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Review
. 2022 Dec:132:16-26.
doi: 10.1016/j.semcdb.2022.06.005. Epub 2022 Jun 25.

Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15)

Inbar Magid Gold  1 , Noa Reis  1 , Fabian Glaser  2 , Michael H Glickman  3
Affiliations
Review

Coronaviral PLpro proteases and the immunomodulatory roles of conjugated versus free Interferon Stimulated Gene product-15 (ISG15)

Inbar Magid Gold et al. Semin Cell Dev Biol. 2022 Dec.

Abstract

Ubiquitin-like proteins (Ubls) share some features with ubiquitin (Ub) such as their globular 3D structure and the ability to attach covalently to other proteins. Interferon Stimulated Gene 15 (ISG15) is an abundant Ubl that similar to Ub, marks many hundreds of cellular proteins, altering their fate. In contrast to Ub, , ISG15 requires interferon (IFN) induction to conjugate efficiently to other proteins. Moreover, despite the multitude of E3 ligases for Ub-modified targets, a single E3 ligase termed HERC5 (in humans) is responsible for the bulk of ISG15 conjugation. Targets include both viral and cellular proteins spanning an array of cellular compartments and metabolic pathways. So far, no common structural or biochemical feature has been attributed to these diverse substrates, raising questions about how and why they are selected. Conjugation of ISG15 mitigates some viral and bacterial infections and is linked to a lower viral load pointing to the role of ISG15 in the cellular immune response. In an apparent attempt to evade the immune response, some viruses try to interfere with the ISG15 pathway. For example, deconjugation of ISG15 appears to be an approach taken by coronaviruses to interfere with ISG15 conjugates. Specifically, coronaviruses such as SARS-CoV, MERS-CoV, and SARS-CoV-2, encode papain-like proteases (PL1pro) that bear striking structural and catalytic similarities to the catalytic core domain of eukaryotic deubiquitinating enzymes of the Ubiquitin-Specific Protease (USP) sub-family. The cleavage specificity of these PLpro enzymes is for flexible polypeptides containing a consensus sequence (R/K)LXGG, enabling them to function on two seemingly unrelated categories of substrates: (i) the viral polyprotein 1 (PP1a, PP1ab) and (ii) Ub- or ISG15-conjugates. As a result, PLpro enzymes process the viral polyprotein 1 into an array of functional proteins for viral replication (termed non-structural proteins; NSPs), and it can remove Ub or ISG15 units from conjugates. However, by de-conjugating ISG15, the virus also creates free ISG15, which in turn may affect the immune response in two opposite pathways: free ISG15 negatively regulates IFN signaling in humans by binding non-catalytically to USP18, yet at the same time free ISG15 can be secreted from the cell and induce the IFN pathway of the neighboring cells. A deeper understanding of this protein-modification pathway and the mechanisms of the enzymes that counteract it will bring about effective clinical strategies related to viral and bacterial infections.

Keywords: Coronavirus; DUB; Deubiquitinating enzymes; HERC5; ISG15; Interferon; MERS-CoV; PLpro; Papain-like proteases; SARS-CoV; SARS-CoV2; USP; Ubiquitin; Ubiquitin E3 ligases; Ubiquitin-Like proteins; Ubiquitin-specific proteases.

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Conflict of interest statement

Declaration of Competing Interest The authors state that we do not have any financial and personal relationships with other people or organizations that could inappropriately influence our work or this manuscript that we have written.

