Porcine Deltacoronavirus Nucleocapsid Protein Suppressed IFN-β Production by Interfering Porcine RIG-I dsRNA-Binding and K63-Linked Polyubiquitination

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  Figure 1. PDCoV N protein inhibited porcine IFN-β and ISGs expression. (A–C) PK-15 cells cultured in 24-well plates were transfected with porcine IFN-β-Luc (pGL3-pIFN-β) or porcine NF-κB-Luc (pGL3-pNF-κB) plasmid and pRL-TK plasmid with PDCoV N expression plasmid (pcDNA3.1-HA-PDCoV-N). At 18 h after transfection, cells were treated or untreated with VSV-GFP or Poly(I:C) (LMW). The cells were then subjected to dual-luciferase assays at 16 h post-treatment. The relative firefly luciferase activity was relative to that of an empty vector control. The Renilla reniformis luciferase activity was used to normalize. The expression of PDCoV N protein was detected by western blot with HA antibody. β-actin was detected as the loading control. (D,E) PK-15 cells cultured in 12-well plates were transfected with pcDNA3.1-HA-PDCoV-N or empty plasmid for 24 h, and then transfected with Poly(I:C) (LMW). (D) After 16 h post transfection, the cells were lysed by TRZOL to extract the total RNA. The qRT-PCR was used to detect the relative expression of porcine ISGs mRNA. (E) At 12 h post transfection, the VSV-GFP (MOI = 0.1) infected the PK-15 cells for another 16 h. The GFP was detected by fluorescence…

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  Figure 2. PDCoV N protein inhibited porcine IFN-β promoter activation by the porcine RLR signaling pathway. PK-15 cells were cotransfected with pGL3-pIFN-β,pRL-TL, and pcDNA3.1-HA-PDCoV-N along with constructed expression porcine RIG-I/RIG-IN (pRIG-I/pRIG-IN) (A), porcine MDA5 (pMDA5) (B), porcine MAVS (pMAVS) (C), porcine TBK1 (pTBK1) (C), or porcine IRF3 (pIRF3) (D). Dual-luciferase assays were performed at 24 h post transfection. The relative firefly luciferase activity was relative to that of an empty vector control. The Renilla reniformis luciferase activity was used to normalize. Western blot was used to detect the protein expression of PDCoV N with HA antibody. The β-actin was as the loading control protein. All the experiments were independently performed three times. **p < 0.01.

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  Figure 3. PDCoV N interacts with both pRIG-I and pTRAF3. HEK293T cells were co-transfected with HA-tagged PDCoV-N and Flag-tagged pRIG-I (A–C), Flag-tagged pMDA5 (A), Flag-tagged TBK1 (B), Flag-tagged pMAVS (B), Flag-tagged pTRAF3 (B,D) or Flag-tagged pIRF3 (B), respectively. After 28 h of transfection, cells were lysed for co-immunoprecipitation (Co-IP) assay with anti-Flag (IP: Anti-Flag) or anti-HA (IP: Anti-HA) affinity gel. The WCLs and immunoprecipitants were detected by western blot with anti-Flag, anti-HA or anti-β-action antibody. (E) PK-15 cells were cotransfected with HA-tagged PDCoV-N and Flag-tagged empty plasmid as control or Flag-tagged pRIG-I, respectively. At 24 h after transfection, cells were fixed for IFA to detect the PDCoV-N (green) and pRIG-I (Red) with anti-HA and anti-Flag antibodies, respectively. 4′, 6-diamidino-2-phenylindole (DAPI, blue) stained the cell nuclei. Fluorescent images were acquired with a confocal laser scanning microscope (scar bar: 10 μm). (F) Schematic representation of PDCoV N protein fragments used for Co-IP analyses (up). HEK293T cells were cotransfected with Flag-tagged pRIG-I and HA-tagged full-length or truncated fragments of the PDCoV N…

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  Figure 4. PDCoV-N suppressed the porcine RIG-I-binding dsRNA. (A) HEK293T cells were transfected with Flag-tagged pRIG-I or HA-tagged PDCoV expression plasmid. Cells were lysed for Co-IP by poly(I:C) (pIC) or poly(C)(pC)-beads at 28 h post-transfection. Western blot was used to detect the protein. (B) HEK293T cells were cotransfected with Flag-tagged pRIG-I with different dose HA-tagged PDCoV expression plasmid. After 28 h of transfection, the cells were lysed for immunoprecipitation by poly(I:C)-beads. Western blot was used to detect the immunoprecipitants and WCLs protein.

