Grin et al. report the first known secretion of a viral protease in infection. 3C-like viral proteases (3CL) are utilized by coronaviruses, including severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), to cleave the viral polyprotein into individual functional proteins necessary for viral replication. In addition to these essential intracellular cleavage events, over 160 intracellular host substrates by the SARS-CoV-2 3CLpro (also known as main protease Mpro) have been characterized and identified (Pablos et al. 2021, Cell Reports).

In a recent Cell Reports paper from the Overall Lab, Grin et al. reveal the unconventional secretion of the SARS-CoV-2 main protease, 3CL^pro, from infected cells into the extracellular milieu through gasdermin (GSDM)-D and GSDME pores activated by caspases. These pores are the terminal effectors of pyroptosis¾a form of cell death that is activated during infection. Grin et al. show that while pores form conduits for 3CL^pro and also viral nucleocapsid protein release from infected cells, excessive pore formation kills the host cell by pyroptosis, thus inhibiting 3CL^pro expression and secretion. To this end, 3CL^pro performs a delicate balance: it regulates its own secretion through GSDMD pores by cleaving at LH²⁷⁰↓N to activate pore formation, but later also cuts at LQ²⁹↓S and LQ¹⁹³↓G to inhibit pore formation. By balancing the levels of pore formation to release 3CLpro, while still maintaining cell function by preventing pyroptosis, selected host and viral proteins are secreted by GSDMD and also GSDME pores. 

What functions does 3CL^pro exert extracellularly?

Through hypothesis-driven investigations, the authors identified and characterized two inactivating cleavages in IFN-l1 by 3CL^pro using amino terminal-oriented mass spectrometry. Such inactivation of IFN-l1 is hypothesized to be an immune escape mechanism for SARS-CoV-2.

The authors further show that 3CL^pro retained ~70% of its activity when incubated in human serum that contains high levels of endogenous protease inhibitors. 3CL^pro also inhibited platelet activation and aggregation in response to the platelet agonist thrombin. These newly described extracellular functions of 3CL^pro may promote the spread of SARS-CoV-2 infection to distal tissues, such as the brain, liver, kidneys, and heart.

This discovery of the regulated secretion of a viral protease with extracellular activity opens the door for future studies to investigate whether release of viral proteases from infected cells occurs with other coronaviruses or diverse virus families. 

Source Publication:

• Grin, P. M., Baid, K., de Jesus, H. C., Kozarac, N., Bell, P. A., Jiang, S. Z., Kappelhoff, R., Butler, G.S., Leborgne, N.G.F., Pan, C., Pablos, I., Machado, Y., Vederas, J.C., Kim, H., Benarafa, C., Banerjee, A., & Overall, C. M. (2024). SARS-CoV-2 3CLpro (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CLpro. Cell Reports, 43(12).