SARS-CoV-2 3CL (main protease) regulates caspase activation of gasdermin-D/E pores leading to secretion and extracellular activity of 3CL.

SARS-CoV-2 3C-like protease (3CL or M) cleaves the SARS-CoV-2 polyprotein and >300 intracellular host proteins to enhance viral replication. By lytic cell death following gasdermin (GSDM) pore formation in cell membranes, antiviral pyroptosis decreases 3CL expression and viral replication. Unexpectedly, 3CL and nucleocapsid proteins undergo unconventional secretion from infected cells via caspase-activated GSDMD/E pores in the absence of cell lysis. Bronchoalveolar lavage fluid of wild-type SARS-CoV-2-infected mice contains 3CL, which decreases in GsdmdGsdme mice. We identify new 3CL cut-sites in GSDMD at LQ↓SS, which blocks pore formation by 3CL cleavage at LH↓N lying adjacent to the caspase activation site (NFLTD↓G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CL secretion. Extracellular 3CL retains activity in serum, dampens platelet activation and aggregation, and inactivates antiviral interferon-λ1. Thus, in countering gasdermin pore formation and pyroptosis in SARS-CoV-2 infection, 3CL is secreted with extracellular pathological sequelae.