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

Abstract

SARS-CoV-2 3C-like protease (3CL^pro or M^pro) 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^pro expression and viral replication. Unexpectedly, 3CL^pro 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^pro, which decreases in Gsdmd^-/-Gsdme^-/- mice. We identify new 3CL^pro cut-sites in GSDMD at LQ²⁹↓³⁰SS, which blocks pore formation by 3CL^pro 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 3CL^pro secretion. Extracellular 3CL^pro 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^pro is secreted with extracellular pathological sequelae.

Keywords: COVID-19; CP; Immunology; caspase; gasdermin; infection; interferon; platelet; protease; proteolysis; unconventional protein secretion; virus.