Coronaviruses exploit a host cysteine-aspartic protease for replication

Chu, Hin; Hou, Yuxin; Yang, Dong; Wen, Lei; Shuai, Huiping; Yoon, Chaemin; Shi, Jialu; Chai, Yue; Yuen, Terrence Tsz-Tai; Hu, Bingjie; Li, Cun; Zhao, Xiaoyu; Wang, Yixin; Huang, Xiner; Lee, Kin Shing; Luo, Cuiting; Cai, Jian-Piao; Poon, Vincent Kwok-Man; Chan, Chris Chung-Sing; Zhang, Anna Jinxia; Yuan, Shuofeng; Sit, Ko-Yung; Foo, Dominic Chi-Chung; Au, Wing-Kuk; Wong, Kenneth Kak-Yuen; Zhou, Jie; Kok, Kin-Hang; Jin, Dong-Yan; Chan, Jasper Fuk-Woo; Yuen, Kwok-Yung

doi:10.1038/s41586-022-05148-4

Download PDF

Article
Published: 03 August 2022

Coronaviruses exploit a host cysteine-aspartic protease for replication

Nature volume 609, pages 785–792 (2022)Cite this article

22k Accesses
19 Citations
199 Altmetric
Metrics details

Subjects

Abstract

Highly pathogenic coronaviruses, including severe acute respiratory syndrome coronavirus 2 (refs. ^1,2) (SARS-CoV-2), Middle East respiratory syndrome coronavirus³ (MERS-CoV) and SARS-CoV-1 (ref. ⁴), vary in their transmissibility and pathogenicity. However, infection by all three viruses results in substantial apoptosis in cell culture^5,6,7 and in patient tissues^8,9,10, suggesting a potential link between apoptosis and pathogenesis of coronaviruses. Here we show that caspase-6, a cysteine-aspartic protease of the apoptosis cascade, serves as an important host factor for efficient coronavirus replication. We demonstrate that caspase-6 cleaves coronavirus nucleocapsid proteins, generating fragments that serve as interferon antagonists, thus facilitating virus replication. Inhibition of caspase-6 substantially attenuates lung pathology and body weight loss in golden Syrian hamsters infected with SARS-CoV-2 and improves the survival of mice expressing human DPP4 that are infected with mouse-adapted MERS-CoV. Our study reveals how coronaviruses exploit a component of the host apoptosis cascade to facilitate virus replication.

Necroptosis blockade prevents lung injury in severe influenza

Article 10 April 2024

Avishekh Gautam, David F. Boyd, … Siddharth Balachandran

Long COVID: major findings, mechanisms and recommendations

Article 13 January 2023

Hannah E. Davis, Lisa McCorkell, … Eric J. Topol

Mechanisms of SARS-CoV-2 entry into cells

Article 05 October 2021

Cody B. Jackson, Michael Farzan, … Hyeryun Choe

Main

A common feature of coronaviruses is their propensity to induce apoptosis in infected target cells. We recently demonstrated that inhibition of apoptosis with a pan-caspase inhibitor significantly attenuated MERS-CoV replication^11,12, suggesting a previously unrecognized connection between the apoptosis cascade and coronavirus replication. Previous studies have revealed that caspase-6 can mediate the cleavage of nucleocapsid (N) protein from a number of coronaviruses, including SARS-CoV-1, transmissible gastroenteritis coronavirus (TGEV) and porcine epidemic diarrhea virus^13,14,15 (PEDV), but the biological significance of caspase-6-mediated N cleavage is unknown. In this study, we identify caspase-6—a cysteine-aspartic protease that serves as an executor caspase of the apoptosis cascade—as a host factor that facilitates efficient coronavirus replication.

Caspase-6 aids coronavirus replication

We recently demonstrated that MERS-CoV infection triggers substantial apoptosis, and inhibition of apoptosis with the pan-caspase inhibitor z-VAD-fmk significantly limits MERS-CoV replication^11,12 (Extended Data Fig. 1a,b). z-VAD-fmk similarly limited the replication of other human pathogenic coronaviruses, including SARS-CoV-2, SARS-CoV-1, HCoV-229E and HCoV-OC43 (Extended Data Fig. 1b,c), suggesting that the dependency on apoptosis or caspase activity for efficient virus replication is conserved for coronaviruses. Caspases are cysteine-aspartic proteases that regulate the host apoptosis cascade¹⁶. To investigate which caspase is most responsible for modulating coronavirus replication, we used MERS-CoV as a model virus to evaluate virus replication in the presence of specific inhibitors against individual caspases, and demonstrated that caspase-6 inhibition had the greatest effect on limiting MERS-CoV replication (Fig. 1a,b). The inhibitory effect on MERS-CoV replication by caspase-6 inhibition was conserved across different cell types with the exception of Vero E6 cells (Extended Data Fig. 1d). Inhibition of caspase-6 with its specific inhibitor, z-VEID-fmk, attenuated the replication of all tested coronaviruses, including that of SARS-CoV-2 and SARS-CoV-1, but did not affect the replication of influenza virus (H1N1) or enterovirus (enterovirus A71) (Fig. 1c and Extended Data Fig. 1e). According to quantitative PCR with reverse transcription (RT–qPCR) and median tissue culture infectious dose (TCID₅₀) assays, the half-maximal inhibitory concentration (IC₅₀) of z-VEID-fmk versus coronaviruses ranged from 3.3 μM for SARS-CoV-2 to 21.1 μM for MERS-CoV in the cell lysate samples and 1.2 μM for SARS-CoV-2 to 30.6 μM for HCoV-OC43 in the supernatant samples (Fig. 1d and Extended Data Fig. 1f).

**Fig. 1: Caspase-6 inhibition limits coronavirus replication.**

Next, we used MERS-CoV and SARS-CoV-2 as model coronaviruses and evaluated the effect of caspase-6 inhibition on coronavirus replication in infected human ex vivo lung tissues, human intestinal organoids and animal models. In human lung tissues, caspase-6 inhibition with z-VEID-fmk significantly reduced MERS-CoV N protein and N gene expression (Fig. 2a,b). Similarly, z-VEID-fmk inhibited MERS-CoV replication in human intestinal organoids and inhibited the production of infectious virus particles by approximately 80% (P < 0.0001) at 24 hours post-infection (hpi) (Fig. 2c–f). As caspase-6 is largely conserved among mammals and the z-VEID-fmk-binding pocket is conserved among humans, mice and hamsters, we can evaluate the effect of caspase-6 inhibition with z-VEID-fmk in these animals (Extended Data Fig. 2a,b). To evaluate the effect of caspase-6 inhibition on MERS-CoV replication in vivo, we infected human DPP4 knock-in (hDPP4-KI) mice with mouse-adapted MERS-CoV¹⁷ (MERS-CoV_MA) and treated them with z-VEID-fmk or DMSO (Fig. 2g). z-VEID-fmk effectively reduced MERS-CoV_MA replication in the lungs of infected mice at both 2 and 4 days post-infection (dpi) (Fig. 2h–j), and significantly attenuated the expression of genes encoding pro-inflammatory cytokines and chemokines (Fig. 2k and Extended Data Fig. 2c). Of note, the z-VEID-fmk treatment largely inhibited body weight loss and significantly improved the survival of infected hDPP4-KI mice from 33.3% to 80% (3 out of 9 versus 8 out of 10; P = 0.0388) (Fig. 2l,m).

**Fig. 2: Caspase-6 inhibition attenuates MERS-CoV replication in human lung tissues and human intestinal organoids, and improves the survival of hDPP4-KI mice.**

We next tested whether z-VEID-fmk could similarly inhibit SARS-CoV-2 replication in vivo using intranasal challenge of golden hamsters with SARS-CoV-2 and treatment with z-VEID-fmk or DMSO (Fig. 3a). Caspase-6 inhibition with z-VEID-fmk significantly reduced SARS-CoV-2 replication in the lungs (Fig. 3b,c), including small airways (Extended Data Fig. 2d) and alveoli (Fig. 3d), and ameliorated the expression of virus-induced pro-inflammatory cytokines and chemokines (Fig. 3e). The attenuated virus replication and expression of pro-inflammatory markers resulted in significant improvements of body weight of infected hamsters (Fig. 3f). Furthermore, we collected lung tissues from hamsters at 4 dpi to evaluate histological changes. Lungs from infected hamsters in the mock treatment group showed severe bronchiolar epithelial cell death and desquamation, extensive mononuclear cell infiltration of alveolar space, protein-rich fluid exudation, alveolar haemorrhage and severe destruction of alveolar structure. Pulmonary blood vessel wall inflammation and endothelium infiltration were frequently observed (Fig. 3g, middle). These histopathological changes were consistent with our previous report on SARS-CoV-2-infected hamsters¹⁸. By contrast, all categories of tissue damage, including bronchiolitis, alveolitis and vasculitis, were significantly ameliorated in the lungs of z-VEID-fmk-treated hamsters. Although the z-VEID-fmk treatment substantially inhibited immune cell infiltration of alveolar space, some degree of haemorrhage was still visible in the treatment group (Fig. 3g, bottom). Semi-quantitative histopathological examination of bronchioles, alveoli and blood vessels using our previously described methods¹⁹ showed that z-VEID-fmk treatment significantly ameliorated lung damage in infected hamsters (Fig. 3h). Together, our results demonstrate that caspase-6 inhibition attenuates coronavirus replication in cell culture, human lung tissue, organoid and animal settings.

