Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2024 Feb 20;98(2):e0168223.
doi: 10.1128/jvi.01682-23. Epub 2024 Jan 30.

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

Haixin Huang #  1   2 , Xiaoxiao Lei #  2 , Chenchen Zhao  2 , Yan Qin  2 , Yuying Li  2 , Xinyu Zhang  2 , Chengkai Li  2 , Tian Lan  2 , Baopeng Zhao  2 , Wenchao Sun  2 , Huijun Lu  3 , Ningyi Jin  1   3
Affiliations

Porcine deltacoronavirus nsp5 antagonizes type I interferon signaling by cleaving IFIT3

Haixin Huang et al. J Virol. .

Abstract

Porcine deltacoronavirus (PDCoV) has caused enormous economic losses to the global pig industry. However, the immune escape mechanism of PDCoV remains to be fully clarified. Transcriptomic analysis revealed a high abundance of interferon (IFN)-induced protein with tetratricopeptide repeats 3 (IFIT3) transcripts after PDCoV infection, which initially implied a correlation between IFIT3 and PDCoV. Further studies showed that PDCoV nsp5 could antagonize the host type I interferon signaling pathway by cleaving IFIT3. We demonstrated that PDCoV nsp5 cleaved porcine IFIT3 (pIFIT3) at Gln-406. Similar cleavage of endogenous IFIT3 has also been observed in PDCoV-infected cells. The pIFIT3-Q406A mutant was resistant to nsp5-mediated cleavage and exhibited a greater ability to inhibit PDCoV infection than wild-type pIFIT3. Furthermore, we found that cleavage of IFIT3 is a common characteristic of nsp5 proteins of human coronaviruses, albeit not alphacoronavirus. This finding suggests that the cleavage of IFIT3 is an important mechanism by which PDCoV nsp5 antagonizes IFN signaling. Our study provides new insights into the mechanisms by which PDCoV antagonizes the host innate immune response.IMPORTANCEPorcine deltacoronavirus (PDCoV) is a potential emerging zoonotic pathogen, and studies on the prevalence and pathogenesis of PDCoV are ongoing. The main protease (nsp5) of PDCoV provides an excellent target for antivirals due to its essential and conserved function in the viral replication cycle. Previous studies have revealed that nsp5 of PDCoV antagonizes type I interferon (IFN) production by targeting the interferon-stimulated genes. Here, we provide the first demonstration that nsp5 of PDCoV antagonizes IFN signaling by cleaving IFIT3, which affects the IFN response after PDCoV infection. Our findings reveal that PDCoV nsp5 is an important interferon antagonist and enhance the understanding of immune evasion by deltacoronaviruses.

