SARS-CoV-2 Nonstructural Proteins 1 and 13 Suppress Caspase-1 and the NLRP3 Inflammasome Activation

Abstract

Viral infection-induced activation of inflammasome complexes has both positive and negative effects on the host. Proper activation of inflammasome complexes induces down-stream effector mechanisms that inhibit viral replication and promote viral clearance, whereas dysregulated activation has detrimental effects on the host. Coronaviruses, including SARS-CoV and MERS-CoV, encode viroporins that activate the NLRP3 inflammasome, and the severity of coronavirus disease is associated with the inflammasome activation. Although the NLRP3 inflammasome activation is implicated in the pathogenesis of coronaviruses, these viruses must evade inflammasome-mediated antiviral immune responses to establish primary replication. Screening of a complementary DNA (cDNA) library encoding 28 SARS-CoV-2 open reading frames (ORFs) showed that two nonstructural proteins (NSPs), NSP1 and NSP13, inhibited caspase-1-mediated IL-1β activation. NSP1 amino acid residues involved in host translation shutoff and NSP13 domains responsible for helicase activity were associated with caspase-1 inhibition. In THP-1 cells, both NSP1 and NSP13 significantly reduced NLRP3-inflammasome-induced caspase-1 activity and IL-1β secretion. These findings indicate that SARS-CoV-2 NSP1 and NSP13 are potent antagonists of the NLRP3 inflammasome.

Keywords: caspase-1; inflammasome; nonstructural protein; severe acute respiratory syndrome coronavirus 2.

Figure 1

Screening of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) open reading frames (ORFs) that regulate caspase-1-mediated IL-1β production. Human embryonic kidney 293T (HEK 293T) cells were co-transfected with vectors expressing caspase-1 and pro-IL-1β, plus either pEF1α-DEST or pEF1α-DEST expressing complementary DNAs (cDNAs) encoding 28 SARS-CoV-2 ORFs. After 24 h, mature IL-1β in culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). * p < 0.001 by unpaired two-tailed Student’s t-tests. Data shown are representative of three independent experiments.

Figure 2

Determination of the amino acid residues of NSP1 critical for caspase-1 inhibition. (A and B) HEK 293T cells were co-transfected with vectors expressing caspase-1 and pro-IL-1β plus either pEF1α-DEST or pEF1α-DEST expressing c-Myc-tagged NSP1 wild type (WT) or NSP1 containing the mutations R124A/K125A, K164A/H165A, and both R124A/K125A and K164A/H165A (quadruple mutant (QM)) and a deletion of amino acids 160–173 (∆ 160–173). After 24 h, (A) the concentrations of mature IL-1β secreted in culture supernatants were measured by ELISA, and (B) levels of caspase-1 and NSP1 (c-Myc) proteins and the cleavage of caspase-1 into its p20 subunit were determined by Western blotting. ELISA data shown are representative of three independent experiments. * p < 0.001 by unpaired two-tailed Student’s t-tests.

Figure 3

Determination of the NSP13 domain required for caspase-1 inhibition. (A) Diagrammatic representation of NSP13 deletion mutants. (B and C) HEK 293T cells were co-transfected with vectors expressing caspase-1 and pro-IL-1β plus either pEF1α-DEST or pEF1α-DEST expressing c-Myc-tagged NSP13 WT or mutants carrying deletions of zinc-binding domains (ZBD) (∆ZBD), the stalk domain (∆S), the 1B domain (∆1B), the two RecA-like domains, 1A (∆1A) and 2A (∆2A) and both 1A and 2A (∆1A/2A). After 24 h, (B) the concentrations of mature IL-1β secreted in culture supernatants were measured by ELISA, and (C) levels of caspase-1 and NSP13 (c-Myc) proteins and the cleavage of caspase-1 into its p20 subunit were determined by Western blotting. ELISA data are representative of three independent experiments. * p < 0.001 by unpaired two-tailed Student’s t-tests.

Figure 4

Effects of NSP1 and NSP13 on the NLRP3 inflammasome in THP-1 cells. THP-1 cells were electroporated with either pEF1α-DEST or pEF1α-DEST expressing c-Myc-tagged NSP1 WT, NSP1 QM, NSP13 WT or NSP13 ∆1A/2A. Twenty hours later, the cells were differentiated by incubation with phorbol-12-myristate 13-acetate (PMA), followed by treatment with lipopolysaccharide (LPS) for 15 h and adenosine triphosphate (ATP) for 1 h. (A) Caspase-1 activity measured by Caspase Glo^®1 Inflammasome assays. (B) Mature IL-1β secretion in culture supernatants measured by ELISA. (C and D) Protein levels of NLRP3, ASC, caspase-1, caspase-1 p20, IL-1β, c-Myc and tubulin in THP-1 cells expressing (C) NSP1 WT and QM and (D) NSP13 WT and ∆1A/2A determined by Western blotting. The results of Caspase Glo^®1 inflammasome assays and IL-1β ELISA are representative of three independent experiments. * p < 0.001 by unpaired two-tailed Student’s t-tests. Sup, supernatant.