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Swine acute diarrhoea syndrome coronavirus (SADS-CoV) Nsp5 antagonizes type I interferon signaling by cleaving DCP1A.

ABSTRACT: Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which is a recently discovered enteric coronavirus, is the major aetiological agent that causes severe clinical diarrhoea and intestinal pathological damage in pigs, and it has caused significant economic losses to the swine industry. Nonstructural protein 5, also called 3C-like protease, cleaves viral polypeptides and host immune-related molecules to facilitate viral replication and immune evasion. Here, we demonstrated that SADS-CoV nsp5 significantly inhibits the Sendai virus (SEV)-induced production of IFN-β and inflammatory cytokines. SADS-CoV nsp5 targets and cleaves mRNA-decapping enzyme 1a (DCP1A) via its protease activity to inhibit the IRF3 and NF-κB signaling pathways in order to decrease IFN-β and inflammatory cytokine production. We found that the histidine 41 and cystine 144 residues of SADS-CoV nsp5 are critical for its cleavage activity. Additionally, a form of DCP1A with a mutation in the glutamine 343 residue is resistant to nsp5-mediated cleavage and has a stronger ability to inhibit SADS-CoV infection than wild-type DCP1A. In conclusion, our findings reveal that SADS-CoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by alpha coronaviruses.

SUBMITTER: Huang HX

PROVIDER: S-EPMC10239798 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Swine acute diarrhoea syndrome coronavirus (SADS-CoV) Nsp5 antagonizes type I interferon signaling by cleaving DCP1A.

Huang Hai-Xin HX Zhao Chen-Chen CC Lei Xiao-Xiao XX Zhang Xin-Yu XY Li Yu-Ying YY Lan Tian T Zhao Bao-Peng BP Lu Jing-Yi JY Sun Wen-Chao WC Lu Hui-Jun HJ Jin Ning-Yi NY

Frontiers in immunology 20230522

Swine acute diarrhoea syndrome coronavirus (SADS-CoV), which is a recently discovered enteric coronavirus, is the major aetiological agent that causes severe clinical diarrhoea and intestinal pathological damage in pigs, and it has caused significant economic losses to the swine industry. Nonstructural protein 5, also called 3C-like protease, cleaves viral polypeptides and host immune-related molecules to facilitate viral replication and immune evasion. Here, we demonstrated that SADS-CoV nsp5 s ...[more]

PMID: 37283741

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Project description:Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The first outbreak of PDCoV was announced from the United States in 2014, followed by reports in Asia. The nonstructural protein nsp5 is a 3C-like protease of coronavirus, and our previous study showed that PDCoV nsp5 inhibits type I interferon (IFN) production. In this study, we found that PDCoV nsp5 significantly inhibited IFN-stimulated response element (ISRE) promoter activity and transcription of IFN-stimulated genes (ISGs), suggesting that PDCoV nsp5 also suppresses IFN signaling. Detailed analysis showed that nsp5 cleaved signal transducer and activator of transcription 2 (STAT2) but not Janus kinase 1 (JAK1), tyrosine kinase 2 (TYK2), STAT1, and interferon regulatory factor 9 (IRF9), key molecules of the JAK-STAT pathway. STAT2 cleavage was dependent on the protease activity of nsp5. Interestingly, nsp5 cleaved STAT2 at two sites, glutamine 685 (Q685) and Q758, and similar cleavage was observed in PDCoV-infected cells. As expected, cleaved STAT2 impaired the ability to induce ISGs, demonstrating that STAT2 cleavage is an important mechanism utilized by PDCoV nsp5 to antagonize IFN signaling. We also discussed the substrate selection and binding mode of PDCoV nsp5 by homologous modeling of PDCoV nsp5 with the two cleaved peptide substrates. The results of our study demonstrate that PDCoV nsp5 antagonizes type I IFN signaling by cleaving STAT2 and provides structural insights for comprehending the cleavage mechanism of PDCoV nsp5, revealing a potential new function for PDCoV nsp5 in type I IFN signaling.IMPORTANCE The 3C-like protease encoded by nsp5 is a major protease of coronaviruses; thus, it is an attractive target for development of anticoronavirus drugs. Previous studies have revealed that the 3C-like protease of coronaviruses, including PDCoV and porcine epidemic diarrhea virus (PEDV), antagonizes type I IFN production by targeting the NF-κB essential modulator (NEMO). Here, for the first time, we demonstrate that overexpression of PDCoV nsp5 also antagonizes IFN signaling by cleaving STAT2, an essential component of transcription factor complex ISGF3, and that PDCoV infection reduces the levels of STAT2, which may affect the innate immune response.

Project description:Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus. The nonstructural protein nsp5, also called 3C-like protease, is responsible for processing viral polyprotein precursors in coronavirus (CoV) replication. Previous studies have shown that PDCoV nsp5 cleaves the NF-κB essential modulator and the signal transducer and activator of transcription 2 to disrupt interferon (IFN) production and signaling, respectively. Whether PDCoV nsp5 also cleaves IFN-stimulated genes (ISGs), IFN-induced antiviral effector molecules, remains unclear. In this study, we screened 14 classical ISGs and found that PDCoV nsp5 cleaved the porcine mRNA-decapping enzyme 1a (pDCP1A) through its protease activity. Similar cleavage of endogenous pDCP1A was also observed in PDCoV-infected cells. PDCoV nsp5 cleaved pDCP1A at glutamine 343 (Q343), and the cleaved pDCP1A fragments, pDCP1A1-343 and pDCP1A344-580, were unable to inhibit PDCoV infection. Mutant pDCP1A-Q343A, which resists nsp5-mediated cleavage, exhibited a stronger ability to inhibit PDCoV infection than wild-type pDCP1A. Interestingly, the Q343 cleavage site is highly conserved in DCP1A homologs from other mammalian species. Further analyses demonstrated that nsp5 encoded by seven tested CoVs that can infect human or pig also cleaved pDCP1A and human DCP1A, suggesting that DCP1A may be the common target for cleavage by nsp5 of mammalian CoVs.IMPORTANCE Interferon (IFN)-stimulated gene (ISG) induction through IFN signaling is important to create an antiviral state and usually directly inhibits virus infection. The present study first demonstrated that PDCoV nsp5 can cleave mRNA-decapping enzyme 1a (DCP1A) to attenuate its antiviral activity. Furthermore, cleaving DCP1A is a common characteristic of nsp5 proteins from different coronaviruses (CoVs), which represents a common immune evasion mechanism of CoVs. Previous evidence showed that CoV nsp5 cleaves the NF-κB essential modulator and signal transducer and activator of transcription 2. Taken together, CoV nsp5 is a potent IFN antagonist because it can simultaneously target different aspects of the host IFN system, including IFN production and signaling and effector molecules.

