Elsevier

Veterinary Microbiology

Volume 251, December 2020, 108892
Veterinary Microbiology

Short communication
The accessory protein ORF3 of porcine epidemic diarrhea virus inhibits cellular interleukin-6 and interleukin-8 productions by blocking the nuclear factor-κB p65 activation

https://doi.org/10.1016/j.vetmic.2020.108892Get rights and content

Highlights

  • PEDV ORF3 inhibits the productions of pro-inflammatory cytokines IL-6 and IL-8.

  • PEDV ORF3 inhibits NF-κB activation through blockage of p65 activation.

  • Our study links PEDV ORF3 to inhibition of pro-inflammatory cytokines.

Abstract

Porcine epidemic diarrhea virus (PEDV) is an enveloped, single-stranded positive-sense RNA virus that belongs to a porcine entero-pathogenic alphacoronavirus, causing lethal watery diarrhea in piglets. Despite existing study reports the sole accessory protein ORF3 identified as NF-κB antagonist, the contribution of PEDV ORF3 to production of the pro-inflammatory cytokines mediated by NF-κB signaling remains largely unknown. Thus in this present study, we showed that PEDV ORF3 protein significantly inhibited the productions of pro-inflammatory cytokines interleukin-6 (IL-6) and IL-8. The phosphorylation of IκBα was inhibited by ORF3 protein, but no degradation of IκBα was induced in ORF3-expressing cells. Furthermore, PEDV ORF3 inhibited NF-κB activation through preventing nuclear factor p65 phosphorylation and down-regulating p65 expression level, as well as interfering nuclear translocation of p65, eventually resulting into the inhibition of IL-6 and IL-8 production. Our study definitely links PEDV ORF3 to inhibition of pro-inflammatory cytokines production, which will provide new insight into the molecular mechanisms of NF-κB activity inhibited by PEDV proteins to facilitate virus evasion of host innate immune.

Section snippets

Short communication

The porcine epidemic diarrhea virus (PEDV), as a member of the genus Alphacoronavirus within the family Coronaviridae of the order Nidovirales, is one of the most important causative agents of lethal watery diarrhea in piglets (Huang et al., 2013; Beall et al., 2016). The PEDV genome is a single stranded, positive-sense RNA of 28 k nucleotides (nt) in length with a 5′-cap, 3′-polyadenylated tail, and at least seven open reading frames (ORF1a, ORF1b, and ORFs 2–6) (Chen et al., 2012; Lin et al.,

Declaration of Competing Interest

There is no conflict of interest among the contributors of this paper.

Acknowledgements

This work is supported by State Key Laboratory of Veterinary Biotechnology Foundation, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences(SKLVBF201913), National Natural Science Foundation of China (NSFC, Grant No. 31570159and31200121), Program for Young Scholars with Creative Talents in HeiLongJiang BaYi Agricultural University(CXRC2016-12), Heilongjiang Bayi Agricultural University Young Creative Talents Scholars Support Program of San Heng San Zong (

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      Porcine epidemic diarrhea virus (PEDV), the causative agent of PED, is an enveloped, single-stranded, positive-sense RNA virus that belongs to the member of Alphacoronavirus genus of Coronaviridae family in order Nidovirales (Zou et al., 2019). The PEDV genome is approximately 28,000 nucleotide (nt) that consists of at least 7 open reading frames (ORF1a, ORF1b, and ORF2–6), flanked with 5′-untranslated region (5’-UTR) and 3’-UTR (Wu et al., 2020). ORF1a and ORF1b encode 16 nonstructural proteins (nsp), nsp1-nsp16, and ORF3 encodes only one nonstructural accessory protein (ORF3), as well as the remaining 4 ORFs encoding four structural proteins including spike glycoprotein (S), envelope protein (E), membrane glycoprotein (M), and nucleocapsid protein (N) (Xu et al., 2019).

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      Previous studies have confirmed that PEDV E protein induced ER stress and significantly activated NF-κB which consequently caused the promotion of IL-8 expression (Xu et al., 2013). Recently, the accessory protein ORF3 of PEDV inhibits cellular IL-6 and IL-8 production by blocking the NF-κB p65 activation (Wu et al., 2020). However, the mechanisms underlying the transcriptional regulation of PDCoV-induced IL-8 production is still unclear.

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      While the RIG-I (Fig. 2G), MAVS (Fig. 2I) and IRF3 (Fig. 2K) protein expressions were significantly down-regulated when cells over-expressed PEDV E protein. Densitometry analysis were performed according to our previous study (Wu et al., 2020), and the results showed a 40%–60% decreasing in RIG-I (Fig. 2H), MAVS (Fig. 2J) and IRF3 (Fig. 2L) protein expressions. In order to further confirm the inhibition of PEDV E protein on RIG-I signaling, the expressions of endogenous antiviral proteins were monitored by Western Blot analysis probed with specific corresponding antibodies in IPEC-J2 cells either transfected with pCMV-Myc or pCMV-Myc-E for 24 h. Similar with the exogenous expression data, the expression levels of the endogenous RIG-I (Fig. 2M), MAVS (Fig. 2O) and IRF3 (Fig. 2Q) proteins in the presence of PEDV E protein were remarkably down-regulated in IPEC-J2 cells as compared with those in control cells.

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