Keywords: IFIT3; PDCoV; cleavage; nsp5; type I interferon signaling.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig 1
Fig 1
PDCoV and poly(I:C) induced IFIT3 expression. (A) Heatmap showing the fold changes in the expression of genes in PDCoV-infected ST cells compared with mock-infected ST cells at 24 h. (B) Volcano plot showing the global differentially expressed genes in PDCoV-infected ST cells compared with mock-infected ST cells at 24 h. (C) pXJ40-HA-vector, pXJ40-HA-pIFIT3, IFIT3 siRNA, and negative control siRNA were transfected into ST cells. After 24 h of transfection, the cells were infected with PDCoV (MOI = 1) for 6, 12, and 24 h and were then harvested for Western blot analysis. Relative expression levels in the pXJ40-HA-vector, pXJ40-HA-pIFIT3, IFIT3 siRNA, and negative control siRNA groups were determined and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression using ImageJ. (D and E) HEK-293T and ST cells were treated with poly(I:C) (2 µg/mL) for 0, 4, 8, 12, or 24 h. And HEK-293T and ST cells were infected with SeV for 0, 4, 8, 12, or 24 h. The cells were lysed and collected, and the IFIT3 mRNA levels were then measured by RT-qPCR. The protein expression levels of IFIT3 and GAPDH were measured by Western blotting. All data are reported as means ± SDs. For all experiments, **P < 0.01, and ***P < 0.001 were considered to indicate statistical significance.
Fig 2
Fig 2
IFIT3 activates the type I IFN signaling pathway. (A) HEK-293T cells were cotransfected with the IFN-β Luc and pRL-TK plasmids, along with 0.5, 1, or 1.5 µg of the pXJ40-HA-pIFIT3 expression plasmid for 30 h. The cells were lysed and subjected to a dual-luciferase assay. (B) HEK-293T cells were transfected with 0.5, 1, or 1.5 µg of the pXJ40-HA-pIFIT3 for 30 h. The cells were collected, and the IFN-β mRNA level was measured by RT-qPCR. (C) HEK-293T cells were transfected with 0.5, 1, or 1.5 µg of the pXJ40-HA-pIFIT3. After 30 h, the cells were collected, and the ISG56, ISG54, IFIT5, RIG-I, MDA-5, MAVs, IκBα , IRF3, and NF-κB mRNA levels were measured by RT-qPCR. (D) pXJ40-HA-vector, pXJ40-HA-pIFIT3, IFIT3 siRNA, and negative control siRNA were transfected into HEK-293T cells. After 30 h of transfection, the cells were collected and analyzed by Western blotting with anti-P65, anti-phospho-P65, anti-IRF3, anti-phospho-IRF3, anti-STAT1, anti-phospho-STAT1, anti-TBK1, anti-phospho-TBK1, anti-IFIT3, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Relative levels of P65, phospho-P65, IRF3, phospho-IRF3, STAT1, phospho-STAT1, TBK1, and phospho-TBK1 were quantified and normalized to the GAPDH expression level using ImageJ. All data are reported as the means ± SDs. For all experiments, *P < 0.05, **P < 0.01, and ***P < 0.001 were considered to indicate statistical significance. ns, nonsignificant difference.
Fig 3
Fig 3
PDCoV nsp5 inhibits the SeV-induced type I IFN signaling pathway. (A) ST cells were infected with the PDCoV (MOI = 1). After 12 h, the cells were further infected or mock infected with SeV or mock infected for 12 h. Cells were collected at 12 h post infection, and the IFN-β, RIG-I, MDA-5, ISG56, ISG54, IFIT3, ISG15, IRF9, RSAD2, and CXCL10 mRNA levels were measured by RT-qPCR. (B) HEK-293T cells were cotransfected with the IFN-β Luc and pRL-TK plasmids, along with 1.2 µg of pCAGGS-flag-nsp5 or an empty vector. After 24 h, the cells were further infected or mock infected with SeV or mock infected for 12 h. The cells were lysed and subjected to a dual-luciferase assay. (C) ST cells were transfected with 1.2 µg of pCAGGS-flag-nsp5 or an empty vector. After 24 h of initial transfection, the cells were further infected or mock infected with SeV or mock infected. The cells and supernatants were collected at 12 h post infection, and the IFN-β, ISG56, ISG54, IFIT3, and IFIT5 mRNA levels were measured by RT-qPCR. (D) HEK-293T cells were transfected with the pCAGGS-flag-nsp5 for 24 h and stimulated with SeV for 12 h. Cell lysates were analyzed by Western blotting with anti-P65, anti-phospho-P65, anti-IRF3, anti-phospho-IRF3, anti-STAT1, anti-phospho-STAT1, anti-TBK1, anti-phospho-TBK1, anti-IFIT3, and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. Relative levels of P65, phospho-P65, IRF3, phospho-IRF3, STAT1, phospho-STAT1, TBK1, and phospho-TBK1 were quantified and normalized to the GAPDH expression level using ImageJ. All data are reported as the means ± SDs. For all experiments, *P < 0.05, **P < 0.01, and ***P < 0.001 were considered to indicate statistical significance.
