Identification of SARS-CoV-2 Main Protease (Mpro) Cleavage Sites Using Two-Dimensional Electrophoresis and In Silico Cleavage Site Prediction.

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.

Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.