**Fig. 3: Caspase-6 inhibition ameliorates lung pathology and body weight change of golden Syrian hamsters infected with SARS-CoV-2.**

Caspase-6 cleaves nucleocapsid protein

Next, we sought to understand the role of caspase-6 in coronavirus replication. Using MERS-CoV as a model, we first assessed the effect of z-VEID-fmk in a time-of-addition assay. The z-VEID-fmk added during MERS-CoV inoculation did not reduce virus replication (Extended Data Fig. 3a). Consistent with this finding, MERS-CoV entry in caspase-6-stable-knockdown A549 and BEAS2B cells was not compromised (Extended Data Fig. 3b–d). Next, we evaluated MERS-CoV N gene expression in caspase-6 stable-knockdown A549 and BEAS2B cells collected at 24 hpi and found that MERS-CoV replication was significantly reduced in the presence of caspase-6 knockdown (Extended Data Fig. 3e). We then treated the stable scrambled knockdown or caspase-6 knockdown A549 and BEAS2B cells with z-VEID-fmk and showed that the viral inhibitory effect of z-VEID-fmk was largely diminished among the caspase-6 stable-knockdown A549 and BEAS2B cells (Extended Data Fig. 3f). We further investigated the role of caspase-6 in MERS-CoV replication in human primary monocyte-derived macrophages (MDMs). Transient depletion of caspase-6 with siRNA markedly reduced MERS-CoV replication in MDMs (Extended Data Fig. 3g,h). For a more complete depletion of caspase-6, we performed caspase-6 CRISPR knockout (KO) in Huh7 cells and demonstrated that MERS-CoV replication was significantly compromised in caspase-6-KO Huh7 cells compared with caspase-6 intact cell controls (Fig. 4a and Extended Data Fig. 3i). As well as gene-depletion studies, we examined MERS-CoV replication in caspase-6-overexpressing cells and demonstrated that overexpression of caspase-6—but not caspase-3—efficiently promoted MERS-CoV replication (Extended Data Fig. 3j,k). Since the z-VEID-fmk may have cross-reactivity against other caspases^20,21, we further validated the in vivo importance of caspase-6 on coronavirus replication by performing caspase-6 knockout in hDPP4-KI mice (Extended Data Fig. 4a,b) followed by MERS-CoV_MA challenge. Our results demonstrated that MERS-CoV_MA replication and MERS-CoV_MA-induced lung damage were significantly reduced in lungs of hDPP4-KI caspase-6-KO mice compared with hDPP4-KI/caspase-6 WT mice (Fig. 4b–e and Extended Data Fig. 4c–e). Collectively, these results indicate that caspase-6 is required for efficient MERS-CoV replication in vitro and in vivo.

**Fig. 4: Caspase-6-mediated cleavage of coronavirus N interferes with IFN signalling.**

N fragments modulate IFN response

Together with caspase-3 and caspase-7, caspase-6 is one of the three executor caspases that execute apoptosis by proteolytic cleavage of host substrates²². Caspase-6 is activated by cleavage when apoptosis is induced, but can also undergo autoactivation²³ (Extended Data Fig. 5a,b). Previous studies have revealed that caspase-6 can cleave the N protein of SARS-CoV-1, TGEV and PEDV^13,14,15. We co-expressed caspase-6 with various MERS-CoV components and evaluated viral protein cleavage by caspase-6. We used staurosporine (STS) to trigger apoptosis to mimic the apoptotic environment in MERS-CoV-infected cells. Our results demonstrated that caspase-6 mediated the cleavage of MERS-CoV N but not other viral components (Extended Data Fig. 5c,d). Cleavage of N was completely abolished in the presence of the specific caspase-6 inhibitor z-VEID-fmk (Extended Data Fig. 5e). Of note, N cleavage was readily detected in cells infected by MERS-CoV (Fig. 4f and Extended Data Fig. 5f), and was similarly inhibited by caspase-6 inhibition in infected cells (Fig. 4f). In addition, N was cleaved by capsase-6, but not by the key executor caspase, caspase-3 (Extended Data Fig. 5g). Together, these results demonstrate that caspase-6 specifically cleaves MERS-CoV N. Next, we investigated how caspase-6-mediated N cleavage modulates coronavirus replication. We previously demonstrated that caspase-6 inhibition attenuated MERS-CoV replication in all evaluated cell types with the exception of Vero E6 cells (Extended Data Fig. 1d), which are deficient in interferon (IFN) signalling owing to a homozygous deletion in the type-I IFN gene cluster^24,25. We then treated MERS-CoV-infected Huh7 cells with z-VEID-fmk in the presence of DMSO, filgotinib (JAK inhibitor), ruxolitinib (JAK inhibitor) or IFNα-IFNAR-IN-1 hydrochloride (IFNAR inhibitor). Our results demonstrated that the antiviral effect of z-VEID-fmk diminished when the IFN pathway was inactivated by JAK or IFNAR inhibition (Fig. 4g). These results hinted that caspase-6-mediated N cleavage might modulate coronavirus replication by regulating IFN signalling. To test this hypothesis, we evaluated the role of caspase-6 and MERS-CoV N in regulating IFN response with IFNβ reporter assays. Notably, we demonstrated that MERS-CoV N co-expressed with caspase-6 suppressed IFNβ reporter activation in a dose-dependent manner (Fig. 4h and Extended Data Fig. 6a). Co-expression of caspase-3 and MERS-CoV N or caspase-6 and MERS-CoV envelope (E) protein did not significantly impact IFNβ reporter activation (Fig. 4i). In addition to IFNβ reporter activity, co-expression of caspase-6 and MERS-CoV N similarly reduced the expression of IFNβ and representative IFN-stimulated genes, including IFIT1, IFIT2, IFIT3, IFITM3, TRIM22 and OAS1 (Extended Data Fig. 6b). Recent studies have identified MERS-CoV ORF4a, ORF4b and membrane (M) protein as potent IFN antagonists^26,27,28. Our data showed that caspase-6 did not modulate the IFN antagonism of these known IFN antagonists (Extended Data Fig. 6c). Moreover, caspase-6-cleaved N modulated IFN response at least as potently as these known IFN antagonists (Extended Data Fig. 6d). Notably, we further demonstrated that caspase-6 similarly mediated N cleavage of other human pathogenic coronaviruses, including that of SARS-CoV-2 and SARS-CoV-1 (Extended Data Fig. 7a,b), which is in agreement with our findings that caspase-6 inhibition attenuated the replication of these coronaviruses (Fig. 1c,d). We next expressed the N genes of these coronaviruses together with caspase-6, which revealed that the co-expression of coronavirus N with caspase-6 antagonized IFNβ reporter activation and reduced the expression of representative IFN-stimulated genes, including IFIT3 and OAS1 (Extended Data Fig. 7c,d). Collectively, these results suggest that caspase-6-mediated N cleavage modulates coronavirus replication by regulating IFN signalling.

To further explain how caspase-6-mediated N cleavage regulates IFN response, we analysed the potential caspase-6 cleavage sites on MERS-CoV N on the basis of known caspase-6 substrate specificity²⁹ and the size of the cleavage fragments, and generated the corresponding N mutants that potentially interfered with caspase-6 cleavage (Fig. 5a). Western blot-based cleavage assays demonstrated that caspase-6-mediated cleavage was abolished for the T²³⁹KKA²⁴² mutant, suggesting that caspase-6 cleaves MERS-CoV N at the T²³⁹KKD²⁴² motif (Fig. 5b). The T²³⁹KKD²⁴² motif is located within the intrinsically disordered region (IDR) of MERS-CoV N that bridges the N-terminal and C-terminal domain of N, which are structurally conserved among coronaviruses³⁰. Similar putative caspase-6 cleavage motifs are also present in the IDR of N of other human pathogenic coronaviruses (Supplementary Table 1). In IFNβ reporter assays, caspase-6 and N-mediated IFN antagonism was attenuated when T²³⁹KKA²⁴² was expressed in place of the wild-type N (Fig. 5c). These findings suggested that caspase-6-mediated MERS-CoV N cleavage is essential for its IFN antagonism. Next, we generated N fragments containing residues 1–241 and 242–413, mimicking the N cleavage products (Fig. 5a). We showed that these two fragments were no longer cleaved by caspase-6 (Extended Data Fig. 8a). Consistent with this result, in IFNβ reporter assays and IFNβ ELISA, the two N cleavage products individually limited IFNβ reporter activity but were no longer modulated by caspase-6 (Extended Data Fig. 8b–d). Similarly, we also identified the caspase-6 cleavage site on SARS-CoV-2 N and showed that the fragments of SARS-CoV-2 N also served as potent IFN antagonists (Extended Data Fig. 9a–e). Next, to dissect how the MERS-CoV N fragments modulate IFN signalling, we evaluated the point of action of the MERS-CoV N fragments with IFNβ luciferase reporter assays using RIG-IN (a constitutively active form of RIG-I), MAVS or TBK1 as activators. We demonstrated that MERS-CoV N fragments efficiently reduced the IFNβ luciferase reporter activity when activated by RIG-IN, MAVS or TBK1, suggesting that the N fragments act downstream of RIG-I, MAVS and TBK1 (Extended Data Fig. 8e). Next, we evaluated the capacity of N fragments to interact with different components of the IFN signalling pathway using co-immunoprecipitation assays. We demonstrated that N^1–241 and N^242–413 interacted with IRF3 but not with other components of the IFN signalling pathway (Extended Data Fig. 8f,g). In Huh7 and 293T cells treated with the TLR3 agonist poly(I:C), IRF3 translocated to the cell nuclei, regardless of MERS-CoV N expression. N^1–241 and N^242–413 fragments co-localized with IRF3 and abolished its nuclear translocation (Extended Data Fig. 10a–c).

**Fig. 5: Recombinant MERS-CoV with a single amino change at the caspase-6 cleavage motif on N protein is attenuated in vitro and in vivo.**

To further investigate the in vitro and in vivo roles of caspase-6-mediated N cleavage, we engineered MERS-CoV with a point mutation at the caspase-6 cleavage site on the N protein that abolished caspase-6 cleavage (rMERS-CoV/TKKA) (Extended Data Fig. 11a,b). We then infected A549, Huh7 and Vero E6 cells with rMERS-CoV/TKKA or the wild-type recombinant MERS-CoV (rMERS-CoV/TKKD). We demonstrated that rMERS-CoV/TKKA replication was significantly reduced compared with rMERS-CoV/TKKD in A549 and Huh7 cells, but not in IFN-deficient Vero E6 cells (Fig. 5d and Extended Data Fig. 11c). Next, we infected Huh7 and A549 cells with rMERS-CoV/TKKA or rMERS-CoV/TKKD, followed by z-VEID-fmk treatment. Our results demonstrated that although z-VEID-fmk remained effective against the replication of rMERS-CoV/TKKD, the replication of rMERS-CoV/TKKA was not limited by z-VEID-fmk (Extended Data Fig. 11d). Finally, we infected hDPP4-KI mice with rMERS-CoV/TKKA or rMERS-CoV/TKKD to evaluate virus replication in vivo. This showed that in hDPP4-KI mice, the single amino acid substitution at the caspase-6 cleavage site on N significantly reduced rMERS-CoV/TKKA replication in the lungs as well as lung damage compared with rMERS-CoV/TKKD (Fig. 5e–g and Extended Data Fig. 11e).

Discussion

Our study showed that human pathogenic coronaviruses exploit the host apoptotic pathway through caspase-6-mediated N cleavage to dampen the host IFN response for efficient replication. Inhibition of caspase-6 markedly attenuates coronavirus replication and ameliorates coronavirus-induced lung pathology in vivo, suggesting that caspase-6 inhibition should be further explored as an option for the treatment of highly pathogenic coronaviruses (Extended Data Fig. 12).

