The SARS-CoV-2 virus, responsible for the COVID-19 pandemic, necessitates a thorough understanding of its biology to develop effective treatments and vaccines. Proteolytic cleavage, mediated by both viral and cellular proteases, is crucial for SARS-CoV-2 replication. Viral proteases, specifically the papain-like protease (PLP, nsp3) and main protease (Mpro, nsp5), cleave viral polyproteins to generate functional viral proteins. Beyond their role in viral replication, these proteases can also target cellular proteins, potentially modifying or neutralizing host responses to benefit viral propagation. Previous research has shown that viral proteases cleave proteins involved in innate immune signaling. However, a comprehensive, unbiased identification of novel substrates for these proteases during infection was lacking. Cellular proteases also contribute to viral proteolysis, with the most prominent example being the cleavage of the spike (S) glycoprotein by furin, TMPRSS2, and cathepsins, although precise cleavage sites for each protease remain incompletely characterized. Understanding these cleavage events is critical, especially for spike, the primary antigen in many current vaccines. Modifications like glycosylation, phosphorylation, and proteolytic cleavage during natural infection can impact antigenicity and vaccine efficacy. Various vaccine platforms have varying susceptibilities to altered post-translational modifications. Mass spectrometry-based proteomics offers an unbiased approach to identify proteolytic cleavage sites, offering insights into both viral and cellular processes. This study uses N-terminomics, a mass spectrometry-based approach, to identify novel cleavage and processing sites within viral and cellular proteins during SARS-CoV-2 infection, with the goal of identifying new therapeutic targets.

Previous research has characterized the SARS-CoV-2 proteome, phosphoproteome, ubiquitome, and interactome, revealing some insights into viral-host interactions. Studies have shown that inhibitors targeting viral and cellular proteases can inhibit SARS-CoV-2 replication. However, there was a gap in knowledge regarding a comprehensive, unbiased characterization of proteolysis during SARS-CoV-2 infection. Previous studies identified some cellular targets of SARS-CoV-2 proteases but did not provide a systematic analysis. Several studies focused on the cleavage of the spike glycoprotein by host proteases, but the precise cleavage sites were often unclear. The impact of proteolytic cleavage on other viral proteins and its effect on host cell pathways and processes required further investigation. Mass spectrometry-based proteomics is a powerful tool for studying proteolytic cleavage but had not been fully exploited in the context of SARS-CoV-2 infection. Existing proteomic studies of SARS-CoV-2 have provided valuable information on various aspects of viral infection but lacked a systematic analysis of proteolytic events.

This study used two cell lines, Vero E6 (African Green Monkey kidney cells) and A549-Ace2 (human lung cells overexpressing ACE2), to model SARS-CoV-2 infection. Cells were infected at a multiplicity of infection (MOI) of 1 and harvested at 0, 6, 12, and 24 hours post-infection. Mock-infected samples were also collected. Viral RNA, protein levels, and infectious virus production were quantified by qRT-PCR, mass spectrometry, and plaque assay, respectively. N-terminomics analysis via liquid chromatography-mass spectrometry (LC-MS/MS) was performed on N-terminus-enriched samples. This involved isobaric labeling (TMTpro) for quantification, allowing for sample pooling to reduce variability. The method focused on neo-N-termini (N-termini beginning at amino acid 2 or later), reflecting post-translational modifications and proteolytic cleavage. Data analysis involved principal component analysis (PCA) to separate infected and mock samples. Viral and cellular neo-N-termini showing significant changes in abundance were identified by statistical analysis (two-tailed unpaired t-test, multiple-hypothesis testing correction). To further investigate proteases involved, additional N-terminomics experiments were performed using protease inhibitors (camostat mesylate and calpeptin) added at 12 hours post-infection. A pseudovirus entry assay was used to assess the functional impact of spike glycoprotein mutations near identified cleavage sites. A siRNA screen was conducted to determine the impact of depleting cellular proteins with identified neo-N-termini on viral replication. Finally, a dose-response experiment using inhibitors of putative cellular substrates (SRC and MYLK) evaluated their effect on SARS-CoV-2 replication. Cytotoxicity assays were also performed to determine the safety of these inhibitors.

