Related Experiment Video
Updated: Aug 9, 2025
A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico
Noémi Miltner1, Gergő Kalló1,2, Éva Csősz1,2
1Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4032 Debrecen, Hungary.
Abstract:
The main protease (Mpro) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) plays a crucial role in its life cycle. The Mpro-mediated limited proteolysis of the viral polyproteins is necessary for the replication of the virus, and cleavage of the host proteins of the infected cells may also contribute to viral pathogenesis, such as evading the immune responses or triggering cell toxicity. Therefore, the identification of host substrates of the viral protease is of special interest. To identify cleavage sites in cellular substrates of SARS-CoV-2 Mpro, we determined changes in the HEK293T cellular proteome upon expression of the Mpro using two-dimensional gel electrophoresis. The candidate cellular substrates of Mpro were identified by mass spectrometry, and then potential cleavage sites were predicted in silico using NetCorona 1.0 and 3CLP web servers. The existence of the predicted cleavage sites was investigated by in vitro cleavage reactions using recombinant protein substrates containing the candidate target sequences, followed by the determination of cleavage positions using mass spectrometry. Unknown and previously described SARS-CoV-2 Mpro cleavage sites and cellular substrates were also identified. Identification of target sequences is important to understand the specificity of the enzyme, as well as aiding the improvement and development of computational methods for cleavage site prediction.
Insights
Researchers identified host proteins cleaved by the SARS-CoV-2 main protease (Mpro), crucial for viral replication. This study reveals new Mpro targets, aiding understanding of viral pathogenesis and computational prediction methods.
Area of Science:
- Virology
- Proteomics
- Biochemistry
Background:
- The SARS-CoV-2 main protease (Mpro) is essential for viral replication through polyprotein processing.
- Mpro's interaction with host proteins may influence viral pathogenesis, immune evasion, and cell toxicity.
Purpose of the Study:
- To identify cellular substrates and cleavage sites targeted by SARS-CoV-2 Mpro.
- To enhance understanding of Mpro specificity and improve computational cleavage site prediction tools.
Main Methods:
- Proteomic analysis of HEK293T cells expressing SARS-CoV-2 Mpro using 2D gel electrophoresis.
- Mass spectrometry for identifying candidate substrates and cleavage positions.
- In silico prediction of cleavage sites using NetCorona 1.0 and 3CLP web servers.
- In vitro cleavage assays with recombinant proteins to validate predicted sites.
Main Results:
- Identification of novel cellular substrates cleaved by SARS-CoV-2 Mpro.
- Determination of specific cleavage sites within identified host proteins.
- Validation of previously known and discovery of unknown Mpro cleavage sites and substrates.
Conclusions:
- The study successfully identified cellular targets of SARS-CoV-2 Mpro, providing insights into viral pathogenesis.
- Findings contribute to understanding Mpro enzyme specificity and advancing computational prediction models for protease cleavage sites.