Fig 4
Fig 4
PDCoV nsp5 targets IFIT3 for cleavage. (A) HEK-293T cells were cultured in 6-well plates and cotransfected with the PDCoV nsp5 expression plasmid or empty vector along with 1.2µg of the HA-tagged IFIT1, IFIT2, IFIT3, IFIT5, RSAD2, USP18, OAS1, or TRAF2 expression plasmid. After 30h, the cells were lysed and analyzed by Western blotting with an anti-HA antibody. (B–D) HEK-293T, LLC-PK1, and IPEC-J2 cells were cotransfected with pXJ40-HA-pIFIT3 and various amounts of pCAGGS-flag-nsp5. After 30 h, the cells were lysed for Western blotting. The cleavage products are labeled “Cp.” (E) ST cells were transfected with 1.2 µg/well pXJ40-HA-pIFIT3. After 24 h of transfection, the ST cells were infected with PDCoV at MOIs of 1, 2, and 3. After 24 h, the cells were lysed for Western blotting. (F) ST cells were infected with PDCoV (MOI = 1), and the cells and supernatants were collected at 0, 6, 12, and 24 h for analysis by Western blot analysis. (G) IPEC-J2 cells were infected with PDCoV (MOI = 1), and the cells and supernatants were collected at 0, 12, 24, and 36 h for analysis by Western blot analysis. (H) HEK-293T cells were transfected with expression constructs encoding pCAGGS-flag-nsp5 and pXJ40-HA-pIFIT3. The cells were lysed 30 h after transfection and subjected to immunoprecipitation with an anti-FLAG antibody or anti-HA antibody. The whole-cell lysates (WCLs) and immunoprecipitation (IP) complexes were analyzed by immunoblotting using anti-FLAG, anti-HA, or anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibodies. (I) ST cells were transfected with PDCoV nsp5 and nsp13. After 24 h, the cells were fixed and then stained with a rabbit monoclonal antibody against IFIT3 and a mouse anti-flag tag antibody prior to incubation with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (green) or Alexa Fluor 594-conjugated goat anti-rabbit IgG antibody (red). Nuclei were stained with DAPI (blue).
Fig 5
Fig 5
PDCoV nsp5 cleaves IFIT3 via its protease activity. (A) HEK-293T cells were cotransfected with plasmids expressing the wild-type PDCoV nsp5 or one of protease-defective mutants (C144A and H41A) and the pXJ40-HA-pIFIT3. After 30 h, the cells were lysed, and supernatants were used for Western blotting. (B) HEK-293T cells were cotransfected with pXJ40-HA-pIFIT3 and pCAGGS-flag-nsp5. After 24 h, the cells were mock treated or treated with PF-00835231 (10 µM). After 12 h, the cells were lysed for Western blotting. (C) HEK-293T cells were cotransfected with plasmids expressing the wild-type PDCoV nsp5 and pXJ40-HA-pIFIT3. After 24 h, the cells were treated with MG132 (10 µM), Z-VAD-FMK (25 µM), or 3-MA (10 µM) for 12 h. Cell lysates were prepared and analyzed by Western blotting. (D) HEK-293T and LLC-PK1 cells were cotransfected with the IFN-β-Luc and pRL-TK plasmids, along with 1.2 µg of pCAGGS-flag-nsp5, one of its protease-defective mutants (C144A, H41A, or DM-41-144) , or empty vector. After 30 h, the cells were lysed and subjected to a dual-luciferase assay. (E–G) HEK-293T cells seeded in a 12-well plate were transfected with equal amounts of plasmids encoding PDCoV nsp5 or one of its protease-defective mutants (C144A, H41A, and DM-41-144) for 24 h and were then infected with SeV for 12 h. The cell supernatants were then collected, treated with UV irradiation for 30 min, and added to a new 12-well plate of HEK-293T cells for 24 h. The IFN-treated cells were then inoculated with VSV-GFP for 12 h, and GFP expression was detected by flow cytometry, immunofluorescence staining, and Western blotting. Relative expression levels of GFP were quantified and normalized to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression level using ImageJ. All data are reported as the means ± SDs. For all experiments, **P < 0.01, and ***P < 0.001 were considered to indicate statistical significance. ns, nonsignificant difference.
