Poster Presentation 29th Annual Lorne Proteomics Symposium 2024

Interactomics Identifies Novel Stealth SARS-CoV-2 Intercellular Infection Executed by NSP5/3CLpro Main Protease Rearrangement of the Cytoskeleton (#141)

Chris Overall 1
  1. UBC Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada

SARS-CoV-2 main protease 3CLpro (or Mpro) is essential for viral replication leading to COVID-19. We and others have reported that 3CLpro is active against ~300 intracellular host cell substrates to circumvent antiviral host cell proteins and garner cellular resources essential for viral replication. To further explore how 3CLpro targets and reorganizes the human cellular proteome to promote viral replication, we identified host cell protein interactors of 3CLpro by interactomics employing Inactive Catalytic Domain Capture (ICDC).

Over half of the 259 3CLpro interactors were associated with the cytoskeleton and we confirmed cleavage of 13 interactors as 3CLpro substrates, in SARS-CoV-2-infected human lung cells, and verified that five substrates were depleted in COVID-19 post-mortem lungs. SARS-CoV-2 infection induced the translocation of NUMA1 and TRIM28 to epithelial cell adherens junctions and YAP1 to the nucleus. 3CLpro cleavage of adherens junction proteins facilitates cytoskeletal rearrangement and signaling that was associated with increased formation of tunnelling nanotubes following SARS-CoV-2 infection of human lung epithelial cells. These intercellular channels are conduits for the SARS-CoV-2 virus to promote immune-protected “stealth” infection of nearby cells, which may provide additional therapeutic avenues to treat COVID-19.

3CLpro activity also triggers unconventional protein secretion mechanisms enabled by gasdermin-D and GSDME pore formation that was associated with pyroptosis cell death. In synergy, 3CLpro secretion through GSDME is facilitated by endogenous caspase activation, with the caspase inhibition significantly reducing 3CLpro secretion. Extracellular 3CLpro cleaved and inactivated antiviral IFN-lambda 1 with decreased pSTAT1-Y701 signaling that was confirmed in the BALF of C57Bl/6J mice infected with mouse-adapted SARS-CoV-2 and in K18-hACE2 transgenic mice infected with SARS-CoV-2 (Wuhan S-D614G). The new intracellular and extracellular activities of SARS-CoV-2 3CLpro reveal new mechanisms underpinning the complexity of the multifaceted COVID-19 disease and afford new sites for antiviral protease drug targeting.

 

  1. Pablos, I., et al and Overall, C.M. 2021. Mechanistic Insights into COVID-19 by Global Analysis of the SARS-CoV-2 3CLpro Substrate Degradome. Cell Reports 37, 1–17 DOI:https://doi.org/10.1016/j.celrep.2021.109892.
  2. Bell, P.A., and Overall, C.M. (2023). No Substrate Left Behind—Mining of Shotgun Proteomics Datasets Rescues Evidence of Proteolysis by SARS-CoV-23CLpro Main Protease. International Journal of Molecular Science 24, 8,723 – 8,739. https://doi.org/10.3390/ijms24108723.