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. 2019 May:265:57-66.
doi: 10.1016/j.virusres.2019.03.005. Epub 2019 Mar 5.

PEDV nsp16 negatively regulates innate immunity to promote viral proliferation

Peidian Shi  1 , Yanxin Su  1 , Ruiqiao Li  1 , Zhixuan Liang  2 , Shuren Dong  3 , Jinhai Huang  4
Affiliations

PEDV nsp16 negatively regulates innate immunity to promote viral proliferation

Peidian Shi et al. Virus Res. 2019 May.

Abstract

Type-I IFNs (IFN-I) provide a key mediator of innate antiviral response during virus proliferation. Porcine epidemic diarrhea virus (PEDV), which causes diarrhea in swine of all ages, is a worldwide-distributed alphacoronavirus with economic importance. Here, we screened PEDV RNA modification enzymes involved in regulating antiviral response. Whereas the PEDV nsp13 barely regulates type I IFN, inflammatory cytokines (IL-6, TNF-a) and MHCII, nsp16 and nsp14 (to a lesser extent) down-regulate these antiviral effectors. Importantly, we found nsp16 KDKE tetrad appears to play a key role in interferon inhibition by mutating the D129 catalytic residue. Mechanistically, nsp16 down-regulates the activities of RIG-I and MDA5 mediated IFN-β and ISRE. In turn, the mRNA levels of IFIT family members (IFIT1, IFIT2, IFIT3) was inhibited in cells overexpressing nsp16. In addition, nsp10 enhanced the inhibitory effect of nsp16 on IFN-β. Altogether these results indicate PEDV nsp16 negatively regulates innate immunity to promote viral proliferation. Findings from this study provides novel perspective to advance the understanding in the pathogenesis of PEDV.

Keywords: 2'O-MTase; IFIT; IFN-β; PEDV; nsp16.

