The Importance of the ISG15 Protein in COVID-19 Research

Historically, the ISG15 protein hasn’t been thoroughly investigated by researchers. We know it can function as a cytokine outside the cell and is involved in inflammatory response. Inside the cell, it can act as a post-translational modification, influencing function and activation. It has been implicated in one rare genetic disorder, ISG15-deficiency, which can have serious effects among the small population afflicted. Now, however, new data suggests that this is another big potential target of interest in the ongoing fight against COVID-19.

ISG15 in the SARS-CoV-2 Virus

Specifically, the publication of the article “Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity” provides evidence of ISG15’s* significance. We now know that the coronavirus has two different proteases: The main protease and the papain-like protease (PLpro). Both proteases are required for processing viral polyproteins to generate a functional replicase complex and enable viral replication. The PLpro has also been implicated in the cleavage of proteinaceous post-translational modifications on host proteins, which serves as an evasion mechanism against host antiviral immune response.

SCoV2-PLpro (COVID-19) and SCoV-PLpro (SARS) share an 83% sequence identity yet exhibit different preferences for host substrate; SCoV2-PLpro favors cleavage of the ubiquitin-like interferon-stimulated gene 15 protein (ISG15), whereas SCoV-PLpro predominantly targets ubiquitin chains. During infection, SCoV2-PLpro contributes to the cleavage of ISG15 from interferon responsive factor 3 (IRF3) and attenuates type I interferon responses.

Using GRL-0617 (a cell penetrant and potent inhibitor) to inhibit SCoV2-PLpro impairs the virally induced cytopathogenic effect, maintaining the antiviral interferon pathway, and reducing viral replication in infected cells. These findings reveal a potential dual therapeutic strategy in which targeting SCoV2-PLpro could suppress SARS-CoV-2 infection and promote overall antiviral immunity.

The data show some differences between SCoV2-PLpro and SCoV-PLpro, showing how differing affinity for ISG15 between these two proteases can influence our antiviral immunity. These findings highlight a possible explanation why SCoV2 is spreading much faster and heavily compared to SCoV. In addition, in the future this knowledge can be used for a potential therapeutic strategy. ISG15 and ubiquitin can act as post-translational modifications on proteins, and after treatment with trypsin, mediate protein cleavage, leaving behind the k-ε-gg motif.

A new strategy to identify ISG15 sites was performed using the CST PTMScan^® Ubiquitin Remnant Motif (K-ε-GG) Kit #5562. Recently, Radoshevich et al. published a novel approach to identify sites of ISGylation.² This method is based on the comparison of wild-type sample (normal level of ISG15) with ISG15-deficient sample (absence of ISG15) and mutated USP18 sample (Increased level of ISG15). USP18 is the only deconjugating protease with specificity to ISG15 that has been identified to date.

Cell Signaling is proud to support the research of the Radoshevich laboratory in studying COVID-19 in vivo. Of course, more research in this space is necessary. Discovering new ISG15-modified proteins is difficult. If you use an antibody for analysis (classic immunoprecipitation followed by mass spectrometry) you will pull down just the free ISG15 that is in the cell. Using PTMScan^® Ubiquitin Remnant Motif (K-ε-GG) Kit #5562, you can focus only on the ISG15 linked to other proteins.  

Select References

1. Shin D, Mukherjee R, Grewe D, et al. Papain-like protease regulates SARS-CoV-2 viral spread and innate immunity. Nature. 2020;587(7835):657-662. doi:10.1038/s41586-020-2601-5
2. Zhang Y, Thery F, Wu NC, et al. The in vivo ISGylome links ISG15 to metabolic pathways and autophagy upon Listeria monocytogenes infection. Nat Commun. 2019;10(1):5383. Published 2019 Nov 26. doi:10.1038/s41467-019-13393-x

*Interferon-stimulated gene 15 (ISG15) is a 17 kDA secreted protein that, in humans, is encoded by the ISG15 gene. ISG15 is induced by type I interferon (IFN) and serves several roles, acting as both an extracellular cytokine and an intracellular protein modifier. ISG15 is linked covalently by its C-terminal LRLRGG motif to lysine residues on newly synthesized proteins. This process, termed ISGylation, is catalyzed by a series of conjugating enzymes.