By TAILS N-terminomics (1) and interactomics (2), we identified and validated a diverse array of >200 host cell substrates of SARS-CoV-2 3CLpro/Main protease (Mpro), which we mechanistically investigated to reveal new pathways to evade host cell antiviral defences.145 interactors were associated with the cytoskeleton of which we determined kcat/kM for 139 3CLpro cut-sites, from which 29 are efficiently cleaved substrates in vitro, in SARS-CoV-2-infected human lung cells, and in mouse infection models. 3CLpro cleavage of adherens junction proteins rapidly initiated cytoskeletal rearrangement, activated zonular signalling and removed nuclear localization motifs, triggering translocation of nuclear TRIM28 and NUMA1 to adherens junctions, and PTBP1 and YAP1 to the nucleus. Together, these activities form tunnelling nanotubes that connect lung cells and contain virus colocalized with 3CLpro substrates. TNTs are conduits for the viral transmission to non-infected recipient cells that are concealed from protective antibody and immune cell recognition.
Gasdermin-D (GSDMD) is activated by caspase cleavage at (GSDMD 1–275) to form membrane pores for unconventional secretion of cytokines. We establish that 3CLpro is secreted from SARS-CoV-2 infected cells by unconventional protein secretion through GSDMD and GSDME pores formed by viral 3CLpro in concert with host caspase cleavages. MS/MS identified 3CLpro cleaves gasdermin-D (GSDMD) at LH270↓271NF, 5 amino-acids away, generating a pore-forming N-terminal domain (GSDMD 1–270) that leads to 3CLpro and nucleocapsid protein secretion from infected cells. 3CLpro also cleaves and inactivates GSDMD at two sites to self-regulate and prevent excessive pore formation and pyroptosis. 3CLpro was abundant in conditioned media of SARS-Cov-2-infected cells, detected in bronchoalveolar lavage fluid of wild-type SARS-Cov-2-infected mice and decreased in Gsdmd–/–Gsdme–/– mice. Extracellular 3CLpro retained proteolytic activity in serum, dampened platelet activation and aggregation, and inactivated interferon-lambda1 by two cleavages. Linking intracellular 3CLpro cleavage of GSDMD with 3CLpro secretion and extracellular activity reveals unexpected viral protease-driven immune evasion mechanisms.