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Figures

Fig. 1
Fig. 1
PEDV nsp16 is an antagonist of interferon. (a) IPEC-J2 cells were transfected with pFlag-CMV2 (empty vector) or pFlag-CMV2-nsp13 (Flag-nsp13), pFlag-CMV2-nsp14 (Flag-nsp14), pFlag-CMV2-nsp16 (Flag-nsp16) plasmid respectively, the plasmid expression was detected by western blotting. (b–d) IPEC-J2 cells were transfected with empty vector or Flag-nsp13 (b), Flag-nsp14 (c), Flag-nsp16 (d) plasmid respectively. At 12 h post-transfection, cells were infected with 0.5 MOI PEDV for 12 h. The levels of IFN-β, IL-6, TNF-α, MHCI and MHCII mRNA were analyzed by qRT-PCR. (e) IPEC-J2 cells were transfected with empty vector, Flag-nsp13, Flag-nsp14, Flag-nsp16 plasmid respectively and stimulated with 0.1 MOI SeV or PBS (mock) for 12 h, IFN-β mRNA levels were analyzed by qRT-PCR. (f) HEK293 T cells were transfected with Flag-nsp16 and stimulated with 0.1 MOI SeV for 12 h, IFN-β mRNA levels were analyzed by qRT-PCR. (g) Increasing amounts (100 ng, 300 ng, 500 ng) of Flag-nsp16 or control plasmid together with IFN-β-luc reporter (50 ng), the LacZ (50 ng) were co-transfected in HEK293 T cells. At 12 h post-transfection, cells were infected with SeV (MOI 0.5) or not. After 12 h, the infected cells were collected and IFN-β activation was analyzed by the luciferase reporter system. *P < 0.05**P < 0.01; ***P < 0.001 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments.
Fig. 2
Fig. 2
The KDKE tetrad is necessary for the ability of PEDV nsp16 to inhibit IFN induction. (a) Schematic representation of the genomic RNA structure of PEDV. Replicase (ORF1a and ORF1b.), structural regions, and the position of the 5′-capped and 3′ poly(A) tract and the ribosomal frameshift site between ORF1a and ORF1b is shown at the top. Each box represents a protein product, 16 non-structural protein; the envelope proteins, the N protein, and the accessory ORF3 protein; S, spike; E, envelope; M, membrane; N, nucleocapsid; PLpro, papain-like cysteine protease; 3CLpro, the main 3C-like cysteine protease; Hel, helicase; N7-MTase, N7-methyltransferase; 2′-O-MTase, ribose-2′-O-methyltransferase. Colors indicate the level of amino-acid identity with the best-matching protein of other coronaviruses. (b) Sequence comparison of nsp16 and homologs from other viruses. Percent similarity is expressed as a color code from white to brown and shows precise similarity within each element of the matrix. (c) Nsp16 protein 3D structure simulation was performed using Zhang Lab's I-TASSER and PyMOL software. The key amino acid sites that can bind to the SAM substrate are labeled with different colors. (d) The vector, Flag-nsp16 and Flag-nsp16 (D129A) plasmids was transfected in IPEC-J2 cells respectively and then inoculated without or with 0.1 MOI SeV for 12 h, IFN-β mRNA levels were analyzed by qRT-PCR. (e) HEK293 T cells were transfected with Flag-nsp16 or control plasmid together with IFN-β-luc reporter (50 ng), the LacZ (50 ng), and then inoculated without or with 0.1 MOI SeV for 12 h and luciferase activity was subsequently determined. *P < 0.05**P < 0.01 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments.
Fig. 3
Fig. 3
Nsp16 plays a negative role in RLR-mediated signal pathway regulation. (a–c) The Flag-nsp16 or Flag-nsp16 (D129A) or empty vector plasmids together with IFN-β-luc reporter (50 ng), the LacZ (50 ng) and poly I:C (a) Flag-tagged vector or Flag-tagged RIG-I(N) (b) or Flag-tagged MDA5(N) (c) were co-transfected in HEK293 T cells. IFN-β activation was analyzed by the luciferase reporter system. (d–f) ISRE luciferase activity was analyzed in HEK293 T cells co-transfected with Flag-nsp16 or Flag-nsp16 (D129A) or empty vector plasmids together with ISRE-luc reporter (50 ng), the LacZ (50 ng) together with poly I:C (d) Flag-tagged vector or Flag-tagged RIG-I(N) (e) or Flag-tagged MDA5(N) (f).
Fig. 4
Fig. 4
Nsp10 enhances the inhibitory effect of nsp16 on type I interferon. (a) HEK293 T cells were transfected with Myc-nsp10 or Myc-tagged vector. At 24 h post-transfection, the cells were infected with or without 0.1 MOI SeV for 12 h, then IFN-β mRNA levels were analyzed by qRT-PCR. (b) HEK293 T cells were transfected with Myc-nsp10 or Myc-tagged vector together with IFN-β-luc reporter (50 ng), the LacZ (50 ng) and then infected with SeV (MOI = 0.1). After 12 h stimulation, the IFN-β luciferase activity was subsequently analyzed. (c–e) HEK293 T were transfected with Flag-nsp16(WT) or Flag-tagged vector and Myc-nsp10 or Myc-tagged vector together with IFN-β-luc reporter, the LacZ and poly I:C (c) or Flag-RIG-I(N) (d) or Flag-MDA5(N) (e) for 24 h and then the cells were lysed for luciferase assays. (f–h) HEK293 T were transfected with Flag-nsp16(D129A) or Flag-tagged vector and Myc-nsp10 or Myc-tagged vector together with IFN-β-luc reporter, the LacZ and poly I:C (f) or Flag-RIG-I(N) (g) or Flag-MDA5(N) (h) for 24 h and then the cells were lysed for luciferase assays.
Fig. 5
Fig. 5
Nsp16 can promote PEDV replication. (a–b) The Flag-nsp16 or Flag-tagged vector plasmids were transfected into IPEC-J2 respectively and then infected with 2 MOI PEDV. At 24 h post-infection, the cells were collected. The PEDV N protein was detected by IFA (a). Analysis of PEDV levels by flow cytometry detection of PEDV N (b). (c) IPEC-J2 transfected with vector or Flag-nsp16 or Flag-nsp16 (D129A) plasmids respectively. At 24 h post-transfection, the cells were either mock-infected or infected with 2 MOI PEDV at the indicated times. The mRNA level of PEDV N were tested by qRT-PCR (c) and the PEDV load was tested by TCID50 (d), and analysis of the PEDV N protein expression levels by western blotting (e). *P < 0.05**P < 0.01 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments.
Fig. 6
Fig. 6
Nsp16 promotes virus replication by suppressing cellular antiviral response. (a) HEK293 T were transfected with Flag-nsp16 or Flag-nsp16 (D129A) plasmid, respectively and then infected with 0.1 MOI SeV for 12 h. Next, cells were harvested, mRNA expression of IFIT, IFIT2, IFIT3 were analyzed by qRT-PCR. (b) HEK293 T were transfected with Flag-nsp16 or Flag-nsp16 (D129A) or Flag-tagged vector and then infected with 0.1 MOI VSV-GFP, fluorescence microscopy imaging examined the proliferation of VSV. (c) 3D4/21 cells transfected with control vector or Flag-nsp16 or Flag-nsp16 (D129A) plasmid, respectively. After 24 h post-transfection, the cells were either mock-infected or infected with PRRSV at an 0.5 MOI at the indicated times. The loads of PRRSV N were tested by qRT-PCR, and PRRSV load was tested by TCID50 (d), and western blotting (e). (f) 3D4/21 cells were transfected with vector or Flag-nsp16 or Flag-nsp16 (D129A) plasmid and then inoculated without or with 0.1 MOI SeV for 12 h, the p-IRF3 protein was detected by western blotting. **P < 0.01 (analysis of two-way ANOVA followed by Bonferroni post-test). Data are representative of three independent experiments.
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