Project description:Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose major threats for the horse- and swine-breeding industries worldwide. A previous study suggested that PRRSV nsp4, a 3C-like protease, antagonizes interferon beta (IFN-β) production by cleaving the NF-κB essential modulator (NEMO) at a single site, glutamate 349 (E349). Here, we demonstrated that EAV nsp4 also inhibited virus-induced IFN-β production by targeting NEMO for proteolytic cleavage and that the scission occurred at four sites: E166, E171, glutamine 205 (Q205), and E349. Additionally, we found that, besides the previously reported cleavage site E349 in NEMO, scission by PRRSV nsp4 took place at two additional sites, E166 and E171. These results imply that while cleaving NEMO is a common strategy utilized by EAV and PRRSV nsp4 to antagonize IFN induction, EAV nsp4 adopts a more complex substrate recognition mechanism to target NEMO. By analyzing the abilities of the eight different NEMO fragments resulting from EAV or PRRSV nsp4 scission to induce IFN-β production, we serendipitously found that a NEMO fragment (residues 1 to 349) could activate IFN-β transcription more robustly than full-length NEMO, whereas all other NEMO cleavage products were abrogated for the IFN-β-inducing capacity. Thus, NEMO cleavage at E349 alone may not be sufficient to completely inactivate the IFN response via this signaling adaptor. Altogether, our findings suggest that EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is critical for disarming the innate immune response for viral survival.IMPORTANCE The arterivirus nsp4-encoded 3C-like protease (3CLpro) plays an important role in virus replication and immune evasion, making it an attractive target for antiviral therapeutics. Previous work suggested that PRRSV nsp4 suppresses type I IFN production by cleaving NEMO at a single site. In contrast, the present study demonstrates that both EAV and PRRSV nsp4 cleave NEMO at multiple sites and that this strategy is essential for disruption of type I IFN production. Moreover, we reveal that EAV nsp4 also cleaves NEMO at glutamine 205 (Q205), which is not targeted by PRRSV nsp4. Notably, targeting a glutamine in NEMO for cleavage has been observed only with picornavirus 3C proteases (3Cpro) and coronavirus 3CLpro In aggregate, our work expands knowledge of the innate immune evasion mechanisms associated with NEMO cleavage by arterivirus nsp4 and describes a novel substrate recognition characteristic of EAV nsp4.

Project description:IntroductionSwine acute diarrhea syndrome coronavirus (SADS-CoV) is an enveloped, positive-sense, single-stranded RNA virus that causes clinical symptoms such as vomiting and diarrhea in 10-day-old piglets. SADS-CoV has caused significant economic losses in the swine industry in southern China. Currently, no effective treatments or vaccines are available for this disease, making it crucial to establish a point-of-care testing (POCT) technology for early diagnosis and prevention.MethodsIn this study, we first validated the specificity and immunogenicity of four monoclonal antibodies (mAbs) targeting the nucleocapsid (N) protein of swine acute diarrhea syndrome coronavirus (SADS-CoV). The optimal antibody pair for constructing the fluorescent microsphere-based immunochromatographic assay (FM-ICA) was determined through systematic pairwise screening. Critical parameters of the FM-ICA test strip, including antibody labeling concentration, coating concentration, incubation time, and sample dilution ratio, were subsequently optimized. Analytical performance characteristics of the developed FM-ICA were then rigorously evaluated. Finally, clinical validation was conducted by parallel testing of 72 field samples using both FM-ICA and quantitative PCR (qPCR), followed by concordance rate analysis.ResultsFirst, we demonstrated that all four monoclonal antibodies exhibited favorable immunogenicity and specificity. Subsequently, mAb 12E1 was identified as the coating antibody, and mAb 5G12 was selected as the labeled antibody, forming the optimal combination for FM-ICA preparation. After optimization, the ideal parameters were determined: a labeling concentration of 200 μg/mg for antibodies, a coating concentration of 1 mg/mL, an incubation time of 10 min, and a dilution factor of 10. The FM-ICA exhibited outstanding specificity, sensitivity, reproducibility, and stability, achieving a maximum detectable dilution factor of 1280 and a limit of detection (LOD) of 78 PFU mL⁻¹. Finally, the concordance rate between FM-ICA and qPCR for clinical samples reached 97.22%.DiscussionThese results indicate that FM-ICA is an excellent POCT technology that can be used for the early diagnosis of SADS-CoV, providing support for disease prevention and treatment.

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Swine acute diarrhoea syndrome coronavirus (SADS-CoV) Nsp5 antagonizes type I interferon signaling by cleaving DCP1A.

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Swine acute diarrhoea syndrome coronavirus (SADS-CoV) Nsp5 antagonizes type I interferon signaling by cleaving DCP1A.

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