Identification of Human Host Substrates of the SARS-CoV-2 M^pro and PL^pro Using Subtiligase N-Terminomics

Abstract

The recent emergence of SARS-CoV-2 in the human population has caused a global pandemic. The virus encodes two proteases, Mpro and PLpro, that are thought to play key roles in the suppression of host protein synthesis and immune response evasion during infection. To identify the specific host cell substrates of these proteases, active recombinant SARS-CoV-2 Mpro and PLpro were added to A549 and Jurkat human cell lysates, and subtiligase-mediated N-terminomics was used to capture and enrich protease substrate fragments. The precise location of each cleavage site was identified using mass spectrometry. Here, we report the identification of over 200 human host proteins that are potential substrates for SARS-CoV-2 Mpro and PLpro and provide a global mapping of proteolysis for these two viral proteases in vitro. Modulating proteolysis of these substrates will increase our understanding of SARS-CoV-2 pathobiology and COVID-19.

Data-Independent Acquisition Proteomics and N-Terminomics Methods Reveal Alterations in Mitochondrial Function and Metabolism in Ischemic-Reperfused Hearts

“…The number of identified cleavage sites is low compared to reports in cell extracts from cell lines. , This is the first use of subtiligase N-terminomics in heart tissue, which is highly complex, containing numerous cell types and a connective matrix. Further optimization may be necessary to improve the coverage of proteolytic substrates.…”

SARS-CoV-2 NSP5 antagonizes MHC II expression by subverting histone deacetylase 2

“…The epigenetic reprogramming of the CIITA locus we identified is part of a broader pattern of epigenetic modifications that suppress antiviral interferon responses and to promote the cytokine storm ( Balnis et al, 2021 ; Chlamydas et al, 2021 ). This epigenetic reprogramming occurs, in part, via NSP5 interactions with HDAC2 and BRD2 ( Gordon et al, 2020 ; Luo et al, 2023 ). In addition to altering nuclear epigenetic marks, SARS-CoV-2 NSP5 directly interferes with transcription via cleavage of POLDIP3, a DNA polymerase δ-interacting protein required for transcription of antiviral interferon-induced genes ( Wu et al, 2023 ).…”

“…NSP5 also interferes with IRF3-dependent transcription through blocking IRF3 activation downstream of RIG-I signaling, and via modulating its interaction with HDAC2 ( Naik et al, 2022 ; Zheng et al, 2022 ). A screen for host cell targets of NSP5-mediated proteolysis identified 203 proteins, of which a quarter are nuclear proteins that include DNA polymerases, components of the nuclear pore complex, histones, epigenetic modifiers, and transcription factors ( Luo et al, 2023 ). While it is unknown what effect most of these cleavage events have on host cell function, it is clear that nuclear proteins are an important target for NSP5.…”

Application of N-Terminal Labeling Methods Provide Novel Insights into Endoproteolysis of the Prion Protein in Vivo

“…Only a few studies have used N-terminal labeling strategies to characterize proteolysis in samples of higher complexity, such as blood (enzymatic labeling), pancreatic tumors (chemical labeling), and colon biopsies (chemical labeling) . Subtiligase labeling, despite the limitation of high amounts of starting material (in the mg range), has allowed the generation of valid information about proteolytic events in important biological processes such as viral infections (Zika virus and SARS-CoV-2) or cleavage event at the cell surface . In our study, we explored the applicability of subtiligase-based N-terminomics for the first time in brain samples.…”