The replication of coronaviruses, including MERS-CoV and SARS-CoV-2, is known to depend on the host serine protease transmembrane protease serine 2 (TMPRSS2), which cleaves and activates the spike (S) protein of coronaviruses for efficient cell entry and replication^31,32. By contrast, the role of host cysteine-aspartic protease in coronavirus replication has not been explored. Previous studies have suggested that influenza viruses require caspase-3 for efficient translocation of viral ribonucleoprotein (RNP) complexes across the nuclear membrane, which is essential for efficient virus replication³³. Here we show that caspase-6—but not caspase-3—facilitates coronavirus replication. Caspase-6 is known for its role as an executor caspase and its catalytic role in neurodegeneration in Huntington’s and Alzheimer’s disease³⁴. Of note, caspase-6 has been shown to facilitate inflammasome activation, cell death and host defence during influenza virus infection³⁵. In contrast to coronaviruses, influenza virus replicated more efficiently in the lungs of caspase-6-KO mice compared with wild-type mice³⁵. Here we show that caspase-6 knockout or inhibition attenuates virus replication and disease severity in mice and hamsters infected with highly pathogenic coronavirus. These findings suggest that the inhibition of cysteine-aspartic protease caspase-6 should be explored as a therapeutic option against highly pathogenic coronaviruses, including SARS-CoV-2.

The IFN response is a first-line host defence against coronavirus infection. Genomes of coronaviruses encode multiple viral components^{26,27,28,36,37,38,39,40}, including N protein^41,42,43,44 that can serve as IFN antagonists by targeting different steps of the IFN signalling pathway. Extending these findings, our study further reveals host proteases as factors that can modulate viral antagonism in conjunction with viral components. This suggests that our current understanding of viral components that antagonize IFN signalling may be incomplete, since viral components may be processed by host proteases, thus modulating their capacity to interact with host IFN signalling pathways.

Coronavirus N is an abundantly expressed multi-functional structural protein³⁰ that is required for forming complexes with viral RNA and assembly of virus particles. A complex with uncleaved N is required to carry out these functions. After virus uncoating, increasing intracellular viral RNA is detected by host innate immune sensors, which activates interferon responses and apoptotic pathways. Concomitantly, viral replication increases the amount of N that can serve another important purpose during which the complex structure is cleaved by caspase-6 to antagonize the IFN signalling pathway so that viral replication can continue. This mechanism exemplifies how coronaviruses evolve to maximize the use of their most abundantly expressed protein in a time-dependent manner.

Overall, our study reveals how coronaviruses exploit a component of the apoptosis cascade to facilitate virus replication in order to maximize virus production. This is an elegant example of virus–host interaction that exemplifies the longstanding arms race between humans and coronaviruses.

Methods

Cell lines

A549, BSC1, Caco2, Huh7, Vero E6 and 293T cells were maintained in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin. BEAS2B and Calu3 cells were maintained in DMEM/F12 supplemented with 10% heat-inactivated FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. HFL were maintained in minimum essential medium (MEM) supplemented with 10% heat-inactivated FBS, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Human primary monocytes were obtained from human peripheral blood mononuclear cells (PBMCs) taken from healthy donors, collected from Hong Kong Red Cross Blood Transfusion Service according to a protocol approved by the Institutional Review Board of the University of Hong Kong. MDMs were differentiated from monocytes in RPMI-1640 medium supplemented with 10% heat-inactivated FBS, 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 2 mM glutamine, 1% sodium pyruvate, 1% non-essential amino acids, and 10 ng ml⁻¹ recombinant human granulocyte macrophage colony-stimulating factor (GM-CSF) (R&D Systems) as previously described⁴⁵.

Viruses

The MERS-CoV (EMC/2012) strain of MERS-CoV was provided by R. Fouchier. MERS-CoV_MA was a gift from P. McCray. SARS-CoV-2 wild-type HKU-001a⁴⁶, Omicron BA.1 (GenBank: OM212472) and Omicron BA.2 (GISAID: EPI_ISL_9845731) were isolated from a laboratory-confirmed patient with COVID-19 in Hong Kong. SARS-CoV-1 GZ50, HCoV-229E, HCoV-OC43, enterovirus A71 and influenza A virus strain A/Hong Kong/415742/2009(H1N1)pdm09 were archived clinical isolates at Department of Microbiology, Hong Kong University. All infectious experiments involving MERS-CoV, SARS-CoV-2 and SARS-CoV-1 followed the approved standard operating procedures of the Biosafety Level 3 facility at the Department of Microbiology, Hong Kong University⁴⁷.

Chemical modulators

The pan-caspase inhibitor, z-VAD-fmk, was obtained from Invivogen (tlrl-vad). The caspase-1-to-caspase-10 inhibitor sampler kit was purchased from R&D Systems. The caspase-6 inhibitors, z-VEID-fmk, used for in vitro and in vivo experiments, were obtained from R&D Systems (FMK006) and APExBIO (A1923). The apoptosis enhancer, STS (S5921), was obtained from Sigma. Filgotinib (JAK1 inhibitor) (HY-18300), ruxolitinib (JAK1/2 inhibitor) (HY-50856), and IFNα-IFNAR-IN-1 hydrochloride (HY-12836A) were obtained from MedChemExpress. Poly(I:C) was from R&D Systems (4287/10).

Antibodies for western blots

MERS-CoV N was detected by an in-house guinea pig immune serum (1:100,000). MERS-CoV Spike was detected by an in-house mouse immune serum (1:10,000). SARS-CoV-2 N was detected by an in-house rabbit immune serum (1:10,000). SARS-CoV-1 N was detected by an in-house rabbit immune serum (1:10,000). Primary antibodies, including rabbit anti-caspase-3 (ab32351) (1:5,000), rabbit anti-caspase-6 (ab185645) (1:5,000), rabbit anti-HA (ab9110) (1:5,000), mouse anti-Flag (ab49763) (1:5,000), mouse anti-His (ab18184) (1:3,000) and mouse anti-β-actin (ab8226) (1:10,000), were from Abcam. Secondary antibodies, including goat anti-mouse horseradish peroxidase (HRP) (62-6520) (1:5,000), goat anti-rabbit HRP (65-6120) (1:5,000) and goat anti-guinea pig HRP (A18769) (1:10,000), were from Thermo Fisher Scientific.

Antibodies for immunofluorescence staining

MERS-CoV N was detected by an in-house guinea pig immune serum (1:2,000). SARS-CoV-2 N was detected by an in-house rabbit immune serum (1:1,000). IRF3 was detected by rabbit anti-HA (ab9110) (1:500). Secondary antibodies, including Alexa Fluor 488 goat anti-guinea pig (A-11073) (1:500), Alexa Fluor 488 goat anti-rabbit (A-11034) (1:500) and Alexa Fluor 568 goat anti-rabbit (A-11036) (1:500), were from Thermo Fisher Scientific.

Ex vivo human lung tissues

Human lung tissues for ex vivo studies were retrieved from patients undergoing surgical operations at the Queen Mary Hospital, Hong Kong. All donors gave written consent as approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Normal nonmalignant lung tissue fragments in excess for clinical diagnosis were used. The freshly obtained lung tissues were processed into small rectangular pieces and were rinsed with the primary tissue culture medium, which contained the advanced DMEM/F12 medium supplemented with 2 mM HEPES (Gibco), 1× GlutaMAX (Gibco), 100 U ml⁻¹ penicillin, 100 μg ml⁻¹ streptomycin, 20 μg ml⁻¹ vancomysin, 20 μg ml⁻¹ ciprofloxacin, 50 μg ml⁻¹ amikacin, and 50 μg ml⁻¹ nystatin. The specimens were infected with MERS-CoV at a titre of 1 × 10⁸ PFU ml⁻¹. After 2 h, the inoculum was removed and the specimens were washed thoroughly with the primary tissue culture medium. The infected tissues were then incubated with primary tissue culture medium supplemented with 100 μM caspase-6 inhibitor z-VEID-fmk or DMSO. Tissues were collected at 24 hpi with either 10% neutral-buffered formalin for immunofluorescence staining or with RL buffer (TaKaRa) for RT–qPCR analysis.

Human intestinal organoids

Human intestinal organoids were established using biopsied human intestinal tissues from patients who underwent surgical operations at the Queen Mary Hospital, Hong Kong. All donors had written consent as approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster. Human intestinal organoids were maintained in expansion medium and induced differentiation by incubating with differentiation medium for 5 days. Differentiated intestinal organoids were sheared mechanically and inoculated with MERS-CoV at 1 MOI for 2 h. After the inoculum was removed, the intestinal organoids were rinsed with PBS and embedded in Matrigel and maintained in differentiation medium containing 100 μM z-VEID-fmk or DMSO. At the indicated time points after inoculation, intestinal organoids were collected for the quantification of intracellular viral load and immunofluorescence staining, whereas the cell-free Matrigel and culture medium were combined for viral titration of extracellular virions using standard plaque assays.

Human DPP4 mouse model

The hDPP4-K mice were provided by P. McCray¹⁷. The use of animals complied with all relevant ethical regulations and was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. On the day of infection, hDPP4-KI mice were inoculated intranasally with 2.5 × 10³ PFU mouse-adapted MERS-CoV (MERS-CoV_MA) pre-diluted in 20 μl DMEM, followed by intraperitoneal injection with 12.5 mg kg⁻¹ day⁻¹ z-VEID-fmk or DMSO diluted in 200 μl 0.3% methylcellulose/0.1% tween-80/PBS once per day for 6 days or until sample collection. The health status and body weight of the mice were monitored for 14 days on a daily basis or until the mouse was euthanized because it reached the humane endpoint of the experiment. Mice were euthanized at the designated time points and lung tissues from mice of both treatment and control groups were collected for immunofluorescence staining, RT–qPCR and plaque assay analysis.

Golden Syrian hamster model

Infection of golden Syrian hamsters was performed as described previously¹⁸. Golden Syrian hamsters aged 6–8 weeks old were obtained from the Chinese University of Hong Kong Laboratory Animal Service Centre through the HKU Centre for Comparative Medicine Research (CCMR). The use of animals has complied with all relevant ethical regulations and was approved by the Committee on the Use of Live Animals in Teaching and Research of The University of Hong Kong. On the day of infection, each hamster was inoculated intranasally with 3 × 10³ PFU SARS-CoV-2 pre-diluted in 50 μl DMEM under intraperitoneal ketamine (100 mg kg⁻¹) and xylazine (10mg kg⁻¹) anaesthesia. Infected hamsters were treated with 12.5 mg kg⁻¹ day⁻¹ z-VEID-fmk or DMSO diluted in 600 μl 0.3% methylcellulose/0.1% tween-80/PBS once per day for 4 days. The health status and body weight of the hamsters were monitored on a daily basis or until the animal was euthanized because it reached the humane endpoint of the experiment. Hamsters were euthanized at 4 dpi and lung tissues were collected for immunofluorescence staining, histopathology examination, RT–qPCR and TCID₅₀ assay analysis.

