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

SARS-CoV-2 3C-like protease (3CLpro or Mpro) 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 3CLpro expression and viral replication. Unexpectedly, 3CLpro 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 3CLpro, which decreases in Gsdmd-/-Gsdme-/- mice. We identify new 3CLpro cut-sites in GSDMD at LQ29↓30SS, which blocks pore formation by 3CLpro cleavage at LH270↓N lying adjacent to the caspase activation site (NFLTD275↓G). Cleavage inactivation of GSDMD prevents excessive pore formation, thus countering antiviral pyroptosis and increasing 3CLpro secretion. Extracellular 3CLpro 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, 3CLpro is secreted with extracellular pathological sequelae.