A better understanding of the interactions between SARS-CoV-2 and the host cells it infects could lead to new approaches to treat or prevent infections. Proteolysis is essential to the SARS-CoV-2 life cycle; two viral polyproteins must be cleaved to generate components of the viral replication/transcription complex. The polyproteins are snipped apart by two viral proteases, the papain-like protease (PLP) and main protease (Mpro).
PLP and Mpro also cleave cellular proteins, which may help the virus by modifying the activities of these targets. Protease inhibitors have demonstrated antiviral activity in cell culture and are a subject of anti-coronavirus drug development. To this end, understanding the complete set of proteolytic changes that occur upon viral infection would be important.
Meyer et al carried out an unbiased study of proteolysis of viral and cellular proteins during viral infection in cell culture. Proteins were extracted from infected and uninfected cells, differentially labeled, and analyzed by mass spectrometry in an N-terminomics approach that identified neo-N-termini. In doing so, several cellular targets of viral proteases were identified as well as new viral protein cleavage sites, which hold potential for future vaccine or antibody development.”
To confirm their findings, both in vitro and cell-based assays were used to observe the process of SARS-CoV-2 proteases cleaving cellular proteins. Specifically, SARS-CoV-2 Mpro was expressed in cells with the subsequent cleavage of two target proteins being confirmed via chemiluminescent Western blotting imaged on an Azure c600.
The authors found that the cleavage of cellular proteins was important for viral infectivity. To test for this, RNAi was used to deplete the levels of 14 potential viral protease substrates. Surprisingly, reducing each of the 14 proteins resulted in reduced viral titers, suggesting that viral proteases target and cleave cellular proteins to increase pro-viral activities. Consistent with this, two inhibitors of cellular targets were found to potently inhibit infection of cells by SARS-CoV-2, indicating the activity of these cellular proteins is important to viral infectivity.
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This study demonstrates the enormous potential of characterizing proteolysis in the context of viral infection to aid in the development of targeted anti-viral strategies.
In addition to chemiluminescence imaging, the Azure 600 imager provides multicolor fluorescence, white light, and two-channel near-infrared imaging of blots, gels, and more.