黑鲤RNF115在先天免疫中限制irf3 /7介导的抗病毒信号

IF 5.1 Q1 ENVIRONMENTAL SCIENCES

Water Biology and Security Pub Date : 2025-01-01 DOI:10.1016/j.watbs.2024.100310

Yixuan He , Qun Wang , Lili Xiao , Hui Wu , Jun Xiao , Jun Zou , Hao Feng

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引用次数: 0

摘要

真正有趣的新基因（RING）泛素E3连接酶家族包括大量成员，在抗病毒过程中起着至关重要的作用。RING finger protein 115 (RNF115)，也被称为BCA2、Rabring7或ZNF364，是一种新的RING结构域蛋白。本文从青鱼（Mylopharyngodon piceus）中克隆了RNF115同源基因并对其进行了鉴定。黑鱼RNF115开放阅读框包含933个核苷酸，编码310个氨基酸。RNF115的c端RING结构域在不同的同源物种中高度保守。免疫荧光分析显示了RNF115在鲤病毒（SVCV）春季病毒血症存在或不存在情况下的细胞质和细胞核分布。RNF115过表达会损害干扰素（IFN）和相关干扰素刺激基因（ISG） mRNA的表达，同时上调SVCV复制。体外敲除RNF115可增强宿主细胞的抗病毒信号。在体内敲除RNF115也增强了黑鱼的抗病毒能力。此外，双荧光素酶报告试验、斑块试验和qRT-PCR试验的结果表明，RNF115与IRF3/7共转染可降低IRF3/7诱导的IFN转录和抗病毒能力。通过免疫共沉淀法和免疫荧光法检测RNF115和IRF3/7之间的相关性。RNF115与IRF3/7共转染也降低了IRF3/7的蛋白水平，IRF3/7被MG132拯救。RNF115共转染条件下，IRF3/7的k48连锁泛素化增强，暗示了RNF115催化的泛素/蛋白酶体降解途径。RING结构域的半胱氨酸238和241是RNF115的主要酶活性位点，突变体C238/241A失去了大部分限制IRF3/7的能力。综上所述，黑鱼RNF115通过促进IRF3/7的泛素化和降解来抑制IRF3/7介导的IFN信号通路，从而揭示了IFN信号通路的调控机制。

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Black carp RNF115 restricts IRF3/7-mediated antiviral signaling in innate immunity

The Really Interesting New Gene (RING) ubiquitin E3 ligase family comprises a large number of members and plays a crucial role in the antiviral process. RING finger protein 115 (RNF115), also known as BCA2, Rabring7, or ZNF364, is a novel RING domain protein. In this paper, we cloned the RNF115 homologue from black carp (Mylopharyngodon piceus) and characterized it. The open reading frame of black carp RNF115 contains 933 nucleotides and encodes 310 amino acids. The C-terminal RING domain of RNF115 is highly conserved among various homologous species. Immunofluorescence assays revealed the cytoplasmic and nuclear distribution of RNF115 in the presence or absence of spring viremia of carp virus (SVCV) infection. Overexpression of RNF115 impaired interferon (IFN) and the related interferon-stimulated gene (ISG) mRNA expression, while upregulating SVCV replication. Ex vivo knockdown of RNF115 offered the host cells enhanced antiviral signaling. In vivo knockdown of RNF115 also strengthened black carp's antiviral capacity. Additionally, the results of a dual-luciferase reporter assay, plaque assay, and qRT-PCR assay demonstrated that co-transfection of RNF115 with IRF3/7 reduced IRF3/7-induced IFN transcription and antiviral ability. The association between RNF115 and IRF3/7 was detected by co-immunoprecipitation and immunofluorescence assays. Co-transfection of RNF115 with IRF3/7 also reduced the protein levels of IRF3/7, which were rescued by MG132. The enhanced K48-linked ubiquitination of IRF3/7 under the condition of RNF115 co-transfection implied the ubiquitin/proteasome degradation pathway catalyzed by RNF115. Cysteine 238 and 241 in the RING domain are the main enzyme active sites for RNF115, and the mutant C238/241A lost most of its ability to restrict IRF3/7. In conclusion, black carp RNF115 dampens IRF3/7-mediated IFN signaling through facilitating the ubiquitination and degradation of IRF3/7, which sheds light on the regulation of IFN signaling.

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Water Biology and Security

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4.10

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