Generation and infection of caspase-6 knockout mice

Generation of caspase-6-KO mice on the hDPP4-KI mouse background performed with help from the Centre for Comparative Medicine Research at the University of Hong Kong. In brief, three sgRNA purchased from Synthego (5′-CCCTCATCTTCAATCACGAGAGG-3′, 5′-CTCCTGCTCAAAATTCACGAGGG-3′, and 5′-TGGCGTCGTATGCGTAAACGTGG-3′) were designed to target caspase-6 gene exon 3, exon 4, and exon 5, respectively (Extended Data Fig. 4a). The Cas9 protein was purchased from Invitrogen (TrueCut Cas9 Protein v2). First, the sgRNAs (0.5 μg μl⁻¹) and Cas9 protein (0.4 μg μl⁻¹) were diluted in Opti-MEM (Gibco). The ribonucleoprotein (RNP) was formed by vortexing the reagent mixture, followed by a 20 min incubation at room temperature. Second, in vitro fertilization was performed to generate homozygous hDPP4 embryos required for generating double mutant by electroporation. Five-week-old homozygous hDPP4 females were super-ovulated with intraperitoneal injection of 7.5 IU pregnant mare serum gonadotropin (PMSG), followed by intraperitoneal injection of 7.5 IU human chorionic gonadotropin (hCG) 48 h later. Twelve hours after hCG injection, three-month-old homozygous hDPP4 stud males were euthanized. Sperm was released from cauda epididymis into one well of a 4-well plate. The sperm was allowed to capacitate in modified human tubal fluid medium (mHTF) for 45 min in an incubator (5% CO₂, 37 °C). Oocytes cumulus were collected from the oviducts of the super-ovulated females and were added into the sperm well. Four hours after the in vitro fertilization, embryos were washed twice with mHTF medium and twice with potassium-supplemented simplex optimized medium (KSOM). Embryos were cultured in KSOM in an incubator until the electroporation. Third, the RNP was delivered into pronuclear stage embryos using a square wave electroporator (CUY21 EDIT II, BEX). Five microlitres of the RNP were pipetted into the chamber of the platinum electrode with a 1 mm gap (LF501PT1-5, BEX). The parameters were set as followed, five poring pulses: 200 V, 2 ms length, 50 ms interval, 4 times, 10% voltage decay, + polarity, followed by five transfer pulses: 20 V, 50 ms length, 50 ms interval, 40% voltage decay, alternating + and − polarity. The impedance was adjusted below 0.17 ohm by adding small volume of Opti-mem into the gap. The electroporated embryos were immediately transferred back to KSOM and were incubated for 30 min. The embryos were washed twice with fresh KSOM and were incubated overnight. 2-cell embryos were transferred into the oviduct of 0.5 days post-conception pseudo-pregnant ICR (CD-1) females in the next morning. Twenty days after the embryo transfer, pups carrying the knockout allele were born. The pups were weaned after 3 weeks of age. Ear punch samples were collected for caspase-6 genotyping. On the day of infection, hDPP4-KI caspase-6 KO mice and hDPP4-KI caspase-6 wild-type littermate controls were concurrently infected by intranasal inoculation with 2.5 × 10³ PFU MERS-CoV_MA pre-diluted in 20 μl DMEM. Mice were euthanized at 2 dpi and 4 dpi and lung tissues from mice of both groups were collected for RT–qPCR, plaque assay analysis, H&E staining and immunofluorescence staining.

Generation and infection of MERS-CoV recombinant virus

The cDNA from the MERS-CoV strain EMC/2012 (GenBank accession number JX869059), assembled into the pBAC (pBAC-SA-FL), was used as the background to generate the TKKA mutant (rMERS-CoV/TKKA) with a D to A change at the TKKD motif of the MERS-CoV N gene⁴⁸. The mutation was introduced into the pBAC by red recombineering⁴⁹. The mutation was confirmed by Sanger sequencing. The recombinant clones with mutant site were transformed into DH10B electrocompetent cells, followed by plasmids extraction to acquire ultrapure and high quality of full-length cDNA clone. Infectious virus is recovered by transfection of BHK21 cells with 5 μg of the full-length cDNA clone using Lipofectamine 3000 as transfection reagent. After 6 h post-transfection, the transfected BHK21 cells were re-seeded and co-cultured with Huh7 cells. After 72 h, the supernatant was used to inoculate Huh7 cells for viral passage. The recombinant virus was sent to next generation sequencing to confirm the desired mutation and the absence of undesired mutations in the viral genome. For in vitro infection, Huh7, A549, and Vero E6 cells were infected with rMERS-CoV/TKKA and rMERS-CoV/TKKD. Cell lysate and supernatant samples were collected from the infected cells at 2, 24 and 48 hpi for RT–qPCR and TCID₅₀ assays, respectively. Additionally, rMERS-CoV/TKKA- and rMERS-CoV/TKKD-infected Huh7 cells were collected at 1, 24, and 48 hpi for western blot analysis of N protein expression. For in vivo infection, hDPP4-KI mice were intranasally inoculated with 3.5 × 10⁴ PFU rMERS-CoV/TKKA or rMERS-CoV/TKKD pre-diluted in 20 μl DMEM. Mice were euthanized at 2 dpi and 4 dpi and lung tissues from mice of both groups were collected for RT–qPCR, plaque assay analysis, H&E staining and immunofluorescence staining.

Caspase-6 CRISPR–Cas9 knockout in vitro

Caspase-6 CRISPR–Cas9 KO plasmids were purchased from Santa Cruz Biotechnology. Knockout of caspase-6 in Huh7 cells was operated by co-transfection with HDR plasmid according to the manufacturer’s protocol (https://datasheets.scbt.com/protocols/CRISPR_protocol). In brief, Huh7 cells were seeded in a 6-well plate, followed by transfection with 3 μg caspase-6 CRISPR–Cas9 KO plasmids and 3 μg HDR plasmid, or non-targeting control plasmids using Lipofectamine 3000. At 72 h post-transfection, caspase-6 knockout targeting cells were selected by medium containing 1.2 μg ml⁻¹ puromycin. Caspase-6 knockout was verified with western blots. On the day of infection, Huh7 cells were infected by MERS-CoV at 1 MOI. At 2, 24 and 48 hpi, the cell lysates and supernatants were collected for RT–qPCR quantification and TCID₅₀ assays.

RNA extraction and RT–qPCR

Cells were lysed in RL buffer and extracted with the MiniBEST Universal RNA Extraction Kit (TaKaRa). Viral RNA in the supernatant was extracted with the MiniBEST Viral RNA/DNA Extraction Kit (TaKaRa). RT–qPCR was performed with Transcriptor First Strand cDNA Synthesis Kit and LightCycler 480 master mix from Roche. Data was collected through LightCycler96 (version SW1.1). All primer and probe sequences were provided in Supplementary Table 2.

Plaque assays and TCID₅₀ assays

Infectious titres of MERS-CoV and SARS-CoV-2 were determined with standard plaque assays⁴⁶. In brief, Vero E6 cells were seeded in 24-well plates 1 day before the experiment. The collected supernatant samples were serially diluted and inoculated to the cells for 2 h at 37 °C. After inoculation, the cells were washed with PBS 3 times, and covered with 2% agarose/PBS mixed with 2× DMEM/2%FBS at 1:1 ratio. The cells were fixed after incubation at 37 °C for 72 h. Fixed samples were stained with 0.5% crystal violet in 25% ethanol/distilled water for 10 min for plaque visualization. In some experiments, infectious titres of coronaviruses were determined with standard TCID₅₀ assays. In brief, Vero E6 cells were seeded in 96-well plates 1 day before the experiment. The collected supernatant samples were serially diluted and inoculated to the cells for 2 h at 37 °C. After inoculation, the cells were washed with PBS 3 times and incubated at 37 °C. After 72 hpi, virus titre was calculated using the Muench and Reed method.

siRNA and shRNA knockdown

On-Targetplus caspase-6 siRNA was obtained from Dharmacon (L-004406-00-0005). Transfection of siRNA on MDMs was performed using Lipofectamine RNAiMAX (Thermo Fisher Scientific). In brief, the cells were transfected with 50 nM caspase-6 siRNA for two consecutive days. At 24 h after the second siRNA transfection, the cells were collected in RIPA buffer for western blot analysis. In parallel, siRNA-transfected cells were challenged with MERS-CoV at 1 MOI for 1 h at 37 °C. Following the inoculation, the cells were washed with PBS and incubated for 24 h. The virus copy number at 24 hpi was determined with RT–qPCR. pLKO.1 lentiviral caspase-6 shRNA plasmid was obtained from Dharmacon. Transfection of caspase-6 shRNA plasmid, psPAX2 packaging plasmid and pMD2.G envelope plasmid on 293T cells was performed using Lipofectamine 3000 (Thermo Fisher Scientific) following manufacturer’s manual. In brief, 293T cells in 10 cm dishes were transfected with 6 μg caspase-6 shRNA plasmid, 4.5 μg packaging plasmid, and 1.5 μg envelope plasmid in FBS-supplemented DMEM medium, followed by aspirating supernatant at 6 h post-transfection and replacing with FBS-free medium. On the next day, the supernatant containing caspase-6 shRNA lentivirus particles was collected and was used to transduce A549 and BEAS2B cells. A549 and BEAS2B caspase-6 stable knockdowns cells were selected by 0.5 μg ml⁻¹ and 0.7 μg ml⁻¹ puromycin, respectively. The selected cells were challenged with MERS-CoV at 0.1 MOI for 1h at 37 °C. The virus copy number at 1 and 24 hpi was determined with RT–qPCR.

Caspase-6 activity assay

Huh7 cells were infected with MERS-CoV at 1 MOI for 12 h. In parallel, Huh7 cells were stimulated with STS at 1 μM for 6 h. Caspase-6 activity in the cell lysate was determined with the caspase-Glo-6 assay kit (Promega). The luminescence signal of caspase-6 activity was measured following manufacturer’s manual with a multilabel plate reader Victor X3 (Perkin-Elmer). Data was analysed with Kaleido (version 1.2) software.

