Key Laboratory of Zoonosis, Ministry of Education, College of Veterinary Medicine, Jilin Universitygrid.64924.3d, Changchun, China.
Key Laboratory of Zoonosis, Ministry of Education, Institute of Zoonosis, Jilin Universitygrid.64924.3d, Changchun, China.
J Virol. 2021 Sep 9;95(19):e0015321. doi: 10.1128/JVI.00153-21.
Orf virus (ORFV) is a highly epitheliotropic parapoxvirus with zoonotic significance that induces proliferative lesions in the skin of sheep, goats, and humans. Several viral proteins carried by ORFV, including nuclear factor-κB (NF-κB) inhibitors, play important roles in hijacking host-associated proteins for viral evasion of the host innate immune response. However, the roles of proteins with unknown functions in viral replication and latent infection remain to be explored. Here, we present data demonstrating that the ORF120, an early-late ORFV-encoded protein, activates the NF-κB pathway in the early phase of infection, which implies that ORFV may regulate NF-κB through a biphasic mechanism. A DUAL membrane yeast two-hybrid system and coimmunoprecipitation experiments revealed that the ORF120 protein interacts with Ras-GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1). The overexpression of the ORF120 protein can efficiently increase the expression of G3BP1 and nuclear translocation of NF-κB-p65 in primary ovine fetal turbinate (OFTu) and HeLa cells. The knockdown of G3BP1 significantly decreased ORF120-induced NF-κB activation, indicating that G3BP1 is involved in ORF120-induced NF-κB pathway activation. A dual-luciferase reporter assay revealed that ORF120 could positively regulate the NF-κB pathway through the full-length G3BP1 or the domain of G3BP1. In conclusion, we demonstrate, for the first time, that the ORF120 protein is capable of positively regulating NF-κB signaling by interacting with G3BP1, providing new insights into ORFV pathogenesis and a theoretical basis for antiviral drug design. As part of the host innate response, the nuclear factor-κB (NF-κB) pathway plays a partial antiviral role in nature by regulating the innate immune response. Thus, the NF-κB pathway is probably the most frequently targeted intracellular pathway for subversion by anti-immune modulators that are carried by a wide range of pathogens. Various viruses, including poxviruses, carry several proteins that prepare the host cell for viral replication by inhibiting cytoplasmic events, leading to the initiation of NF-κB transcriptional activity. However, NF-κB activity is hypothesized to facilitate viral replication to a great extent. The significance of our research is in the exploration of the activation mechanism of NF-κB induced by the Orf virus (ORFV) ORF120 protein interacting with G3BP1, which helps not only to explain the ability of ORFV to modulate the immune response through the positive regulation of NF-κB but also to show the mechanism by which the virus evades the host innate immune response.
口疮病毒（ORFV）是一种具有致瘤性的正痘病毒，具有动物源性，可引起绵羊、山羊和人类皮肤的增殖性病变。ORFV 携带的几种病毒蛋白，包括核因子-κB（NF-κB）抑制剂，在劫持宿主相关蛋白以逃避宿主固有免疫反应方面发挥重要作用。然而，在病毒复制和潜伏感染中具有未知功能的蛋白质的作用仍有待探索。在这里，我们提供的数据表明，ORF120，一种早期晚期 ORFV 编码的蛋白，在感染的早期阶段激活 NF-κB 途径，这意味着 ORFV 可能通过双相机制调节 NF-κB。一个双膜酵母双杂交系统和免疫共沉淀实验表明，ORF120 蛋白与 Ras-GTPase-activating protein（SH3 结构域）结合蛋白 1（G3BP1）相互作用。ORF120 蛋白的过表达可以有效地增加主要绵羊胎儿鼻甲（OFTu）和 HeLa 细胞中 G3BP1 的表达和 NF-κB-p65 的核转位。G3BP1 的敲低显著降低了 ORF120 诱导的 NF-κB 激活，表明 G3BP1 参与了 ORF120 诱导的 NF-κB 途径激活。双荧光素酶报告基因检测显示，ORF120 可以通过全长 G3BP1 或 G3BP1 结构域正向调节 NF-κB 途径。总之，我们首次证明 ORF120 蛋白通过与 G3BP1 相互作用，能够正向调节 NF-κB 信号通路，为 ORFV 发病机制提供了新的见解，并为抗病毒药物设计提供了理论依据。作为宿主固有反应的一部分，核因子-κB（NF-κB）途径通过调节先天免疫反应，在自然状态下发挥部分抗病毒作用。因此，NF-κB 途径可能是被广泛的病原体携带的免疫调节剂进行免疫逃避的最常被靶向的细胞内途径之一。包括痘病毒在内的各种病毒都携带几种蛋白，通过抑制细胞质事件为病毒复制做准备，从而启动 NF-κB 转录活性。然而，NF-κB 活性被假设在很大程度上促进了病毒的复制。我们的研究意义在于探索 ORF 病毒（ORFV）ORF120 蛋白与 G3BP1 相互作用诱导的 NF-κB 激活的机制，这不仅有助于解释 ORFV 通过 NF-κB 的正调控调节免疫反应的能力，还展示了病毒逃避宿主固有免疫反应的机制。