Fig 6
Fig 6
PDCoV nsp5 recognizes and cleaves IFIT3 at residue Q406. (A) HEK-293T cells were transfected with expression constructs encoding pXJ40-HA-pIFIT3, the pIFIT31-293 truncated mutant, and pIFIT31-371 truncated mutant and were collected after 30 h for Western blotting. (B) Sequence logo of the polyprotein junctions that are cleaved by PDCoV nsp5. An amino acid sequence logo of the substrate was generated by WebLogo version 3. (C) Schematic representation of pIFIT3 and its mutation sites. (D) HEK-293T cells were cotransfected with the pCAGGS-flag-nsp5 along with expression constructs encoding wild-type pIFIT3 or pIFIT3 mutants (pIFIT3-Q384A, pIFIT3-Q406A, and pIFIT3-Q436A). The cells were then lysed after 30 h and analyzed by Western blotting. (E) HEK-293T cells were co-transfected with the PDCoV nsp5 expression plasmid along with wild-type IFIT3 or pIFIT3-Q406A and collected after 30 h for Western blotting. (F and G) ST cells were transfected with empty vector, pIFIT3, pIFIT3-Q406A, pIFIT31-406, or pIFIT3406-510. After 24 h, the cells were infected with PDCoV (MOI = 1) for 24 h and were then lysed and collected for analysis by Western blotting and RT-qPCR. All data are reported as the means ± SDs. For all experiments, *P < 0.05 and ***P < 0.001 were considered to indicate statistical significance. ns, nonsignificant difference.
Fig 7
Fig 7
The efficiency of Nsp5-mediated cleavage of IFIT3 differs across coronaviruses. (A) Porcine, human, monkey, canine. and bovine sequences of IFIT3 were aligned with DNAMAN8. (B) HEK-293T cells were cultured in 12-well plates and cotransfected with pXJ40-HA-pIFIT3 and a plasmid encoding the nsp5 of SADS-CoV, SARS-CoV-2, SARS-CoV, MERS-CoV, or PDCoV. After 30 h, the cells were lysed and analyzed by Western blotting. (C) HEK-293T cells were cotransfected with PDCoV nsp5 and pXJ40-HA-B-IFIT3 (bovine), pXJ40-HA-M-IFIT3 (monkey), pXJ40-HA-D-IFIT3 (canine), pXJ40-HA-P-IFIT3 (porcine), and pXJ40-HA-H-IFIT3 (human). After 30 h, the cells were lysed and analyzed by Western blotting.
Fig 8
Fig 8
PDCoV nsp5 inhibits host innate immunity by directly cleaving IFIT3. Mechanistic diagram showing the antagonism of the IFIT3-mediated type I IFN signaling pathway by Nsp5 of PDCoV. The schematic was drawn using the Biorender website.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Woo PCY, Lau SKP, Lam CSF, Lau CCY, Tsang AKL, Lau JHN, Bai R, Teng JLL, Tsang CCC, Wang M, Zheng B-J, Chan K-H, Yuen K-Y. 2012. Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol 86:3995–4008. doi:10.1128/JVI.06540-11 - DOI - PMC - PubMed
    1. Ma Y, Zhang Y, Liang X, Lou F, Oglesbee M, Krakowka S, Li J, Parker J, Griffin DE. 2015. Origin, evolution, and virulence of porcine deltacoronaviruses in the United States. mBio 6:e00064. doi:10.1128/mBio.00064-15 - DOI - PMC - PubMed
    1. Liang Q, Zhang H, Li B, Ding Q, Wang Y, Gao W, Guo D, Wei Z, Hu H. 2019. Susceptibility of chickens to porcine deltacoronavirus infection. Viruses 11:573. doi:10.3390/v11060573 - DOI - PMC - PubMed
    1. Lednicky JA, Tagliamonte MS, White SK, Elbadry MA, Alam MM, Stephenson CJ, Bonny TS, Loeb JC, Telisma T, Chavannes S, Ostrov DA, Mavian C, Beau De Rochars VM, Salemi M, Morris JG. 2021. Emergence of porcine delta-coronavirus pathogenic infections among children in Haiti through independent zoonoses and convergent evolution. medRxiv:2021.03.19.21253391. doi:10.1101/2021.03.19.21253391 - DOI
    1. Homwong N, Jarvis MC, Lam HC, Diaz A, Rovira A, Nelson M, Marthaler D. 2016. Characterization and evolution of porcine deltacoronavirus in the United States. Prev Vet Med 123:168–174. doi:10.1016/j.prevetmed.2015.11.001 - DOI - PMC - PubMed

MeSH terms

Substances

Supplementary concepts

LinkOut - more resources

-