SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species

Abstract

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease) that are responsible for cleaving viral polyproteins during replication. NSP3 and NSP5 of SARS-CoV are also known interferon antagonists and it was shown that PLpro of SARS-CoV and SARS-CoV-2 cleave post-translational modifications on host proteins involved in anti-viral responses. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We designed a fluorescent based cleavage assay to rapidly screen the protease activity of NSP3 and NSP5 on a library of 71 human innate immune proteins (HIIPs), covering most pathways involved in human innate immunity. By expressing each of these HIIPs with a genetically encoded fluorophore in a cell-free system and titrating in the recombinant protease domain of NSP3 or NSP5, we could readily detect cleavage of cognate HIIPs on SDS-page gels. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in the mouse NLRP12 protein, the second recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for in-depth studies into the pathophysiology of COVID-19 and should facilitate the search or development of more effective animal models for severe COVID-19.