within its secondary and tertiary structure, ISG15 contains two global domains similar to ubiquitin, which were connected by a hinged region[2]. At its C-terminus, ISG15 has a sequence of LRLRGG, which is critical for its UBL modifications. Through its glycine reside of C-terminus, ISG15 covalently conjugates with the lysine reside of substrates, which is also termed as ISGylation[2-3].

As an important component of the natural immune system, ISG15 activates interferon to play an essential role in anti-viruses pathway[1]. However, some studies have suggested the evasive ability of SARS-CoV-2 to escape the antiviral immune effect of ISG15[4]. Therefore, understanding the molecular mechanism of the antiviral effect of ISG15 is also important for the anti-COVID-19 medicine study.

1 ISGylation regulation

Similar to ubiquitination, ISGylation also requires E1 activating enzyme, E2 binding enzyme and E3 ligase, which mediate the three-step cascade. Compared with ubiquitination, which involves hundreds of E1-E2-E3 enzymes, ISGylation often involves unique enzymes[5].For example, ISGylation enzymes in human include specific E1 activating enzymes (UBA7/UBE1L), E2 binding enzyme (UBE2E2/ UBCH8) and E3 ligase (HECT and RLD domain E3 ubiquitin ligase 5, HERC5). First, ISG15 is activated by binding to the C-terminus of the E1 enzyme UBE1Lviaan ATP-dependent high-energy thioester bond, which is then transferred to the cysteine residue of the active site of the E2 enzyme UBCH8 though thioester bond. The E3 enzyme then cleaves the thioester bond and ISG15 is attached to the designated target proteinviaa lysine residue[5]. Similarly, the ISG15 modification process can also be removed by ISG15-specific de-uquitinase 18 (USP18), a specific isopeptidase of ISG15[6]. USP18 is an ISG15-specific protease that does not affect ubiquitination process. USP18 specifically binds to ISG15 through the ISG15-binding box1 and box2 (IBB-1 and IBB-2) regions[6]. In addition to its deISGylation activity, USP18 can also function independent of its isopeptidase activity as a molecule to regulate interferon-mediated immune processes by binding to STAT-2, or the receptor of Type I interferon (IFN-I), inhibiting JAK/STAT signaling pathway. Therefore, maintaining the homeostasis of ISG15 and USP18 plays a critical role in physiological activities[6].

2 Immune regulation and antiviral effect of ISG15

IFN-I, double-stranded RNAs, or viral infection (pathogen-associated molecular patterns, PAMPs), can activate IFN and JAK/STAT signaling, which upregulate ISG15 expression[4]. By covalently modifying the substrate proteins, or by binding with the key ligands as a secretory cytokine, ISG15 molecule regulates the immune process (Fig. 1). In conjugated form, ISG15 covalently binds with the target protein through the C-terminus, affecting the ubiquitination process of substrates, for example, ISGylation can inhibit the E2, E3 enzymes of ubiquitination, or competitively suppress ubiquitination of the substrate proteins, thus blocking the ubiquitin-proteasome system and eventually inhibiting the degradation of substrates. Whereas in non-conjugated form, ISG15 molecule can function as an extracellular cytokine, secreted by fibroblasts, neutrophils, monocytes or lymphocytes, and bind to the integrin receptor LFA-1 of immune cell surface, promoting NK cells and T lymphocytes to secrete IFN-γ, enhancing the lysis ability of NK cells[7].

Notes:The activation of the JAK/STAT pathway promotes the increased expression of interferon regulatory elements and a series of ISG components. Similar to ubiquitination, ISGylation also requires E1 activating enzyme, E2 binding enzyme and E3 ligase, which mediate the three-step cascade. As a specific isopeptidase of ISG15, USP18 can remove the covalently conjugated ISG15 tags from the substrate proteins. Protein ISGylation regulation plays critical roles in antiviral, antitumor and immunomodulatory effects in the cell. P, phosphorylation.Fig.1 Three-step of ISGylation enzymatic cascade and USP18 mediated deISGylation regulation

In addition to its regulation on immune system, ISG15 also plays a critical role in anti-viral infection. In the study of Sindbis virus, for the first time researchers observed that ISG15 has an anti-viral effect[8]. So far, ISG15 has been found to have anti-viral effects on a variety of DNA and RNA viruses, such as influenza virus, hepatitis B virus, respiratory virus, human immunodeficiency virus type I, vesicular stomatitis virus, Ebola virus, SARS-CoV and SARS-CoV-2[9-10].ISG15 is involved in the interferon-mediated antiviral immune cascade response. After the virus invades the host cells, the viral RNA receptor retinoic acid inducible factor receptor (RIG-I-like receptor, RLRs) is activated, and the nucleus-encoding interferon factor IFNα/β gene responds rapidly, upregulating a series of interferon stimulating genes (ISGs), which are transcriptionally controlled by interferon stimulating response elements (ISRE). In addition, the ISGylation E1/E2/E3 enzymes are also induced, such as UBE1L, UBCH8 and HERC5, which promote the ISGylation of viral proteins, as well as a series of host proteins, producing antiviral effects (Fig.2)[9, 11].