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  Figure 5. PDCoV N protein inhibited the pRIG-I K63-linked polyubiquitination. (A,B) HEK293T or PK-15 were cotransfected with Flag-tagged pRIG-I and Myc-tagged PDCoV N or empty control plasmid with HA-tagged ubiquitin (HA-Ub) or empty control plasmid. (C,D) HEK293T cells were cotransfected Flag-tagged pRIG-I and Myc-tagged PDCoV N or empty control plasmid with HA-Ub K48R, K63R, K48 only, or K63 only mutant plasmid or empty control plasmid, respectively. After 28 h of transfection, the cells were lysed for Co-IP by Flag-affinity gel. Western blot was used to detect the immunoprecipitants and WCLs protein.

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  Figure 6. PDCoV N repressed porcine Riplet-induced pRIG-I activation. (A) The sequence alignment of porcine TRIM25 (pTRIM25) and Riplet (pRiplet) with human TRIM25 (hTRIM25) and Riplet (hRiplet), respectively, by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/). (B) HEK293T cells were transfected with the HA-tagged pTRIM25 or HA-tagged pRiplet expression plasmid. After 28 h of transfected, the WCLs were detected with anti-HA antibody by western blot. (C) PK-15 cells were co-transfected with pRIG-I, pGL3-pIFN-β, and pRL-TK plasmid with pTRIM25 or pRiplet, the empty plasmid as the control. Dual-luciferase assays were performed at 24 h post transfection. (D) PK-15 cells were co-transfected with pRIG-I, pGL3-pIFN-β, and pRL-TK plasmid with pRiplet and Myc-PDCoV-N, the empty plasmid as the control. Dual-luciferase assays were performed at 24 h post transfection. All the experiments were independently performed three times. (E,G) HEK293T cells were cotransfected with HA-tagged pRiplet or HA-tagged pTRIM25 or HA-empty plasmid as the control with Myc-tagged PDCoV-N expression plasmid, respectively. (F,H) HEK293T cells were co-transfected with Myc-tagged PDCoV (Myc-PDCoV-N) or Myc-empty…

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  Figure 7. PDCoV N protein inhibited pRiplet-induced pRIG-I K63-polyubiquitination. (A,B) HEK293T cells were co-transfected with HA-tagged pRiplet or Flag-tagged pRIG-I or empty plasmid as the control with Flag-tagged pRIG-I or HA-tagged PDCoV-N expression plasmid, respectively. After 28 h of transfection, the cells were lysed for Co-IP by Flag-affinity gel or HA-affinity gel. (C) Schematic representation of porcine RIG-I fragments used for Co-IP analyses. (D,G) HEK293T cells were co-transfected with HA-tagged pRiplet (D) or HA-tagged PDCoV-N (G) with three porcine RIG-I fragments (Flag-pRIG-I-2′CARD, Flag-pRIG-I-HEL, Flag-pRIG-I-CTD), or expression plasmid, respectively. After 28 h of transfection, the cells were lysed for Co-IP by Flag-affinity gel. (E,F) HEK293T cells were co-transfected with Flag-tagged pRIG-I and HA-Ub or HA-Ub-K63 with Myc-tagged PDCoV-N or HA-tagged pRiplet or empty control plasmid, respectively. After 28 h of transfection, the cells were lysed for Co-IP by Flag-affinity gel. (H) HEK293T cells were co-transfected with HA-tagged pRiplet and Flag-tagged pRIG-I with Myc-tagged PDCoV-N or empty plasmid as the control expression plasmid, respectively. After 28 h of…

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  Figure 8. The mechanism of PDCoV N protein interfered with the type I IFN response. PDCoV infect the host cells by attaching to the celluar receptor porcine APN. In the cytoplasm, PDCoV release the viral genomic RNA and then complete the transcription and replication process. The ssRNA as well as dsRNA as replicative intermediate are sensed by porcine innate nucleic acids sensors in the cytoplasm to induce the IFNs production, such as porcine RIG-I. The E3 ubiquitin ligase Riplet is shown to active RIG-I by K63-linked polyubiqutination. Activation of JAK-STAT pathway by IFNs binding its receptor induces the production of ISGs, such as OAS1, ISG15. In the present, PDCoV N protein can block the porcine RIG-I dsRNA-binding and interferes with porcine Riplet-mediated porcine RIG-I K63-linked polyubiquitination to restrain the type I IFN response. PDCoV N protein also interacts with porcine TRAF3. However, the mechanism of the association is unclear. The other viral structure proteins are unclear in regulating type I IFN response.