Abstract 112: The 3CL Protease Of SARS-CoV2 Induces Sarcomeric Degradation Through Cleavage Of The Giant Protein Obscurin

Abstract

We hypothesized that the 3CL protease (3CLPro) is responsible for the sarcomere degradation observed in cardiomyocytes infected by SARS-COV2. Overexpression of 3CLPro, but not a catalytically inactive mutant, resulted in breakdown of sarcomeres characterized by intact Z-disk/thin filament subunits that has been recently reported (Perez-Bermejo, et al, 2021) in SARS-COV2 infection. To identify potential host protein targets of 3CLPro in an unbiased fashion we screened the human proteome using a cut-site scoring algorithm that we developed. Scoring, ie likelihood of 3CL protease cleavage, was based off experimental data (Chuck et al, 2010) previously published on the highly homologous (96%) 3CL protease from SARS-COV. This scoring was followed by refinement by secondary structure prediction to identify cut-sites that lie in unstructured regions that are thus thus more likely to be accessible to the protease. Using this method, we identified >1000 potential high-likelihood cut sites across the proteome. Further filtering by proteins with cardiomyocyte expression showed 5 high-likelihood sites within the giant sarcomeric protein, Obscurin (OBSCN), as well as many other structural and signaling proteins which we experimentally validated. Expression of 3CLPro in IPSC cardiomyocytes resulted in significantly reduced OBSCN staining without alterations in Z-disk, thin filament, or thick filament proteins by both western blot and immunocytochemistry. In addition, imaging showed loss of OBSCN at sites with intact Z-disk/thin filament subunits. Thus we propose that activity of 3CLPro is a significant contributor to sarcomere breakdown in SARS-COV2 infection via degradation of Obscurin.