key: cord-317523-idji1l0a
authors: Xu, Huanzhou; Chitre, Siddhi A.; Akinyemi, Ibukun A.; Loeb, Julia C.; Lednicky, John A.; McIntosh, Michael T.; Bhaduri-McIntosh, Sumita
title: SARS-CoV-2 viroporin triggers the NLRP3 inflammatory pathway
date: 2020-10-27
journal: bioRxiv
DOI: 10.1101/2020.10.27.357731
sha: 
doc_id: 317523
cord_uid: idji1l0a

Cytokine storm resulting from a heightened inflammatory response is a prominent feature of severe COVID-19 disease. This inflammatory response results from assembly/activation of a cell-intrinsic defense platform known as the inflammasome. We report that the SARS-CoV-2 viroporin encoded by ORF3a activates the NLRP3 inflammasome, the most promiscuous of known inflammasomes. ORF3a triggers IL-1β expression via NFκB, thus priming the inflammasome while also activating it via ASC-dependent and -independent modes. ORF3a-mediated inflammasome activation requires efflux of potassium ions and oligomerization between NEK7 and NLRP3. With the selective NLRP3 inhibitor MCC950 able to block ORF3a-mediated inflammasome activation and key ORF3a residues needed for virus release and inflammasome activation conserved in SARS-CoV-2 isolates across continents, ORF3a and NLRP3 present prime targets for intervention. Summary Development of anti-SARS-CoV-2 therapies is aimed predominantly at blocking infection or halting virus replication. Yet, the inflammatory response is a significant contributor towards disease, especially in those severely affected. In a pared-down system, we investigate the influence of ORF3a, an essential SARS-CoV-2 protein, on the inflammatory machinery and find that it activates NLRP3, the most prominent inflammasome by causing potassium loss across the cell membrane. We also define key amino acid residues on ORF3a needed to activate the inflammatory response, and likely to facilitate virus release, and find that they are conserved in virus isolates across continents. These findings reveal ORF3a and NLRP3 to be attractive targets for therapy.

Worldwide reports of COVID-19 indicate that effective management of severely ill individuals 67 will require both antiviral and anti-inflammatory strategies. Indeed, during the second week of cells. Importantly, we find that although the CoV-2 ORF3a protein has diverged somewhat from 94 its homologs in other CoVs, some of these newly divergent residues are essential for activating the 95 NLRP3 inflammasome and are perfectly conserved in virus isolates across continents. 

With lung as the predominant site of pathology along with established tropism for kidney and other 100 organs 18 , we introduced ORF3a into lung origin A549 cells and for comparison, kidney origin 101 HEK-293T cells, cell types that readily support SARS-CoV-2 infection 19 , and found induction of 102 pro-IL-1in both cell types, consistent with priming of the inflammasome. Compared to empty 103 vector-exposed cells, ORF3a also increased the levels of cleaved, i.e. the active form of the pro-104 inflammatory caspase, caspase 1, as well as the cleaved form of the caspase 1 substrate, pro-IL-105 1, indicating activation of the inflammasome, again in both cell types (Fig.1A) . Priming by 106 ORF3a resulted from NFB-mediated expression of IL-1 message (Fig.1B) as indicated by 107 increased IB phosphorylation and enrichment of NFB p65 at the IL-1 promoter in ORF3a-108 exposed cells (Figs.1C-E). ORF3a also caused cleavage/activation of Gasdermin D, the 109 pyroptosis-inducing caspase 1-substrate, indicated by an increase in the N-terminal fragment of Gasdermin D (Fig.1F ). This was accompanied by ORF3a-mediated increased cleavage/activation 111 of caspase 3 and cell death, likely secondary to both pyroptosis and apoptosis (Figs.1G and H).

Thus, ORF3a primes the inflammasome by triggering NFB-mediated expression of pro-IL-1 113 while also activating the inflammasome to cleave pro-caspase-1, pro-IL-1, and the pore-forming 114 Gasdermin D, inducing cell death.

ORF3a activates the NEK7-NLRP3 inflammasome via ASC-dependent and independent 117 modes. 118 In probing the mechanism of ORF3a-mediated activation of the inflammasome, we found that it NIMA-related kinase NEK7 recently linked to NLRP3 activation 16 , we also depleted NEK7 and 125 found that ORF3a was impaired in its ability to cause cleavage of caspase 1, i.e. unable to activate 126 the inflammasome (Fig.2D) . The NLRP3 inflammasome is activated by a variety of cell-extrinsic 127 and -intrinsic stimuli that trigger the assembly of the inflammasome machinery wherein NLRP3 128 oligomerizes with the adaptor protein ASC (Apoptosis-associated speck-like protein containing a 129 CARD) leading to recruitment of pro-caspase 1 which is then activated by proximity-induced 130 intermolecular cleavage. Given ORF3a-mediated inflammasome activation in HEK-293T cells 131 that lack ASC (Fig.2E) , we asked if ORF3a activated the inflammasome solely in an ASC-132 independent manner. We found that ORF3a's ability to activate pro-caspase 1 was substantially 133 impaired upon depletion of ASC in A549 cells (Fig.2F) , supporting the idea that ORF3a activates 134 the inflammasome in both ASC-dependent and -independent ways. To assess if ORF3a also 135 mediates activation of other prominent inflammasomes including NLRP1 and NLRC4, we 136 depleted each of these molecules but were unable to block cleavage of pro-caspase 1 (Fig.2G) , 137 indicating that ORF3a predominantly activates the NLRP3 inflammasome. ORF3a triggers NLRP3 inflammasome assembly via K + efflux. 140 With NEK7 a key mediator of NLRP3 activation downstream of potassium efflux, and efflux of 141 potassium ions a central mechanism of NLRP3 activation, particularly by ion channel-inducing 142 viroporins 15,16,21 , we investigated the effect of blocking potassium efflux by raising the 143 extracellular concentration of K + and found that ORF3a-mediated caspase 1 cleavage was 144 abrogated (Fig.3A) . To identify the type of K + channel formed by ORF3a, we employed known 145 pharmacologic inhibitors including quinine, barium, iberiotoxin, and tetraethylammonium to block 146 two-pore domain K + channels, inward-rectifier K + channels, large conductance calcium-activated 147 K + channels, and voltage gated K + channels, respectively 22 . Mimicking the ability of barium to dampened response by the inflammasome (Fig.4B) . Thus, SARS-CoV-2 ORF3a has retained some 170 of the key residues needed for virus release and inflammasome activation but it has acquired 171 additional changes that support a functionally consequential divergence from earlier CoVs.

Nonetheless, this domain bearing the abovementioned residues that is essential for forming ion 173 channels for virus release has remained remarkably well conserved throughout the pandemic, 174 thereby maintaining its ability to activate the inflammasome. In summary, an essential viroporin required for release of SARS-CoV-2 from infected cells is also 178 able to prime and activate the NLRP3 inflammasome, the machinery responsible for much of the 

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CoV-2 reference strain Wuhan-Hu-1 (GenBank NC_045512.2) and 100% identity with Co-Immunoprecipitation (Co-IP) 127 Co-IP was performed as described previously 7 . Cells were lysed in ice-cold IP Lysis