Notes:Once the JAK/STAT pathway is activated by antiviral immune signaling, interferon regulatory factor 9 (IRF9) is induced, binding with STAT1 and STAT2. Then STAT1 and STAT2 subsequently migrate into the nucleus, up-regulating the expression of downstream ISGs, such as UBE1L, UBE2L6 and HERC5, which mediate protein ISGylation, such as viral proteins and MDA5. The ISGylated MDA5 then upregulates nuclear interferon gene, improving the overall IFN-I content, which eventually increases the antiviral immune reaction of the cell. P, phosphorylation. M, methylation.Fig.2 Antiviral mechanism of ISGylation

ISG15 also produces specific immunity against viral infection by regulating the innate immune process. During the immune process, according to the different types of viruses and the different stages of infection, ISG15 can specifically modify certain viral proteins to have effect. Through different ways, such as affecting the cytosolic localization of viral proteins, inhibiting virus recombination and generation, or stabilizing key viral proteins, which promote viral proteins presentation, ISGylation may be beneficial to virus identification and anti-viral immunity[12]. For example, in the immune process against influenza A virus, ISG15 modifies the non-structural viral protein 1 (NS1) to prevent its dimerization, which blocks the ability of NS1 to inhibit the antiviral ability of host cells[12]. Moreover, ISG15 can also exert anti-infection effect by activating interferon signaling pathway. For example, ISGylation of interferon regulator 3 (IRF3) blocks the binding of IRF3 with PIN1 protein, thus inhibits the polyubiquitination degradation of IRF3, which ensures that host cells can successfully activate type I interferon signaling pathway after virus invasion[13]. In the immune process against HIV, ISGylation of viral Gag protein prevents the early assembly of HIV. Whereas at late stage of viral synthesis, ISG15 can acylate Gag proteins by HERC E3 ligase, which inhibits virus budding[9, 11].

Moreover, ISG15 can also function by controlling the expression of cytokines and related receptors in the interferon signaling pathways. ISG15 regulates the expression of interferon factor IFN- I by binding with the cellular viral RNA sensor - MDA5 (melanoma differentiation-associated gene 5). Through HERC5, ISG15 is covalently conjugated with MDA5, and ISGylated MDA5 will form oligomers, enhancing the expression of interferon factor IFN-I, which thus strengthens the host’s immune function against pathogens[7, 14]. In conclusion, ISG15 plays a broad antiviral role by directly modifying viral proteins, or by indirectly affecting the expression of intracellular proteins such as MDA5 and IRF3, promoting interferon anti-viral immune pathways[14].

3 Antiviral effect of ISG15 against coronavirus

Coronavirus belongs to theCoronaviridaefamily, which includes a group of enveloped, positive-strand RNA viruses that infect a wide variety of animals. The coronavirus is a positive-sense single-stranded RNA virus, and until now seven coronaviruses infectious to human have been identified, including MERS-CoV, SARS (SARS-CoV) and SARS-CoV-2, which have caused the pandemics in the 21st century[15-16].When the coronavirus attaches to the host, the spike glycoprotein on the viral surface binds to the receptor of host cells. Then the viral envelope fuses with the host cell membrane and the virus invades into the host through endocytosis[17]. After invasion, the viral antisense RNA is synthesized by the host enzymes to form the template of viral genome, with viral proteins are subsequently expressed[17].

SARS-CoV-2 has 95% similarity in protein homology with SARS-CoV, which caused the pandemic in 2003[16]. Some viral proteases of SARS-CoV and SARS-CoV-2, such as main proteinase (M-pro) and papain-like protease (PLpro), play critical roles in the virus life cycle. For example, PLpro is involved in viral replication and transcription, regulation of protein post-translational modification, cleavage of polyproteins and immune escape of virus, which make PLpro as one of the most promising antiviral drug targets[18]. PLpro of coronavirus belongs to the family of cysteine peptidases C16. After invasion of virus into host cells, PLpro and other viral proteases cleave the first translated viral polyproteins PP1A and PP1AB into non-structural proteins (NSPs), to ensure that subsequent processes such as viral RNA replication and viral packaging proceed smoothly. During the synthesis of virus-encoded proteins, PLpro specifically recognizes the L-X-G-G sequence between NSP1 and NSP2, NSP2 and NSP3, and NSP3 and NSP4, hydrolyzes the carboxyl side chain of glycine and releases NSP1-4. Among these NSPs, NSP3 is the largest coding protein synthesized by the virus, which forms PLpro molecule after cleavage, known as NSP3 self-processing (Fig. 3).