Figures

Fig. 1
Fig. 1
Conservation of ubiquitin and ISG-15 across vertebrates. Sequence alignment of ubiquitin from zebrafish, mice, and humans indicates perfect conservation of ubiquitin. The ISG15 protein contains two ubiquitin-like domains (UBLs). Although the 3D structure of each of the ISG15 domains resembles the 3D fold of Ub, a comparison of each domain of human ISG15 to the Ub sequence results in roughly 33 % sequence identity by BLAST . In comparison to ubiquitin, ISG15 is less conserved across vertebrates, dropping to 66 % and 40 % identity between humans and mice or zebrafish, respectively.
Fig. 2
Fig. 2
Factors that balance free and conjugated ISG15 during the immune response. 1. Binding of interferon (IFN) to the interferon receptor (IFNR) results in a conformational change that enables the transphosphorylation of associated Janus Kinase (JAK). The activated JAK then phosphorylates adjacent signal transducer and activator of transcription (STAT) proteins. 2. Phosphorylated STAT dimers migrate to the nucleus where they bind to interferon-sensitive response elements (ISRE) inducing hundreds of interferon-stimulated genes (ISGs), among them are ISG15, ISG15 conjugating enzymes (e.g. UBE1L, UBE2L6, HERC5), and its specific deISGylase (DIG), USP18). 3. HERC5, the major ISG15 E3 ligase may interact with the ribosome where it massively labels synthesized proteins (from both host or viral transcripts) as they emerge from the ribosome (3a). An as-yet-unidentified E3 may interact with a ubiquitous protein signal such as ubiquitin to generate mixed Ub-ISG15 modifications on target proteins (3b). 4. ISGylation contributes to innate immune response and inhibits viral replication, though the precise mechanism remains elusive. 5. ISGylation is overturned by USP18, a DIG that can remove ISG15 from a substrate releasing free ISG15. 6. The free form of ISG15 can be conjugated to new targets (by HERC5 and other E3 ligases), or stabilize USP18, a known inhibitor of the STAT-JAK pathway (unrelated to its catalytic DIG activity) thereby inhibiting the innate immune response. Free ISG15 can also be secreted from the cell and act as a cytokine-like protein thereby enhancing the host immune response. 7. Some viruses evolved the ability to bypass the ISG15 pathway. Coronaviruses encode a PLpro enzyme that can cleave Ub or ISG15 from conjugates (in addition to cutting the viral polypeptide precursors, PP1a, PP1ab, into an array of functional proteins (NSPs) initiating the replication of the virus). 8. By counteracting the conjugating pathway, the virus generates free ISG15. |Deconjugating ISG15 may benefit the virus to bypass the host's innate immune response, but the formation of free ISG15 could also alert neighboring cells (6).
Fig. 3
Fig. 3
Structural alignment of viral PLpro and select USP enzymes. A. Ribbon illustration of apoenzyme 3D structures: i) MERS-PLpro (pdb:4rna), ii) SARS-CoV-PLpro (pdb:2fe8), iii) SARS-CoV2-PLpro (pdb:6wrh), iv) hUSP18 (AF-Q9UMW8-F1; [130]), and v) hUSP14 (pdb:6iik+1wgg). PDB entries were taken from https://www.rcsb.org/ and AlphaFold models from https://alphafold.ebi.ac.uk/. A full 3D structure of hUSP14 was generated by superimposing the USP domain (6iik) and N-terminal UBL domain (1wgg) onto the PLpro structure. A model of hUSP18 was generated by Alphafold . vi) Superimposition of all five enzymes highlights structurally equivalent residues shared by all five enzymes (i.e. the common core shown in pink) calculated by mTM-align . B. Ribbon illustration of PLpro and USP 3D structures in complex with substrate, either Ub (hUSP14) or proximal domain of ISG15: i) MERS-PLpro (pdb:5w8u), ii) SARS-CoV-PLpro (pdb:5tl7), iii) SARS-CoV2-PLpro (pdb:6xa9), iv) hUSP18 (AlphaFold model:AF-Q9UMW8-F1 +PDB:2hj8), and v) hUSP14 (PDB:2ayo+1wgg). Full 3D structure of Ub-bound hUSP14 was generated by the superimposition of 2ayo for the Ub-aldehyde domain bound to the USP domain and 1wgg for the N-terminal UBL domain of hUSP14, onto the structure of PLpro. A model of ISG15-bound hUSP18 was generated by Alphafold for hUSP18 and the solution structure of the proximal domain of hISG15 (PDB:2hj8) superimposed on complexed mUSP18-mISG15 (PDB:5CHV). vi) Superimposition of substrate-bound enzymes defines the common core. Structurally equivalent residues of all five enzymes are shown in pink as computed by mTM-align . C. Superimposition of the catalytic core of hUSP18 and ISG15-bound SARS-CoV2-PLpro (as determined in panel A). The shared residues of both these proteases calculated by mTM-align are shown in pink; left. The catalytic triad residues overlay precisely in the active site; right. The narrow cleft leading to this active site restricts access to substrates with flexible stretches (shown in blue). Cleavage is limited to the presence of Glycine at P1 and P2 sites. Images were generated using ChimeraX .
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