Immunofluorescence and histology

Infected human and animal lung tissues were fixed overnight in 10% formalin⁵⁰. The fixed samples were then embedded in paraffin with a TP1020 Leica semi-enclosed benchtop tissue processor and sectioned at 5 μm. Tissue sections were fished and dried to fix on Thermo Fisher Scientific Superfrost Plus slides at 37 °C overnight. Antigen retrieval was performed by heating the slides in antigen unmasking solution (Vector Laboratories) for 90 s. MERS-CoV and SARS-CoV-2 were detected with an in-house guinea pig anti-MERS-CoV N immune serum and an in-house rabbit anti-SARS-CoV-2-N immune serum, respectively. Cell nuclei were labelled with DAPI nucleic acid stain (Thermo Fisher Scientific). Alexa Fluor secondary antibodies were obtained from Thermo Fisher Scientific. Mounting was performed with the Diamond Prolong Antifade Mountant from Thermo Fisher Scientific. Images were captured with an Olympus BX53 fluorescence microscope (Olympus Life Science) or a Carl Zeiss LSM 780 confocal microscope in the faculty core facility of HKU using Olympus cellSens Dimension (version 1.17) and ZEISS (ZEN 2) microscope software, respectively. For H&E staining, mice and hamster lung tissue sections were stained with Gill’s H&E Y (Thermo Fisher Scientific). Images were captured with an Olympus BX53 fluorescence microscope. H&E-stained lung tissue sections were blinded for the identities of experimental settings and examined by a trained histopathologist. Lung pathology was graded on a scale of 0 (normal) to 4 (most severe) according to a grading system as previously described¹⁹.

Western blot

Cells were lysed with RIPA buffer (Thermo Fisher Scientific) with protease inhibitor (Roche, Basel, Switzerland). Proteins were separated by SDS–PAGE and transferred to PVDF membranes (Thermo Fisher Scientific). Specific primary antibodies were incubated with the blocked membranes at 4 °C overnight, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Thermo Fisher Scientific) for 1 h at room temperature. The signal was developed with Immobilon Crescendo Western HRP Substrate (Merck Millipore) and detected using an automatic X-ray film processor (Advansta) or an Alliance Q9 Advanced imager (Uvitec). Raw western blot images were collected using Alliance Q9 software (v17-02) and provided as Supplementary Figs. 1–4.

Flow cytometry

BEAS2B cells were infected with MERS-CoV at 1 MOI. At 24 hpi, the cells were detached with 10 mM EDTA in PBS, fixed in 4% paraformaldehyde, followed by immunolabelling with an in-house guinea pig anti-MERS-CoV N immune serum and a rabbit anti-active caspase-3 antibody (560626, BD Biosciences) (1:500). Flow cytometry was performed using a BD FACSCanto II flow cytometer (BD) and data were analysed using FlowJo (version vX 0.7) (BD). The gating strategy was demonstrated in Supplementary Fig. 5.

IFNβ luciferase reporter assays

IFNβ luciferase reporter assays were performed as previously described^26,27. In brief, 500 ng IFNβ luciferase reporter plasmid, 10 ng transfection efficiency control plasmid (pNL1.1.TK, Promega), 1 μg coronavirus N plasmids, 3 μg caspase-6 expression plasmid, together with or without 5 μg Poly(I:C) were co-transfected into 293T cells for 24 h. On the next day, the cells were collected for luciferase measurement with the dual-luciferase reporter assay system kit (Promega) according to the manufacturer’s protocol using a multilabel plate reader Victor X3 (Perkin-Elmer). Data were analysed with Kaleido (version 1.2) software. To investigate the point of action of the N fragments, IFNβ luciferase reporter plasmid pNL1.1.TK and MERS-CoV N(1–241) or MERS-CoV N(242–413) were co-transfected into 293T cells together with expression plasmids for RIG-IN, MAVS or TBK1. At 24 h post-transfection, the cells were collected for luciferase measurement.

Alignment of human, mouse and hamster caspase-6

Caspase-6 protein sequences of Homo sapiens (Uniprot ID: P55212), Mus musculus (Uniprot ID: O08738) and Mesocricetus auratus (Uniprot ID: A0A1U7QNN7) were downloaded from UniProt. Multiple sequence alignment was performed with MUSCLE (v3.8.31). The crystal structure of caspase-6 and VEID complex was retrieved from the Protein Data Bank (PDB code: 3OD5). Caspase-6 residues within 4 Å of VEID were defined as the binding sites and visualized with PyMOL (version 2.5).

Prediction of potential caspase-6 cleavage sites

The protein sequence of MERS-CoV N (NC_019843) was used for caspase-6 cleavage site analysis. Potential caspase-6 cleavage motifs on MERS-CoV N was determined based on published substrate specificity of caspase-6²⁹. The amino acid pattern [TVILENYF]..D was searched against the N sequence, where ‘.’ represented any amino acid.

Statistical analysis

Data in figures are mean ± s.d. Statistical comparison between different groups was performed by one-way ANOVA, two-way ANOVA, Student’s t-test or log-rank (Mantel–Cox) test using GraphPad Prism 9. Differences were considered statistically significant at P < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Source data are provided with this paper.

References

Chan, J. F. et al. A familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster. Lancet 395, 514–523 (2020).
Article CAS PubMed PubMed Central Google Scholar
Zhou, P. et al. A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270–273 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Zaki, A. M., van Boheemen, S., Bestebroer, T. M., Osterhaus, A. D. & Fouchier, R. A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia. N. Engl. J. Med. 367, 1814–1820 (2012).
Article CAS PubMed Google Scholar
Peiris, J. S. et al. Coronavirus as a possible cause of severe acute respiratory syndrome. Lancet 361, 1319–1325 (2003).
Article CAS PubMed PubMed Central Google Scholar
Tao, X. et al. Bilateral entry and release of Middle East respiratory syndrome coronavirus induces profound apoptosis of human bronchial epithelial cells. J. Virol. 87, 9953–9958 (2013).
Article CAS PubMed PubMed Central Google Scholar
Chu, H. et al. Middle East respiratory syndrome coronavirus efficiently infects human primary T lymphocytes and activates the extrinsic and intrinsic apoptosis pathways. J. Infect. Dis. 213, 904–914 (2016).
Article CAS PubMed Google Scholar
Zhu, N. et al. Morphogenesis and cytopathic effect of SARS-CoV-2 infection in human airway epithelial cells. Nat. Commun. 11, 3910 (2020).
Article ADS CAS PubMed PubMed Central Google Scholar
Chau, T. N. et al. SARS-associated viral hepatitis caused by a novel coronavirus: report of three cases. Hepatology 39, 302–310 (2004).
Article PubMed Google Scholar
Ding, Y. et al. The clinical pathology of severe acute respiratory syndrome (SARS): a report from China. J. Pathol. 200, 282–289 (2003).
Article PubMed PubMed Central Google Scholar
Li, S. et al. Clinical and pathological investigation of patients with severe COVID-19. JCI Insight https://doi.org/10.1172/jci.insight.138070 (2020).
Yeung, M. L. et al. MERS coronavirus induces apoptosis in kidney and lung by upregulating Smad7 and FGF2. Nat. Microbiol. 1, 16004 (2016).
Article CAS PubMed PubMed Central Google Scholar
Chu, H. et al. Targeting highly pathogenic coronavirus-induced apoptosis reduces viral pathogenesis and disease severity. Sci. Adv. 7, eabf8577 (2021).
Article ADS CAS PubMed PubMed Central Google Scholar
Diemer, C. et al. Cell type-specific cleavage of nucleocapsid protein by effector caspases during SARS coronavirus infection. J. Mol. Biol. 376, 23–34 (2008).
Article CAS PubMed Google Scholar
Eleouet, J. F. et al. The viral nucleocapsid protein of transmissible gastroenteritis coronavirus (TGEV) is cleaved by caspase-6 and -7 during TGEV-induced apoptosis. J. Virol. 74, 3975–3983 (2000).
Article CAS PubMed PubMed Central Google Scholar
Oh, C., Kim, Y. & Chang, K. O. Caspase-mediated cleavage of nucleocapsid protein of a protease-independent porcine epidemic diarrhea virus strain. Virus Res. 285, 198026 (2020).
Article CAS PubMed PubMed Central Google Scholar
McIlwain, D. R., Berger, T. & Mak, T. W. Caspase functions in cell death and disease. Cold Spring Harb. Perspect. Biol. 5, a008656 (2013).
Article PubMed PubMed Central CAS Google Scholar
Li, K. et al. Mouse-adapted MERS coronavirus causes lethal lung disease in human DPP4 knockin mice. Proc. Natl Acad. Sci. USA 114, E3119–E3128 (2017).
CAS PubMed PubMed Central Google Scholar
Chan, J. F. et al. Simulation of the clinical and pathological manifestations of coronavirus disease 2019 (COVID-19) in a golden Syrian hamster model: implications for disease pathogenesis and transmissibility. Clin. Infect. Dis. 71, 2428–2446 (2020).
CAS PubMed Google Scholar
Lee, A. C. et al. Oral SARS-CoV-2 inoculation establishes subclinical respiratory infection with virus shedding in golden Syrian hamsters. Cell Rep. Med. 1, 100121 (2020).
Article CAS PubMed PubMed Central Google Scholar
Groborz, K. et al. Exploring the prime site in caspases as a novel chemical strategy for understanding the mechanisms of cell death: a proof of concept study on necroptosis in cancer cells. Cell Death Differ. 27, 451–465 (2020).
Article PubMed Google Scholar
McStay, G. P., Salvesen, G. S. & Green, D. R. Overlapping cleavage motif selectivity of caspases: implications for analysis of apoptotic pathways. Cell Death Differ. 15, 322–331 (2008).
Article CAS PubMed Google Scholar
Parrish, A. B., Freel, C. D. & Kornbluth, S. Cellular mechanisms controlling caspase activation and function. Cold Spring Harb. Perspect. Biol. 5, a008672 (2013).
Article PubMed PubMed Central CAS Google Scholar
Wang, X. J., Cao, Q., Zhang, Y. & Su, X. D. Activation and regulation of caspase-6 and its role in neurodegenerative diseases. Annu. Rev. Pharmacol. Toxicol. 55, 553–572 (2015).
Article CAS PubMed Google Scholar
Desmyter, J., Melnick, J. L. & Rawls, W. E. Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero). J. Virol. 2, 955–961 (1968).
Article CAS PubMed PubMed Central Google Scholar
Osada, N. et al. The genome landscape of the African green monkey kidney-derived vero cell line. DNA Res. 21, 673–683 (2014).
Article CAS PubMed PubMed Central Google Scholar
Lui, P. Y. et al. Middle East respiratory syndrome coronavirus M protein suppresses type I interferon expression through the inhibition of TBK1-dependent phosphorylation of IRF3. Emerg. Microbes Infect. 5, e39 (2016).
Article CAS PubMed PubMed Central Google Scholar
Siu, K. L. et al. Middle East respiratory syndrome coronavirus 4a protein is a double-stranded RNA-binding protein that suppresses PACT-induced activation of RIG-I and MDA5 in the innate antiviral response. J. Virol. 88, 4866–4876 (2014).
Article PubMed PubMed Central CAS Google Scholar
Yang, Y. et al. The structural and accessory proteins M, ORF 4a, ORF 4b, and ORF 5 of Middle East respiratory syndrome coronavirus (MERS-CoV) are potent interferon antagonists. Protein Cell 4, 951–961 (2013).
Article CAS PubMed PubMed Central Google Scholar
Talanian, R. V. et al. Substrate specificities of caspase family proteases. J. Biol. Chem. 272, 9677–9682 (1997).
Article CAS PubMed Google Scholar
McBride, R., van Zyl, M. & Fielding, B. C. The coronavirus nucleocapsid is a multifunctional protein. Viruses 6, 2991–3018 (2014).
Article PubMed PubMed Central CAS Google Scholar
Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e278 (2020).
Article CAS PubMed PubMed Central Google Scholar
Chu, H. et al. Host and viral determinants for efficient SARS-CoV-2 infection of the human lung. Nat. Commun. 12, 134 (2021).
Article CAS PubMed PubMed Central Google Scholar
Wurzer, W. J. et al. Caspase 3 activation is essential for efficient influenza virus propagation. EMBO J. 22, 2717–2728 (2003).
Article CAS PubMed PubMed Central Google Scholar
Graham, R. K., Ehrnhoefer, D. E. & Hayden, M. R. Caspase-6 and neurodegeneration. Trends Neurosci. 34, 646–656 (2011).
Article CAS PubMed Google Scholar
Zheng, M., Karki, R., Vogel, P. & Kanneganti, T. D. Caspase-6 is a key regulator of innate immunity, inflammasome activation, and host defense. Cell 181, 674–687.e613 (2020).
Article CAS PubMed PubMed Central Google Scholar
Konno, Y. et al. SARS-CoV-2 ORF3b is a potent interferon antagonist whose activity is increased by a naturally occurring elongation variant. Cell Rep. 32, 108185 (2020).
Article CAS PubMed PubMed Central Google Scholar
Yuen, C. K. et al. SARS-CoV-2 nsp13, nsp14, nsp15 and orf6 function as potent interferon antagonists. Emerg. Microbes Infect. 9, 1418–1428 (2020).
Article CAS PubMed PubMed Central Google Scholar
Comar, C. E. et al. Antagonism of dsRNA-induced innate immune pathways by NS4a and NS4b accessory proteins during MERS coronavirus infection. mBio 10, e00319–e00319 (2019).
Article CAS PubMed PubMed Central Google Scholar
Wong, L. R. et al. Middle East respiratory syndrome coronavirus ORF8b accessory protein suppresses type I IFN expression by impeding HSP70-dependent activation of IRF3 kinase IKKε. J. Immunol. 205, 1564–1579 (2020).
Article CAS PubMed Google Scholar
Xia, H. et al. Evasion of type I interferon by SARS-CoV-2. Cell Rep. 33, 108234 (2020).
Article CAS PubMed PubMed Central Google Scholar
Chang, C. Y., Liu, H. M., Chang, M. F. & Chang, S. C. Middle East respiratory syndrome coronavirus nucleocapsid protein suppresses type I and type III interferon induction by targeting RIG-I signaling. J. Virol. 94, e00099–20 (2020).
CAS PubMed PubMed Central Google Scholar
Chen, K. et al. SARS-CoV-2 nucleocapsid protein interacts with RIG-I and represses RIG-mediated IFN-β production. Viruses 13, 47 (2020).
Article PubMed Central CAS Google Scholar
Kopecky-Bromberg, S. A., Martinez-Sobrido, L., Frieman, M., Baric, R. A. & Palese, P. Severe acute respiratory syndrome coronavirus open reading frame (ORF) 3b, ORF 6, and nucleocapsid proteins function as interferon antagonists. J. Virol. 81, 548–557 (2007).
Article CAS PubMed Google Scholar
Oh, S. J. & Shin, O. S. SARS-CoV-2 nucleocapsid protein targets RIG-I-like receptor pathways to inhibit the induction of interferon response. Cells 10, 530 (2021).
Article CAS PubMed PubMed Central Google Scholar
Chu, H. et al. Tetherin/BST-2 is essential for the formation of the intracellular virus-containing compartment in HIV-infected macrophages. Cell Host Microbe 12, 360–372 (2012).
Article CAS PubMed PubMed Central Google Scholar
Chu, H. et al. Comparative tropism, replication kinetics, and cell damage profiling of SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory studies of COVID-19: an observational study. Lancet Microbe 1, e14–e23 (2020).
Article CAS PubMed PubMed Central Google Scholar
Shuai, H. et al. Attenuated replication and pathogenicity of SARS-CoV-2 B.1.1.529 Omicron. Nature 603, 693–699 (2022).
Article ADS CAS PubMed Google Scholar
Almazan, F. et al. Engineering a replication-competent, propagation-defective Middle East respiratory syndrome coronavirus as a vaccine candidate. mBio 4, e00650–00613 (2013).
Article PubMed PubMed Central CAS Google Scholar
Almazan, F., Marquez-Jurado, S., Nogales, A. & Enjuanes, L. Engineering infectious cDNAs of coronavirus as bacterial artificial chromosomes. Methods Mol. Biol. 1282, 135–152 (2015).
Article CAS PubMed Google Scholar
Shuai, H. et al. Emerging SARS-CoV-2 variants expand species tropism to murines. eBioMedicine 73, 103643 (2021).
Article CAS PubMed PubMed Central Google Scholar