The N-terminomics analysis identified several novel cleavage sites in SARS-CoV-2 proteins, including the spike (S) and nucleocapsid (N) proteins. Specifically, a novel cleavage site was identified between Y636 and S637 in spike, located in a flexible loop near the furin cleavage site. The abundance of this neo-N-terminus increased during infection. Analysis showed increased abundance of neo-N-termini from the nucleocapsid protein, consistent with proteolytic cleavage by cellular proteases. Multiple cleavage sites within ORF3a were also observed. Treatment with calpeptin, a cathepsin/calpain inhibitor, altered the abundance of several viral cleavage sites, suggesting involvement of these proteases. The study identified 14 high-confidence cellular substrates of SARS-CoV-2 main and papain-like proteases. These substrates show temporal regulation during infection and are cleaved in vitro by recombinant proteases. SiRNA-mediated depletion of these substrates significantly inhibited SARS-CoV-2 replication. Furthermore, pharmacological inhibition of SRC and MYLK using Bafetinib and ML-7, respectively, demonstrated a dose-dependent reduction in SARS-CoV-2 titres. The analysis of cellular neo-N-termini revealed enrichment of serine residues in the P' region of cleavage sites, suggesting a preference for specific protease activity. In vitro and cell-based assays validated the cleavage of a subset of these substrates by SARS-CoV-2 proteases. Finally, analysis of viral neo-N-termini in the context of current Variants of Concern (VOCs) showed no overall enrichment near variant mutations, although several neo-N-termini were located near known mutations, suggesting potential functional implications.

This study provides a comprehensive analysis of proteolytic events during SARS-CoV-2 infection, revealing numerous novel cleavage sites in both viral and cellular proteins. The identification of novel cleavage sites within viral proteins, especially spike and nucleocapsid, is significant for vaccine development and diagnostic testing. The findings regarding the 14 cellular substrates of SARS-CoV-2 proteases highlight potential therapeutic targets. The fact that siRNA knockdown of these substrates largely inhibited viral replication suggests that these cleavage events are not merely for inactivation but likely modulate the activity of these cellular proteins in a way that benefits the virus. The success of pharmacological inhibition of SRC and MYLK in reducing viral titres further supports this strategy. The study's limitations include the use of two cell lines, which may not fully represent the complexity of human infection. While the findings are promising, further validation in diverse models, in vivo studies, and clinical trials are crucial before translating them into therapeutic interventions. This study demonstrates that proteolysis is a multifaceted process during SARS-CoV-2 infection, involving both viral and cellular proteases. Further investigation into the specific mechanisms of regulation and functional consequences of cleavage events could reveal new strategies for antiviral drug development.

This study provides a detailed N-terminomic analysis of SARS-CoV-2 infection, revealing novel cleavage sites in viral proteins and identifying 14 potential cellular substrates for viral proteases. The findings demonstrate that these substrates are likely pro-viral, and targeting them with drugs shows promise for antiviral therapy. Future studies should focus on the detailed mechanisms of action of these cleavage events, including the investigation of other cell lines and in vivo models to confirm the findings and advance the development of novel therapeutic strategies against COVID-19.

The study primarily used two cell lines (Vero E6 and A549-Ace2), which may not fully capture the complexity of SARS-CoV-2 infection in human tissues. The focus on A549-Ace2 cells for inhibitor assays limits the generalizability of these findings to other cell types. The study primarily focuses on a single SARS-CoV-2 isolate, meaning the identified cleavage sites may not be universal across all strains. The siRNA screen may not have identified all potential substrates, and the specific mechanisms by which the identified substrates influence viral replication warrant further investigation.