SARS-CoV-2 proteases cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species and the search for reservoir hosts

Abstract

The genome of SARS-CoV-2 (SARS2) encodes for two viral proteases (NSP3/ papain-like protease and NSP5/ 3C-like protease or major protease) that are responsible for cleaving viral polyproteins for successful replication. NSP3 and NSP5 of SARS-CoV (SARS1) are known interferon antagonists. Here, we examined whether the protease function of SARS2 NSP3 and NSP5 target 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 IL-6 and inflammatory response observed in COVID-19 patients. Surprisingly, both NLRP12 and TAB1 have each two distinct cleavage sites. We demonstrate that in mice, the second cleavage site of NLRP12 is absent. 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. Finally, we discovered that one particular species of bats, David's Myotis, possesses the five cleavage sites found in humans for NLRP12, TAB1 and IRF3. These bats are endemic from the Hubei province in China and we discuss its potential role as reservoir for the evolution of SARS1 and SASR2.

High-Throughput Screening for Drugs That Inhibit Papain-Like Protease in SARS-CoV-2

“…Many of these screens have used a cell-free system involving purified enzyme and Förster resonance energy transfer (FRET)-based reporters. 16 While these studies have identified different potential inhibitors, they have a crucial drawback: cytotoxicity. By beginning our screen with a cell-based system, we sought to identify compounds that inhibit PLpro but cause minimal cytotoxicity.…”

The papain-like protease of coronaviruses cleaves ULK1 to disrupt host autophagy

“…Of note, we identified the SARS-CoV-2 PL pro cleavage site of ULK1 after glycine (G) 499, follows precisely the consensus sequence recognized by the deubiquitinase. The interferon regulatory factor 3 (IRF3) was also recently reported as a substrate of PL pro following an LGGG consensus motif similar to ULK1 (32). The targeting of ULK1 and possibly other host proteins harboring an LXGG motif therefore underscores potentially a novel mechanism through which betacoronaviruses subvert cellular autophagy.…”

Antagonism of Type I Interferon by Severe Acute Respiratory Syndrome Coronavirus 2

“…Moreover, Dikic and coworkers showed that SARS-CoV-2 nsp3-PLpro limited IFN-I production by cleaving the ubiquitin-like protein ISG15 and decreasing the phosphorylated IRF3 (Shin and others 2020 ). Furthermore, Moustaqil and others ( 2020 ) employed a cell-free cleavage assay to show that SARS-CoV-2 nsp3 was able to directly cleave IRF3, resulting in decreased IRF3 activation and IFN-I production.…”