Download references

Acknowledgements

We thank staff members, including P. S. Li and C. K. Chow at the Transgenic Core Facility of the Centre for Comparative Medicine Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, for their help with generating the caspase-6 KO mice. This work was partly supported by funding from the Health and Medical Research Fund (CID-HKU1-5, COVID1903010-Projects 7 and 14, 16150572, and 20190652), the Food and Health Bureau, The Government of the Hong Kong Special Administrative Region; the General Research Fund (17118621, 17123920, and 17124220), the Collaborative Research Fund (C7060-21G), and the Theme-Based Research Scheme (T11-709/21-N) of Research Grants Council, The Government of the Hong Kong Special Administrative Region; Health@InnoHK, Innovation and Technology Commission, the Government of the Hong Kong Special Administrative Region; National Natural Science Foundation of China Excellent Young Scientists Fund (Hong Kong and Macau) (32122001); National Program on Key Research Project of China (grant no. 2020YFA0707500 and 2020YFA0707504); the Consultancy Service for Enhancing Laboratory Surveillance of Emerging Infectious Diseases and Research Capability on Antimicrobial Resistance for Department of Health of the Hong Kong Special Administrative Region Government, Sanming Project of Medicine in Shenzhen, China (no. SZSM201911014); the High Level-Hospital Program, Health Commission of Guangdong Province, China; the University of Hong Kong Li Ka Shing Faculty of Medicine Enhanced New Staff Start-up Fund; the University of Hong Kong Outstanding Young Researcher Award; the University of Hong Kong Research Output Prize (Li Ka Shing Faculty of Medicine); the Major Science and Technology Program of Hainan Province (ZDKJ202003); the research project of Hainan Academician Innovation Platform (YSPTZX202004); the Hainan Talent Development Project (SRC200003); the Emergency COVID-19 Project (2021YFC0866100), Major Projects on Public Security, National Key Research and Development Program; and the Emergency Collaborative Project of Guangzhou Laboratory (EKPG22-01); and the donations of the Shaw Foundation Hong Kong, R. Yu and C. Yu, M. M. Y. Tam-Mak, M. S.-K. Tong, the Providence Foundation Limited (in memory of the late H. M. Lui), Lee Wan Keung Charity Foundation Limited, M. Hui, H. Hui and Chow Sin Lan Charity Fund Limited, Hong Kong Sanatorium and Hospital, The Chen Wai Wai Vivien Foundation Limited, Chan Yin Chuen Memorial Charitable Foundation, M. M.-W. Lee, the Hong Kong Hainan Commercial Association South China Microbiology Research Fund, the Jessie and George Ho Charitable Foundation, Perfect Shape Medical Limited, K.C. Tong, Foo Oi Foundation Limited, L. K.-M. Tse, B. H.-C. Lee, P. C. So and Lo Ying Shek Chi Wai Foundation. The funding sources had no role in the study design, data collection, analysis, interpretation or writing of the report.

Author information

These authors contributed equally: Hin Chu, Yuxin Hou

Authors and Affiliations

State Key Laboratory of Emerging Infectious Diseases, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, China
Hin Chu, Anna Jinxia Zhang, Shuofeng Yuan, Jie Zhou, Kin-Hang Kok, Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Department of Microbiology, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, China
Hin Chu, Yuxin Hou, Dong Yang, Lei Wen, Huiping Shuai, Chaemin Yoon, Jialu Shi, Yue Chai, Terrence Tsz-Tai Yuen, Bingjie Hu, Cun Li, Xiaoyu Zhao, Yixin Wang, Xiner Huang, Cuiting Luo, Jian-Piao Cai, Vincent Kwok-Man Poon, Chris Chung-Sing Chan, Anna Jinxia Zhang, Shuofeng Yuan, Jie Zhou, Kin-Hang Kok, Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Department of Infectious Disease and Microbiology, The University of Hong Kong-Shenzhen Hospital, Shenzhen, China
Hin Chu, Anna Jinxia Zhang, Shuofeng Yuan, Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Centre for Virology, Vaccinology and Therapeutics, Hong Kong Science and Technology Park, Sha Tin, China
Hin Chu, Vincent Kwok-Man Poon, Chris Chung-Sing Chan, Anna Jinxia Zhang, Shuofeng Yuan, Jie Zhou, Kin-Hang Kok, Dong-Yan Jin, Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Transgenic Core Facility, Centre for Comparative Medicine Research, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, China
Kin Shing Lee
Department of Surgery, School of Clinical Medicine, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, China
Ko-Yung Sit, Dominic Chi-Chung Foo, Wing-Kuk Au & Kenneth Kak-Yuen Wong
School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, China
Dong-Yan Jin
Guangzhou Laboratory, Guangzhou, China
Dong-Yan Jin, Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Carol Yu Centre for Infection, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, China
Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Department of Microbiology, Queen Mary Hospital, Pokfulam, China
Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Academician Workstation of Hainan Province, Hainan Medical University, Haikou, China
Jasper Fuk-Woo Chan & Kwok-Yung Yuen
Hainan Medical University-The University of Hong Kong Joint Laboratory of Tropical Infectious Diseases, The University of Hong Kong, Pokfulam, China
Jasper Fuk-Woo Chan & Kwok-Yung Yuen

