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Global ISGylome analysis to identify substrates of SARS-CoV-2 Nsp3-PLpro

ABSTRACT: Interferon-stimulated gene 15 (ISG15) regulates diverse cellular processes, including antiviral immunity, through its conjugation to proteins (ISGylation). Several viruses, including SARS-CoV-2, reverse the process of protein ISGylation by encoding specialized enzymes harboring deISGylating enzymatic activity. The papain-like protease (PLpro), which is part of the nonstructural protein 3 (Nsp3) of SARS-CoV-2, antagonizes mammalian host innate defense pathways by removing ISG15 from certain cellular substrates. However, the full substrate landscape of Nsp3-PLpro’s deISGylating activity is unknown. Moreover, PLpro, as part of Nsp3, localizes to double-membrane vesicles (DMVs), the sites where coronaviruses replicate inside cells; thus, this unique subcellular localization of Nsp3-PLpro is expected to restrain its substrate specificity. Here, we expressed in human cells a SARS-CoV-2 Nsp3-4 fusion construct that produces mature Nsp3 and Nsp4 via PLpro-mediated autoprocessing and promotes DMV formation, followed by IFNα treatment to mimic an infected state. Cells expressing empty vector with or without IFNα treatment, as well as cells expressing Nsp3-4 without IFNα stimulation were included for comparison. LC-MS/MS analysis identified numerous substrates of PLpro-mediated deISGylation that play roles in several major biological processes, including innate immune signaling, translational control, RNA metabolism, and oxidative stress responses. This global ISGylome dataset provides novel insights into how ISGylation regulates host cells at the molecular level and, further, how SARS-CoV-2 Nsp3-PLpro-mediated deISGylation influences the host ISGylome.

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ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Junji Zhu

LAB HEAD: Michaela Gack

PROVIDER: PXD068808 | Pride | 2026-05-05

REPOSITORIES: Pride

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Project description:SARS-CoV-2 virus and its variants have proven to be a threat to global health, in part due to their capacity for rapid evolution. SARS-CoV-2 variants that emerged throughout the COVID-19 pandemic exhibited variations in virulence, leading to diminished vaccine protection and modulating disease severity. Research efforts have identified the molecular changes resulting from structural protein variants of SARS-CoV-2, particularly in Spike, that have enhanced infectivity. Yet, investigating variants of nonstructural proteins is equally critical to understand viral evolution and their role in manipulating protein-protein interactions within a host. This study focuses on nonstructural protein 3 (nsp3), a viral papain-like protease (PLpro) critical for viral polyprotein cleavage, which is also implicated in host de-ubiquitylation, ISGylation, and formation of double-membrane vesicles. Using affinity-purification mass spectrometry (AP-MS), we identify differential protein interactions caused by mutations in nsp3. Included are variants identified between 2019-2024: Alpha 20I, Beta20H, Delta21I, Delta21J, Gamma20J, Kappa21B, Lambda21G, Omicron21K, Omicron21L. A small set of amino acid substitutions in the cytosolic fragment and N-terminal region of nsp3 (nsp3.1) could be traced to increased interactions with RNA binding proteins, which are vital in viral replication. Nsp3.1 variants from Omicron 21K and 21L exhibit distinct patterns of host interactor enrichment, setting them apart from five other variants. In contrast, variants of the central region of nsp3 (nsp3.2) containing the PL2pro domain were found to share interactions with protein quality control machinery, including ER-associated degradation (ERAD). In this construct, shared trends in interactor enrichment are observed between Omicron21K and Delta21I, distinguishing them from three other variants. Our study highlights that even a small number of amino acid substitutions can significantly alter the interaction partners. Our study provides a roadmap to track the interaction changes driven by SARS-CoV-2 variant evolution, which is essential for elucidating the ongoing evolutionary arms race between the host and virus.

Project description:Human coronaviruses have become an increasing threat to global health; three highly pathogenic strains have emerged since the early 2000s, including most recently SARS-CoV-2, the cause of COVID-19. A better understanding of the molecular mechanisms of coronavirus pathogenesis is needed, including how these highly virulent strains differ from those that cause milder, common-cold like disease. While significant progress has been made in understanding how SARS-CoV-2 proteins interact with the host cell, non-structural protein 3 (nsp3) has largely been omitted from the analyses. Nsp3 is a viral protease with important roles in viral protein biogenesis, replication complex formation, and modulation of host ubiquitinylation and ISGylation. Herein, we use affinity purification-mass spectrometry to study the host-viral protein-protein interactome of nsp3 from five coronavirus strains: pathogenic strains SARS-CoV-2, SARS-CoV, and MERS-CoV, and endemic common-cold strains hCoV-229E and hCoV-OC43. We divide each nsp3 into three fragments and use tandem mass tag technology to directly compare the interactors across the five strains for each fragment. We find that that few interactors are common across all variants for a particular fragment, but we identify shared patterns between select variants, such as ribosomal proteins enriched in the N-terminal fragment (nsp3.1) analysis for SARS-CoV-2 and SARS-CoV. We also identify unique biological processes enriched for individual homologs, for instance nuclear protein important for the middle fragment of hCoV-229E, as well as ribosome biogenesis of the MERS nsp3.1 homolog. Lastly, we further investigate the interaction of the SARS-CoV-2 nsp3.1 N-terminal fragment with ATF6, a regulator of the unfolded protein response. We show that SARS-CoV-2 nsp3.1 directly binds to ATF6 and can suppress the ATF6 stress response. Characterizing the host interactions of nsp3 widens our understanding of how coronaviruses co-opt cellular pathways and presents new avenues for host-targeted antiviral therapeutics.

			Action	DRS
	S1_1_Veck-Mock.d.zip	Other
	S1_1_Veck-Mock.mzML	Mzml
	S1_2_Veck-Mock.d.zip	Other
	S1_2_Veck-Mock.mzML	Mzml
	S1_3_Veck-Mock.d.zip	Other

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Global ISGylome analysis to identify substrates of SARS-CoV-2 Nsp3-PLpro

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