Oral Presentation 27th Annual Lorne Proteomics Symposium 2022

SARS-CoV-2 3CLpro Host Substrates and Interactors Identify Subversive Pathways in the COVID-19 Cellular coup d'état (#29)

I Pablos 1 , Y Machado 1 , HC Ramos de Jesus 1 , Y Mohamud 1 , R Kappelhoff 2 , Cecilia Lindskog 3 , M Vlok 1 , P Bell 2 , G Butler 2 , P Grin 2 , Nestor Solis 2 , H Luo 1 , Eric Jan 1 , J Hirota J Hirota 4 , Arinjay Banerjee 4 , Chris M Overall 2
  1. University of British Columbia, Vancouver, BC, Canada
  2. UBC Centre for Blood Research, University of British Columbia, Vancouver, BC, Canada
  3. Uppsala University, Uppsala, Sweden
  4. McMaster University, Hamilton, Ontario, Canada

The main viral protease (3CLpro) is indispensable for SARS-CoV-2 replication. The opaque contribution of 3CLpro to the overwhelming of host cell machinery remains understudied. We addressed this challenge by employing state-of-the-art TAILS substrate-targeted N-terminomics and substrate winnowing analyses to comprehensively profile the human host cell substrates of 3CLpro.

We analyzed 3CLpro cleavages in native proteome extracts from HEK293 cells (N = 3). Antiviral type I interferons (IFN) induce host cell protection by interferon-stimulated gene responses. We treated human lung epithelial (BEAS-2B) cells with IFN-a (N = 3), IFN-b (N = 3), or vehicle (N = 3). For definitive identification as a substrate heavy-labelled neo-N-termini had to be present solely as a “heavy singleton”. For confident identification, these neo-N-termini had to be identified in ≥ 2/3 independent HEK-293 or ≥ 7/9 independent BEAS-2B human lung cell experiments. Combining the HEK-293 and BEAS-2B datasets, we quantified 1,649 labelled N-termini, including 955 neo-N-termini. Thereby, we identified 292 3CLpro-cleaved neo-N-termini in 229 proteins.

We expanded the 3CLpro substrate landscape to over 100 substrates and 58 additional high confidence candidate substrates in human lung and kidney cells supported by analyses of SARS-CoV-2-infected cells. Enzyme kinetics and molecular docking simulations of 3CLproengaging substrates reveal how noncanonical cleavage sites, which diverge from SARS-CoV, guide substrate specificity. In exploring the consequences of 3CLpro cleavage events, we demonstrate the direct binding of galectin-8 to Spike S1 glycoprotein, with galectin-8 cleavage disengaging CALCOCO2/NDP52 to decouple and escape antiviral-autophagy, also known as xenophagy. Indeed, in post-mortem COVID-19 lung samples, NDP52 rarely colocalizes with galectin-8, unlike healthy lung cells.  Cleavage of four Hippo signalling proteins, including Yes-associated protein-1 (YAP1), cyclic AMP responsive element-binding protein 1 (CREB1) and cyclic AMP-dependent transcription factor 1 (ATF1), with cleavage-inactivation of a Hippo pathway regulator—mitogen-activated kinase-kinase-kinase-kinase 5 (MAP4K5), suggests a route to combat antiviral defences. PPI analyses of 101 3CLpro substrates reveal extensive disruption of cellular protein interaction networks resulting from viral proteolysis leading to the stranding of crucial cellular proteins, amplifying the consequences of proteolysis. Thus, our substrate degradome atlas provides a powerful resource to inspire mechanistic studies of COVID-19 pathobiology.