Department of Preventive Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang, China.
Northeastern Science Inspection Station, China Ministry of Agriculture Key Laboratory of Animal Pathogen Biology, Harbin, Heilongjiang, China.
Virol J. 2019 Aug 5;16(1):97. doi: 10.1186/s12985-019-1206-9.
Transmissible gastroenteritis virus (TGEV), a member of the family Coronaviridae, causes lethal watery diarrhea in piglets. Previous studies have revealed that the coronaviruses develop various strategies to evade the host innate immunity through the inhibition of nuclear factor kappa B (NF-κB) signaling pathway. However, the ability of TGEV to inhibit the host innate immune response by modulating the NF-κB signaling pathway is not clear.
In this study, a dual luciferase reporter assay was used to confirm the inhibition of NF-κB by TGEV infection and to identify the major viral proteins involved in the inhibition of NF-κB signaling. Real-time quantitative PCR was used to quantify the mRNA expression of inflammatory factors. The deubiquitination of Nsp3 domains and its effect on IκBα and p65 were analyzed by western blotting. The ubiquitination level of IκBα was analyzed by immunoprecipitation.
In ST and IPEC-J2 cells, TGEV exhibited a dose-dependent inhibition of NF-κB activity. Individual TGEV protein screening revealed the high potential of non-structural protein 3 (Nsp3) to inhibit NF-κB signaling, and leading to the downregulation of the NF-κB-induced cytokine production. We demonstrated that the inhibitory effect of Nsp3 was mainly mediated through the suppression of IκBα degradation as well as the inhibition of p65 phosphorylation and nuclear translocation. Furthermore, the amino acid residues at positions 590-1,215 in Nsp3 were demonstrated to inhibit the degradation of IκBα by inhibiting the IκBα ubiquitination.
TGEV infection can inhibit the activation of the NF-κB signaling pathway, which is mainly mediated by Nsp3 through the canonical pathway. The amino acid residues at positions 590-1,215 in Nsp3 compose the critical domain that mediates NF-κB inhibition. We speculate that this inhibitory effect is likely to be related to the structure of PLP2 with deubiquitinating enzyme activity of the amino acid residues at positions 590-1,215 in Nsp3. Our study provides a better understanding of the TGEV-mediated innate immune modulation and lays the basis for studies on the pathogenesis of coronavirus.
传染性胃肠炎病毒（TGEV）是冠状病毒科的一个成员，它会导致仔猪致命的水样腹泻。先前的研究表明，冠状病毒通过抑制核因子 kappa B（NF-κB）信号通路来逃避宿主先天免疫。然而，TGEV 通过调节 NF-κB 信号通路来抑制宿主先天免疫反应的能力尚不清楚。
在本研究中，我们使用双荧光素酶报告基因检测来证实 TGEV 感染对 NF-κB 的抑制作用，并鉴定参与抑制 NF-κB 信号的主要病毒蛋白。实时定量 PCR 用于量化炎症因子的 mRNA 表达。通过 Western blot 分析 Nsp3 结构域的去泛素化及其对 IκBα 和 p65 的影响。通过免疫沉淀分析 IκBα 的泛素化水平。
在 ST 和 IPEC-J2 细胞中，TGEV 表现出剂量依赖性的 NF-κB 活性抑制。单独的 TGEV 蛋白筛选表明，非结构蛋白 3（Nsp3）具有很强的抑制 NF-κB 信号的潜力，并导致 NF-κB 诱导的细胞因子产生下调。我们证明，Nsp3 的抑制作用主要是通过抑制 IκBα 的降解以及抑制 p65 的磷酸化和核转位来介导的。此外，Nsp3 的 590-1,215 位氨基酸残基通过抑制 IκBα 的泛素化来抑制 IκBα 的降解。
TGEV 感染可以抑制 NF-κB 信号通路的激活，这主要是通过 Nsp3 通过经典途径介导的。Nsp3 的 590-1,215 位氨基酸残基构成介导 NF-κB 抑制的关键结构域。我们推测，这种抑制作用可能与 Nsp3 的 590-1,215 位氨基酸残基的 PLP2 结构域具有去泛素化酶活性有关。本研究加深了对 TGEV 介导的先天免疫调节的认识，并为冠状病毒发病机制的研究奠定了基础。
