Abstract
Novel therapies to treat infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of respiratory coronavirus disease 2019 (COVID-19), would be of great clinical value to combat the current and future pandemics. Two viral proteases, papain-like protease (PL(pro)) and the main protease 3-chymotrypsin-like protease (3CL(pro)), are vital in processing the SARS-CoV-2 polyproteins (pp1a and pp1ab) and in releasing 16 nonstructural proteins, making them attractive antiviral drug targets. In this study, we investigated the degradation of the SARS-CoV-2 main protease 3CL(pro) by matrix metalloproteinase-14 (MMP14). MMP14 is known to recognize over 10 distinct substrate cleavage sequences. Through sequence analysis, we identified 17 and 10 putative MMP14 cleavage motifs within the SARS-CoV-2 3CL(pro) and PL(pro) proteases, respectively. Despite the presence of potential sites in both proteins, our in vitro proteolysis assays demonstrated that MMP14 selectively binds to and degrades 3CL(pro), but not PL(pro). This selective proteolysis by MMP14 results in the complete loss of 3CL(pro) enzymatic activity. In addition, SARS-CoV-2 pseudovirus replication was inhibited in 293 T cells when either full-length MMP14 or its catalytic domain (cat-MMP14) were overexpressed, presumably due to 3CL(pro) degradation by MMP14. Finally, to prevent MMP14 from degrading off-target proteins, we propose a new recombinant pro-PL-MMP14 construct that can be activated only by another SARS-CoV-2 protease, PL(pro). These findings could open the potential of an alternative therapeutic strategy against SARS-CoV-2 infection.