Authors

Hin Chu
View author publications
You can also search for this author in PubMed Google Scholar
Yuxin Hou
View author publications
You can also search for this author in PubMed Google Scholar
Dong Yang
View author publications
You can also search for this author in PubMed Google Scholar
Lei Wen
View author publications
You can also search for this author in PubMed Google Scholar
Huiping Shuai
View author publications
You can also search for this author in PubMed Google Scholar
Chaemin Yoon
View author publications
You can also search for this author in PubMed Google Scholar
Jialu Shi
View author publications
You can also search for this author in PubMed Google Scholar
Yue Chai
View author publications
You can also search for this author in PubMed Google Scholar
Terrence Tsz-Tai Yuen
View author publications
You can also search for this author in PubMed Google Scholar
Bingjie Hu
View author publications
You can also search for this author in PubMed Google Scholar
Cun Li
View author publications
You can also search for this author in PubMed Google Scholar
Xiaoyu Zhao
View author publications
You can also search for this author in PubMed Google Scholar
Yixin Wang
View author publications
You can also search for this author in PubMed Google Scholar
Xiner Huang
View author publications
You can also search for this author in PubMed Google Scholar
Kin Shing Lee
View author publications
You can also search for this author in PubMed Google Scholar
Cuiting Luo
View author publications
You can also search for this author in PubMed Google Scholar
Jian-Piao Cai
View author publications
You can also search for this author in PubMed Google Scholar
Vincent Kwok-Man Poon
View author publications
You can also search for this author in PubMed Google Scholar
Chris Chung-Sing Chan
View author publications
You can also search for this author in PubMed Google Scholar
Anna Jinxia Zhang
View author publications
You can also search for this author in PubMed Google Scholar
Shuofeng Yuan
View author publications
You can also search for this author in PubMed Google Scholar
Ko-Yung Sit
View author publications
You can also search for this author in PubMed Google Scholar
Dominic Chi-Chung Foo
View author publications
You can also search for this author in PubMed Google Scholar
Wing-Kuk Au
View author publications
You can also search for this author in PubMed Google Scholar
Kenneth Kak-Yuen Wong
View author publications
You can also search for this author in PubMed Google Scholar
Jie Zhou
View author publications
You can also search for this author in PubMed Google Scholar
Kin-Hang Kok
View author publications
You can also search for this author in PubMed Google Scholar
Dong-Yan Jin
View author publications
You can also search for this author in PubMed Google Scholar
Jasper Fuk-Woo Chan
View author publications
You can also search for this author in PubMed Google Scholar
Kwok-Yung Yuen
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

H.C., J.F.-W.C. and K.-Y.Y. designed the study. H.C., Y.H., D.Y., L.W., H.S., C.Y., J.S., Y.C., T.T.-T.Y., B.H., C. Li, X.Z., Y.W., X.H., K.S.L., C. Luo, J.-P.C., V.K.-M.P., C.C.-S.C., A.J.Z., S.Y., K.-Y.S., D.C.-C.F., W.-K.A., K.K.-Y.W., J.Z., K.-H.K. and D.-Y.J. performed experiments, analysed data, or provided key resources. H.C., D.-Y.J., J.F.-W.C. and K.-Y.Y. provided funding support. H.C., Y.H., J.F.-W.C. and K.-Y.Y. wrote the manuscript.

Corresponding authors

Correspondence to Hin Chu, Jasper Fuk-Woo Chan or Kwok-Yung Yuen.

Ethics declarations

Competing interests

J.F.-W.C. has received travel grants from Pfizer Corporation Hong Kong and Astellas Pharma Hong Kong Corporation Limited, and was an invited speaker for Gilead Sciences Hong Kong Limited and Luminex Corporation. The other authors declare no competing interests.

Peer review

Peer review information

Nature thanks Christian Holm and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data figures and tables

Extended Data Fig. 1 Caspase-6 inhibition reduces coronavirus replication in different cell types.

a MERS-CoV-infected Huh7 cells were treated with 100 μM z-VAD-fmk or DMSO. Cells were fixed at 24 hpi and immunolabeled with an in-house guinea pig immune serum against MERS-CoV N. The experiment was repeated two times independently with similar results. b Replication of human-pathogenic coronaviruses treated with 100 μM z-VAD-fmk or DMSO. Samples were harvested at 24 hpi and viral gene expression was quantified with RT-qPCR (n = 3). c BEAS2B, Calu3, Huh7, and BSC1 cells were treated with z-VAD-fmk at the indicated concentrations. Cell viability was quantified with CellTiter-Glo assays at 24 h post treatment (n = 6). d MDM, Caco2, Calu3, A549, and VeroE6 cells were infected with MERS-CoV at 1MOI and were treated with z-VEID-fmk, z-VAD-fmk, or DMSO. Cell lysate and supernatant samples were harvested at 24 hpi. MERS-CoV N gene copy was quantified with RT-qPCR (n = 3). e Huh7 cells were infected with SARS-CoV-2 Omicron BA.1 or BA.2 at 1MOI and were treated with z-VEID-fmk at the indicated concentrations. Cell lysate and supernatant samples were harvested at 24 hpi. SARS-CoV-2 RdRp gene copy was quantified with RT-qPCR (n = 3) and infectious titer was quantified with TCID₅₀ assays (n = 3). f HFL, Calu3, Huh7, and BSC1 cells with or without infection with the indicated coronaviruses were treated with z-VEID-fmk. Cell viability was quantified with CellTiter-Glo assays at 24 h post treatment (n = 3). Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA (c–e) or two-way ANOVA (b, f). Bars in (a) represented 20 μm.

Source data

Extended Data Fig. 2 Caspase-6 inhibition reduces the expression of pro-inflammatory cytokines/chemokine and MERS-CoV N in vivo.

a Multiple sequence alignment of human, mouse and golden Syrian hamster caspase-6 full-length sequences. The indices were labelled according to human caspase-6. Z-VEID-fmk binding sites on caspase-6 were indicated with orange triangles. b z-VEID-fmk binding mode represented in 3D structure. Binding sites on caspase-6 and z-VEID-fmk were shown in green and magenta sticks, respectively. The binding site residues were labelled in red. c hDPP4 KI mice were intranasally inoculated with 2.5x10³PFU MERS-CoV_MA followed by intraperitoneal administration of 12.5 mg/kg/day z-VEID-fmk or DMSO for 6 days or until sample harvest. Mouse lungs were harvested at day 2 and day 4 post infection. Expression of pro-inflammatory cytokines and chemokines were quantified with RT-qPCR (n = 6 for the two infected groups, n = 3 for the mock-infected group). Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA. d Golden Syrian hamsters were intranasally inoculated with 3x10³PFU SARS-CoV-2 followed by intraperitoneal administration of 12.5 mg/kg/day z-VEID-fmk or DMSO for 4 days. Hamsters were sacrificed at day 4 post infection. Viral N protein expression in the small airways of infected hamster lungs with or without z-VEID-fmk treatment was revealed with immunofluorescence staining with the in-house rabbit immune serum against SARS-CoV-2 N. The experiment was repeated two times independently with similar results. Bars in (d) represented 100 μm.

Source data

Extended Data Fig. 3 Caspase-6 facilitates MERS-CoV replication.

a MERS-CoV-infected BEAS2B cells were incubated with z-VEID-fmk in a time of addition assay. MERS-CoV N gene copy in the supernatant was determined with RT-qPCR at 24hpi (n = 4). b, c Caspase-6 gene (n = 3) (b) and protein expression (c) from caspase-6 stable knockdown A549 and BEAS2B cells. d-e Caspase-6 stable knockdown A549 and BEAS2B cells were infected with MERS-CoV at 0.1MOI. Cell lysates were harvested at 1 hpi to quantify virus entry with RT-qPCR against the MERS-CoV N gene (n = 4) (d). Virus replication at 24 hpi were quantified with RT-qPCR against the MERS-CoV N gene (n = 4) (e). f Caspase-6 stable knockdown or scrambled knockdown A549 and BEAS2B cells were infected with MERS-CoV at 0.1MOI followed by treating the cells with z-VEID-fmk at the indicated concentrations. Cell lysate and supernatant samples were harvested at 24 hpi to quantify virus replication with RT-qPCR against the MERS-CoV N gene (n = 3). g Caspase-6 protein expression from caspase-6 siRNA transfected MDMs. h MDMs were treated with caspase-6 or nontargeting siRNA and infected with MERS-CoV. MERS-CoV N gene copy was quantified at 24 hpi (n = 4). i Caspase-6 protein detection from caspase-6 KO or scrambled KO Huh7 cells. j, k Caspase-6- or caspase-3-overexpressed 293T cells were infected with MERS-CoV at 1MOI. MERS-CoV replication was quantified at 1 and 24 hpi with RT-qPCR against the MERS-CoV N gene (n = 4). The cleaved and uncleaved bands in (j) were obtained from the same experiment using different exposure times. The experiments in (c, g, and i) were repeated three times independently with similar results. Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA (a), two-way ANOVA (b, d, f, and k), or two-sided Student’s t-test (e and h).

Source data

Extended Data Fig. 4 Caspase-6 KO in hDPP4 KI mice reduces virus replication and virus-induced lung damage.

a Schematic of caspase-6 KO in hDPP4 KI mice. b Western blot analysis of caspase-6 expression from caspase-6 WT and caspase-6 KO hDPP4 KI mice. c-e hDPP4 KI/caspase-6 KO and hDPP4 KI/caspase-6 WT mice were intranasally inoculated with 2.5x10³PFU MERS-CoV_MA. Mouse lung samples were harvested at day 2 and day 4 post infection. MERS-CoV N antigen expression at 2 dpi was determined with immunofluorescence staining (c). Scale bar in (c) represented 100 μm. Representative images of haematoxylin and eosin (H&E) stained lungs from MERS-CoV_MA-infected hDPP4 KI/caspase-6 KO and hDPP4 KI/caspase-6 WT mice were shown (d). Scale bar in (d) represented 500 μm. Representative images of immunofluorescence staining of lungs from MERS-CoV_MA-infected hDPP4 KI/caspase-6 KO and hDPP4 KI/caspase-6 WT mice at low magnification (e). Scale bar in (e) represented 500 μm. The experiments in (b–e) was repeated three times independently with similar results.