SARS-CoV-2 and SARS-CoV PLpro were 83% identical in primary sequence[19]. According to its structure from the N-terminal to the C-terminal, PLpro has several domains including ubiquitin-like region, thumb region, palm region and zinc finger region. The S1 binding domain overlaps with the zinc finger region, and there are also other three binding sites, S2, S3 and S4. The S4-S1 pocket basically overlaps with the palm region of PLpro, which is also the main region where PLpro and ISG15 bind together (Fig. 3). Recent studies have shown that when binding with PLpro, the N-terminal domain of ISG15 demonstrates great flexibility, whereas PLpro maintains an extended conformation[20].

Notes:The PLpro of SARS-CoV-2 is first translated into a precursor peptide chain containing four pre-polypeptides (NSP1, NSP2, NSP3, NSP4). The pre-polypeptide fragments are linked by a highly conserved LXGG sequence, which can be recognized and cleaved by PLpro. After cleavage and modification, the NSP3 of the products can finally become the mature PLpro molecule, which can participate in the precursor polypeptide producing process.Fig.3 Structure and cleavage process of PLpro (PDB ID：6WUU)

PLpro has deubiqutination and deISGylation enzymatic activities. By hydrolyzing ubiquitin or ISG15 tags from the substrate proteins, it regulates the post-translational modification of proteins in host cells, playing an important role in mediating immune escaping of coronavirus. As mentioned above, ISG15 is an important signal regulating the innate immunity of the host against viral infection,and here the SARS-CoV-2 PLpro can inhibit ISG15-dominated innate immunity by hydrolyzing the ISGylation modification of the substrate proteins. Therefore, the antiviral effect of ISGylation can be thus greatly blocked by PLpro of coronavirus. Intriguingly, although the PLpro from SARS-CoV-2 is highly homologous to SARS-CoV-PLpro, their affinities for ISG15 are quite different. SARS-CoV-PLpro prefers to target ubiquitin chains, in contrast, SARS-CoV-2-PLpro preferentially cleaves ISG15 tags, and its deISGylation activity is much higher than the PLpros from other coronaviruses. As a result, in cells infected by SARS-CoV-2, proteins translated are seldomly ISGylated, which significantly weakens the antiviral immune system of the host cells mediated by ISG15 pathway[19].

PLpro can specifically deISGylate certain key substrate molecules, thereby avoiding interferon-mediated immune cascade and inhibiting antiviral innate immunity of the host. For example, studies have shown that SARS-CoV-2 can deISGylate of MDA5 through PLpro to inhibit the activation of MDA5, thereby enabling the virus to evadethe monitor of foreign RNA receptors of the host cells[14]. In addition, studies have reported that SARS-CoV-2 can bind to ISG15-modified IRF3 through PLpro to hydrolyze ISG15 tags. IRF3 protein without ISG15 modification is less stable, and is more likely to be ubiquitinated and degraded by proteasomes, which further weaken the antiviral immune cascade mediated by interferon pathway[21]. In conclusion, the remove of ISG15 conjugation by PLpro of SARS-CoV-2 greatly inhibits the antiviral immune response mediated by interferon pathway, which may possibly explain the low level of type I interferon in the serum of severe COVID-19 patients[22].

COVID-19 patients might have complication or secondary infections such as septicemia or invasive fungi infection, which increases the difficulty of treatment and the mortality[23]. Probably this is because the antagonistic effect of viral PLpro on ISG15, which inhibits the host's immune response. Moreover, the binding of viral spike protein to ACE2 of host cells, does damage to alveolar epithelial cells, breaking physical defensive barriers of the host[24].

4 Anti-coronavirus strategy targeting PLpro

In order to block the immune evasion ability of coronavirus, targeting PLpro activity has been proven to be an effective therapeutic strategy. At present, most of the reported small-molecule PLpro inhibitors bind to PLpro through the S4 pocket, which prevent PLpro from recognizing and binding to ISG15, antagonizing its deISGylation activity. By now, a series of small molecule compounds and natural products targeting PLpro have been reported (Tab. 1).

Tab.1 Coronavirus’ PLpro inhibitors and research progress

A recent study reported that GRL0617, a non-covalent pilot inhibitor of SARS-CoV- PLpro, could bind to the S4-S3 region and reduce the catalytic activity of PLpro through inducing theself-blocking of PLpro active site. Moreover, due to the high specificity, the inhibitor does not affect host de-ubiquitination activity, therefore the side effects are limited[25]. Recent studies have shown that the inhibitor GRL0617 also has a high inhibitory effect on the PLpro from SARS-CoV-2[26].

