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. 2022 Mar 18;53(1):22.
doi: 10.1186/s13567-022-01043-y.

Duck plague virus UL41 protein inhibits RIG-I/MDA5-mediated duck IFN-β production via mRNA degradation activity

Tianqiong He #  1   2   3 , Mingshu Wang #  1   2   3 , Anchun Cheng  4   5   6 , Qiao Yang  1   2   3 , Ying Wu  1   2   3 , Renyong Jia  1   2   3 , Shun Chen  1   2   3 , Dekang Zhu  2   3 , Mafeng Liu  1   2   3 , Xinxin Zhao  1   2   3 , Shaqiu Zhang  1   2   3 , Juan Huang  1   2   3 , Bin Tian  1   2   3 , Xumin Ou  1   2   3 , Sai Mao  1   2   3 , Di Sun  1   2   3 , Qun Gao  1   2   3 , Yanling Yu  1   2   3 , Ling Zhang  1   2   3 , Yunya Liu  1   2   3
Affiliations

Duck plague virus UL41 protein inhibits RIG-I/MDA5-mediated duck IFN-β production via mRNA degradation activity

Tianqiong He et al. Vet Res. .

Abstract

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are cytosolic pattern recognition receptors that initiate innate antiviral immunity. Recent reports found that duck RLRs significantly restrict duck plague virus (DPV) infection. However, the molecular mechanism by which DPV evades immune responses is unknown. In this study, we first found that the DPV UL41 protein inhibited duck interferon-β (IFN-β) production mediated by RIG-I and melanoma differentiation-associated gene 5 (MDA5) by broadly downregulating the mRNA levels of important adaptor molecules, such as RIG-I, MDA5, mitochondrial antiviral signalling protein (MAVS), stimulator of interferon gene (STING), TANK-binding kinase 1 (TBK1), and interferon regulatory factor (IRF) 7. The conserved sites of the UL41 protein, E229, D231, and D232, were responsible for this activity. Furthermore, the DPV CHv-BAC-ΔUL41 mutant virus induced more duck IFN-β and IFN-stimulated genes (Mx, OASL) production in duck embryo fibroblasts (DEFs) than DPV CHv-BAC parent virus. Our findings provide insights into the molecular mechanism underlying DPV immune evasion.