Extended Data Fig. 5 Caspase-6 cleaves MERS-CoV N but not other MERS-CoV proteins.

a Caspase-6 is activated by apoptosis triggered by MERS-CoV infection or staurosporine (STS) stimulation. Huh7 cells were infected with MERS-CoV at 1MOI for 12 h. In parallel, Huh7 cells were stimulated with STS at 1 μM for 6 h. Caspase-6 activity in the cell lysate was determined with the caspase-Glo-6 assay kit (n = 4). b Autoactivation and STS-mediated activation of caspase-6 were demonstrated with Western blots. c-d Caspase-6 and expression constructs for MERS-CoV proteins were transfected in 293T cells. Caspase-6-mediated cleavage was detected with Western blots. Asterisks at Nsp2 and Nsp14 indicated the predicted protein sizes according to the number of amino acid residues. Caspase-6-specific cleavage product was only observed when N was co-expressed with caspase-6 (arrow). e MERS-CoV N and caspase-6 were co-expressed in 293T cells with or without 100 μM z-VEID-fmk. Cells were harvested for Western blot at 24 h post transfection. 1 μM STS was added as an apoptosis trigger at 6 h before sample harvest. f N cleavage in cell lysates from MERS-CoV-infected Huh7 and BEAS2B cells was compared to the N cleavage in cell lysates from MERS-CoV N and caspase-6 co-transfected 293T cells. g Comparison of MERS-CoV N cleavage in caspase-6-overexpressed and caspase-3-overexpressed cells. Different exposure times were used for cleaved and uncleaved bands. Experiments in (b–d) were repeated two times independently with similar results. Experiments in (e–g) were repeated three times independently with similar results. Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA.

Source data

Extended Data Fig. 6 Caspase-6-mediated N cleavage modulates IFN signaling.

a 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression construct of MERS-CoV N, with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 3). b 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of MERS-CoV N or E and caspase-6 or caspase-3, with or without poly(I:C). Gene expression of IFN-β (n = 6), IFIT1 (n = 6), IFIT2 (n = 6), IFIT3 (n = 6), IFITM3 (n = 4), OAS1 (n = 4), and TRIM22 (n = 4) was quantified with RT-qPCR. c 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6 and MERS-CoV N, ORF4a, ORF4b, or M, with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 3). d 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6 and two different amounts of MERS-CoV N, ORF4a, ORF4b, or M, with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 3 for empty vector control and poly(I:C) only, n = 7 for all other groups). Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA with Tukey’s post hoc tests.

Source data

Extended Data Fig. 7 Caspase-6-mediated N cleavage of human-pathogenic coronaviruses.

a N protein of the indicated human-pathogenic coronaviruses were co-expressed with caspase-6. N cleavage was detected with Western blots. b N protein of the indicated coronaviruses were expressed in 293T cells without caspase-6 transfection. N cleavage was detected with Western blots. c 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6 and coronavirus N, with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 3). d 293T cells were transfected with the same set of plasmids. Gene expression of IFIT3 and OAS1 was quantified with RT-qPCR (n = 4). Experiments in (a, b) was repeated three times independently with similar results. Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA (c, d).

Source data

Extended Data Fig. 8 Caspase-6-mediated MERS-CoV N cleavage generates N fragments that interfere with IFN signalling.

a Caspase-6-mediated cleavage of N, N(1-241), and N(242-413) was evaluated with Western blots. The experiment in was repeated three times independently with similar results. b, c 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6 and MERS-CoV N, N(1-241), or N(242-413), with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 6) (b) or IFN-β ELISA (n = 4) (c). d Huh7 cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6, MERS-CoV N, N(1-241), or N(242-413), with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 3). e 293T cells were transfected with an IFN-β-Luc reporter plasmid and expression constructs of MERS-CoV N(1-241) or N(242-413), with or without RIG-IN, MAVS, or TBK1 plasmids. Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 4). f Interaction between IRF3 and N, N(1-241), or N(242-413) was evaluated with co-immunoprecipitation assays using IRF3 as the bait protein. g Interaction between MAVS, IKKε, TBK1, MDA5, and RIG-I with N, N(1-241), or N(242-413) was evaluated with co-immunoprecipitation assays. The experiments in (f, g) were repeated two times independently with similar results. Data in (b–e) represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA with Tukey’s post hoc tests.

Source data

Extended Data Fig. 9 Caspase-6-cleaved N fragments of SARS-CoV-2.

a Schematic of SARS-CoV-2 N mutants. b Caspase-6-mediated cleavage of SARS-CoV-2 N mutants was evaluated with Western blots. The experiment was repeated two times independently with similar results. c, d 293T (c) and Huh7 (d) cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6 and SARS-CoV-2 N, N(1-215), N(216-419), with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays. n = 5 for 293T cells. For Huh7 cells, n = 3 for the empty vector, poly(I:C) only, and poly(I:C)+caspase-6 groups; n = 6 for all other groups. e 293T cells were transfected with an IFN-β-Luc reporter plasmid, expression constructs of caspase-6, SARS-CoV-2 N, NSP13, NSP15, ORF6, ORF8, or ORF3b, with or without poly(I:C). Cells were incubated for 24 h before harvesting for dual-luciferase reporter assays (n = 3 for the empty vector and poly(I:C) only groups; n = 7 for all other groups). Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA (c–e).

Source data

Extended Data Fig. 10 Caspase-6-cleaved MERS-CoV N fragments colocalize with IRF3 in 293T and Huh7 cells.

a 293T cells were transfected with expression constructs of IRF3, MERS-CoV N, N(1-241), or N(242-413), and poly(I:C). Cells were fixed at 16 h post transfection. Localization of N was detected with an in-house guinea pig anti-N immune serum and IRF3 was detected with a rabbit anti-HA antibody. Cell nuclei were identified with the DAPI stain. Bars in (a) represented 10 μm. b Huh7 cells were transfected with expression constructs of IRF3, MERS-CoV N, N(1-241), or N(242-413), and poly(I:C). Cells were fixed at 16 h post transfection. Localization of N was detected with an in-house guinea pig anti-N immune serum and IRF3 was detected with a rabbit anti-HA antibody. Cell nuclei were identified with the DAPI stain. Bars in (b) represented 10 μm. The experiments in (a, b) was repeated two times independently with similar results. c The Pearson correlation coefficient between MERS-CoV N fragments and IRF3 in Huh7 cells was quantified with the Zeiss ZEN software (n = 5). Data in (c) represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with one way-ANOVA.

Source data

Extended Data Fig. 11 Recombinant MERS-CoV with a single amino acid change at the caspase-6 cleavage motif of N protein.

a rMERS-CoV/TKKA is constructed by introducing a D to A change at the TKKD motif of the MERS-CoV N gene with red recombineering. b Huh7 cells were infected with rMERS-CoV/TKKA or rMERS-CoV/TKKD at 1MOI and cell lysates were harvested at 1, 24, and 48 hpi for Western blot analysis of N protein. The experiment was repeated two times independently with similar results. c A549, Huh7, and VeroE6 cells were infected with rMERS-CoV/TKKA or rMERS-CoV/TKKD at 1MOI (A549 and Huh7) or 0.5MOI (VeroE6). MERS-CoV N gene copies in the cell lysate samples at the indicated time points were quantified with RT-qPCR (n = 3). d Huh7 and A549 cells were infected with rMERS-CoV/TKKA or rMERS-CoV/TKKD at 1MOI and treated with z-VEID-fmk at the indicated concentrations. MERS-CoV N gene copies were quantified with RT-qPCR (n = 3). e hDPP4 KI mice were intranasally inoculated with 3.5x10⁴PFU rMERS-CoV/TKKA or rMERS-CoV/TKKD. Mouse lung samples were harvested at day 2 and day 4 post infection. Representative images of H&E stained lungs from rMERS-CoV/TKKA- or rMERS-CoV/TKKD-infected hDPP4 KI mice were shown. The experiment was repeated two times independently with similar results. Data represented mean and standard deviations from the indicated number of biological repeats. Statistical significance between groups was determined with two-way ANOVA (c, d). Scale bar in (e) represented 500 μm.

Source data

Extended Data Fig. 12 Current model of how caspase-6 facilitates coronavirus replication using MERS-CoV as an example.

a Upon coronavirus infection, the host initiates apoptosis to eliminate infected cells, aiming to terminate virus propagation. The triggered apoptosis cascade leads to activation of executor caspases (caspase-3, -6, -7). b MERS-CoV exploits caspase-6 to cleave its N protein, generating N fragments that bind to IRF3, attenuating the activation of IFN signalling, thus benefits virus replication. c In the presence of caspase-6 inhibition, N is not cleaved and IFN signalling is more intact, resulting in restricted virus replication.

Supplementary information

Supplementary Information

This file contains Supplementary Figs. 1–5 (original images for western blots in main figures and Extended Data figures, and gating strategy for the flow cytometry experiment in Figure 1), Supplementary Table 1 (putative caspase-6 cleavage motif in coronavirus N), and Supplementary Table 2 (primer sequences).

Reporting Summary

Peer Review File

Source data

Source Data Figs. 1–5 and Extended Data Figs. 1–3 and 5–11.

Rights and permissions

Springer Nature or its licensor holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Cite this article

Chu, H., Hou, Y., Yang, D. et al. Coronaviruses exploit a host cysteine-aspartic protease for replication. Nature 609, 785–792 (2022). https://doi.org/10.1038/s41586-022-05148-4

Download citation

Received: 19 March 2021
Accepted: 26 July 2022
Published: 03 August 2022
Issue Date: 22 September 2022
DOI: https://doi.org/10.1038/s41586-022-05148-4

This article is cited by

Proviral role of caspase-6 in coronavirus infections
- Lok-Yin Roy Wong
- Stanley Perlman
Cell Research (2022)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Subjects

Abstract

Similar content being viewed by others

Main

Caspase-6 aids coronavirus replication

Caspase-6 cleaves nucleocapsid protein

N fragments modulate IFN response

Discussion

Methods

Cell lines

Viruses

Chemical modulators

Antibodies for western blots

Antibodies for immunofluorescence staining

Ex vivo human lung tissues

Human intestinal organoids

Human DPP4 mouse model

Golden Syrian hamster model

Generation and infection of caspase-6 knockout mice

Generation and infection of MERS-CoV recombinant virus

Caspase-6 CRISPR–Cas9 knockout in vitro

RNA extraction and RT–qPCR

Plaque assays and TCID50 assays

siRNA and shRNA knockdown

Caspase-6 activity assay

Immunofluorescence and histology

Western blot

Flow cytometry

IFNβ luciferase reporter assays

Alignment of human, mouse and hamster caspase-6

Prediction of potential caspase-6 cleavage sites

Statistical analysis

Reporting summary

Data availability

References

Acknowledgements

Author information

Authors and Affiliations

Contributions

Corresponding authors

Ethics declarations

Competing interests

Peer review

Peer review information

Additional information

Extended data figures and tables

Supplementary information

Source data

Rights and permissions

About this article

Cite this article

Share this article

This article is cited by

Comments

Search

Quick links

Plaque assays and TCID₅₀ assays