Recently, ebselen has entered phase II clinical trial (https://clinicaltrials.gov/, identifier：NCT04483973). Ebselen is an organic selenium compound with low molecular weight. Its binding ability to M-Pro, the main protease of SARS-CoV-2, has been confirmed by virtual molecular docking and X-ray crystallography[27]. Studies indicated that in addition to binding to viral M-Pro, ebselen is also an effective inhibitor of PLpro. Ebselen and its structural analogues can irreversibly inhibit CoV-2-PLPro by covalently binding to the canonical catalytic triplex (Cys111-His272-Asp286) in the active site of CoV-2-PLPro[28]. The selenium atom in ebselen binds with sulfhydryl group to form Se-S bond, occupying the active site and preventing the formation of strong nucleophilic ions, which strongly inhibit the PLpro activity, blocking the inhibitory effect of PLpro on ISGylation[28].

The SARS-CoV-2 PLpro specific inhibitors, VIR250 and VIR251 (both are non-natural amino acid compounds with only one amino acid different), bind to PLpro in a very similar manner. 2-aminobutyric acid in VIR250 and tyrosine in VIR251 can both inhibit PLpro from recognizing and binding of ISG15 and ubiquitin, by occupying the S4-S1 pockets of PLpro near the active site[29]. Virtual molecular docking technology suggests that there are still a number of unoccupied potential binding sites of PLpro after binding with the inhibitor, indicating the possibility of improving efficiency of PLpro inhibitors by further structural modification and drug combination. The experimental results also proved that the two small molecules do not have cross reaction with human deubiquitinases (DUBs), which have similar structures with PLpro[29].

Moreover, another inhibitor Rac5c can not only block the NSP3 self-processing, but also inhibit PLpro binding with ISG15 on PLpro Lys48 site[30]. However, due to the low solubility of Rac5c, it does not perform well in DMSO solvent and therefore requires a large amount of solution, which will undoubtedly bring potential safety risks[30].

Among natural production, abitanditerpenoid tanshinones, including cryptotanshinone, tanshinone IIA and dihydrotanshinone I, which were extracted fromSalviamiltiorrhiza, were reported to have high inhibitory effect on PLpro.IC50values of the first two compounds were 0.8 μmol·L-1and 1.6 μmol·L-1respectively[31]. Another natural product that can effectively antagonize PLpro is diarylheptane, such as curcumin in turmeric and the structural analogues[32-33]. In 2016, Parker et al. found that isoprene substituted chalcone compounds inAngelicasinensishad strong non-competitive inhibitory effects on PLpro, such as xanthoangelol E and xanthoangelol F, withIC50of 1.2 μmol·L-1and 5.6 μmol·L-1respectively[34]. In addition, flavonoid compounds such as quercetin have also been reported to have PLpro inhibitory effect[35]. However, most of the above compounds have only been tested in cell experiments, and further experimental data including preclinical and clinical results are needed to provide more evidence for anti-coronavirus drug development and research.

5 Prospect

ISG15 plays an important role in the antiviral immune cascade response mediated by interferon, and is also a key regulator of the anti-infection immune system. However, the latest research shows that ISG15 function will be impaired in the natural immune response against SARS-CoV-2 by the viral PLpro protein, with its antiviral effects greatly weakened. ISG15 inactivation will also influence the subsequent immune process, such as the presentation of viral antigens and specific immunity[18]. Therefore, using specific small molecule inhibitors to inhibit the PLpro of SARS-CoV-2 may be not only directly block viral replication and cut off the viral life cycle, but also have additional indirect antiviral advantages, such as restoration of ISG15 activity, ether by enhancing ISGylation of critical molecules in antiviral immunity, or reactivating the immune promotive function of ISG15. Due to its critical antiviral function, ISG15 restoration may further enhance immunity and reduce secondary infection risk. In addition, computer-aided drug design (CADD) has been widely used in developing drugs that target PLpro[25-26]. In developing drugs against SARS-CoV-2, CADD technology has been used to screen for specific molecules, such as 6-TG and GRL0617, which can target the pocket region of PLpro and inhibit the binding of ISG15 and PLpro. CADD offers a number of advantages, such as effective simulation of reactions in various environments and virtual calculation of ligand-receptor binding energy, which can speed up and improve the accuracy of target discovery, help researchers find more effective PLpro inhibitors that could possibly facilitate the antiviral immune effects of ISG15[38-39]. Further advanced tools for drug discovery, such as CADD, and in-depth mechanistic study focus on the ISG15 pathway will surely help identifying new drug targets against coronaviruses and provide opportunities to intervene in disease progression.