Keywords: DPV; IFN-β; RLRs; UL41 protein; innate immune response; mRNA.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
The DPV UL41 protein inhibited duck IFN-β signalling activation induced by poly(I:C). All transfected samples were collected at 36 hpt. A The DPV UL41 protein inhibited duck IFN-β production induced by poly(I:C) in DEF cells. DEF cells were transfected with pcaggs-UL41-HA or empty vector and then stimulated with 50 µg/mL poly(I:C) after 12 hpt. The cells were harvested and detected by RT-qPCR after 24 h of stimulation. B The DPV UL41 protein significantly inhibited duck IFN-β-Luc luciferase activity in a dose-dependent manner. DEF cells were co-transfected with the duck IFN-β-Luc luciferase reporter plasmid, pRL-TK, 1 μg/well pcaggs-UL41-HA, 2 μg/well pcaggs-UL41-HA or empty vector and then stimulated with 50 µg/mL poly(I:C) at 12 hpt. The cells were harvested and detected by the dual-luciferase assay at 24 hpt. Protein expression was confirmed by Western blotting. The data were analysed by one-way ANOVA. *p < 0.05, **p < 0.01.
Figure 2
Figure 2
Comparison of the amino acid sequences of the DPV UL41 protein and its homologues in alphaherpesviruses.
Figure 3
Figure 3
Mutation of crucial DPV UL41 residues rescued UL41 protein expression and IFN-β signalling. A HEK293T cells were transfected with the pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector. The cells were then harvested and subjected to RT-qPCR. B DEF cells were transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector and then treated with DMSO, 10 μM MG132, or 20 mM NH4Cl. C HEK293T cells were transfected with the pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector. Immunofluorescence analysis revealed that the fluorescence (green) was significantly stronger in the mUL41 group than in the UL41 group. D DEF cells were transfected with the UL41 or mUL41 expression plasmid or empty vector and then stimulated with 50 µg/mL poly(I:C) at 12 hpt. The cells were harvested and subjected to RT-qPCR after 24 h of stimulation. E DEF cells were co-transfected with IFN-β-Luc, pRL-TK, and 1 μg/well pcaggs-UL41-HA, 2 μg/well pcaggs-UL41-HA, 2 μg/well pcaggs-mUL41-HA or empty vector and then stimulated with 50 μg/mL poly(I:C) at 12 hpt. The cells were harvested and detected by the dual-luciferase assay at 24 hpt. Protein expression was confirmed by Western blotting. The data were analysed by one-way ANOVA. *p < 0.05, **p < 0.01.
Figure 4
Figure 4
The DPV UL41 protein inhibited IFN-β-Luc activation by every important adaptor molecule in the RIG-I/MDA5 innate immune pathway. DEF cells were co-transfected with empty vector, pcaggs-UL41-HA or pcaggs-mUL41-HA together with IFN-β-Luc and plasmids expressing important adaptor proteins in the RIG-I/MDA5 innate immune pathway, namely, RIG-I, MDA5, MAVS, STING, TBK1 and IRF7, and then subjected to luciferase reporter assay to detect IFN-β-Luc promoter activity. Protein expression was confirmed by Western blotting. The data were analysed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 5
Figure 5
The DPV UL41 protein broadly decreased the mRNA levels of important adaptor molecules in the RIG-I/MDA5 innate immune pathway. A The DPV UL41 protein inhibited the expression of important adaptor molecules induced by poly(I:C), including, RIG-I, MDA5, MAVS, STING, TBK1 and IRF7, in DEF cells. DEF cells were transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector and then stimulated with 50 µg/mL poly(I:C) at 12 hpt. The cells were harvested and detected by RT-qPCR after 24 h of stimulation. B DEF cells were co-transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector and the RIG-I, MDA5, MAVS, STING, TBK1 and IRF7 expression plasmids. Then, the cells were harvested and subjected to RT-qPCR and Western blot analysis. C HEK293T cells were co-transfected with pcaggs-UL41-HA, pcaggs-mUL41-HA or empty vector and the RIG-I, MDA5, MAVS, STING, TBK1 and IRF7 expression plasmids. Then, the cells were harvested and subjected to RT-qPCR and Western blot analysis. The data were analysed by one-way ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 6
Figure 6
DPV downregulated duck IFN-β and ISG production via the UL41 protein. DEF cells were infected with the DPV CHv-BAC-G parent virus or the DPV CHv-BAC-G-ΔUL41 mutant virus at an MOI of 5 and then harvested and subjected to RT-PCR analysis at 4 hpi. A Quantification of IFN-β. B Quantification of Mx and OASL. C The viral titers in cytoplasmic samples were determined by TCID50. The data were analysed by one-way ANOVA. **p < 0.01, ***p < 0.001 and ****p < 0.0001.
Figure 7
Figure 7
Knockdown of IRF7 expression increased the replication of the DPV CHv-BAC-ΔUL41 mutant virus. A DEF cells were transfected with pGPU6/GFP/Neo-shIRF7 or pGPU6/GFP/Neo-shNC, and the transcription and expression of endogenous IRF7 was detected by RT-qPCR and Western blotting, respectively, using a rabbit anti-IRF7 antibody (ABclonal, China, 1:1000). B DEF cells were transfected with pGPU6/GFP/Neo-shIRF7 or pGPU6/GFP/Neo-shNC and then infected with the DPV CHv-BAC parent virus or DPV CHv-BAC-ΔUL41 mutant virus at an MOI of 0.1 after 12 hpt. The viral titers were determined by the TCID50 at 24 hpi. The data were analysed by one-way ANOVA. *p < 0.05.
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