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目的 NF-κB通路在宿主抗病毒免疫中发挥重要作用，痘病毒可通过编码多种免疫调节蛋白干预该通路的活化。本研究旨在探究山羊痘病毒（Goatpox virus,GTPV）编码的RING finger蛋白（简称GTPVRF）对NF-κB信号通路的调控作用。方法 通过激光共聚焦显微镜分析了GTPVRF的亚细胞定位；采用双荧光素酶报告系统、Western blot等方法分析了GTPVRF对NF-κB转录活性、p-IκBα降解、NF-κB-p65核移位及IKKα/IκBα磷酸化的影响；利用Co-IP结合质谱筛选GTPVRF的互作蛋白，并分析其互作的关键结构域。结果 GTPVRF定位于细胞质中；GTPVRF对NF-κB的转录活性具有显著的抑制作用（P<0.0001），且抑制作用随GTPVRF表达量的增加有逐步增强的趋势，其KILA-N与RING finger结构域在该抑制作用中均发挥重要作用；GTPVRF能够抑制p-IκBα降解和NF-κB-p65核移位，而不影响IKKα/IκBα的磷酸化水平；GTPVRF与NF-κB通路相关因子IKKα、IκBα、p-IκBα、p65不能发生互作，能够与质谱筛选的UBE2K、G3BP2和CUL4B蛋白互作，UBE2K和CUL4B主要与GTPVRF的RING finger结构域互作，而GTPVRF缺失任一结构域，都无法与G3BP2互作。结论 GTPVRF通过抑制p-IκBα降解与NF-κB-p65核移位来下调NF-κB信号通路，其KILA-N与RING finger结构域均与该调控作用相关，互作蛋白UBE2K、G3BP2、CUL4B可能协同参与其中。本研究首次明确了GTPVRF对NF-κB信号通路的抑制作用，并初步探究了其抑制机制，为阐明痘病毒编码的该类蛋白的功能奠定了基础。
Abstract：Objective The NF-κB pathway plays an important role in host antiviral immunity,and poxviruses can interfere with its activation by encoding various immunomodulatory proteins.This study aimed to investigate the regulatory effect of the RING finger protein encoded by goatpox virus(GTPV),designated GTPVRF,on the NF-κB signaling pathway.Methods The subcellular localization of GTPVRF was analyzed by confocal laser scanning microscopy.The effects of GTPVRF on NF-κB transcriptional activity,p-IκBα degradation,NF-κB-p65 nuclear translocation,and IKKα/IκBα phosphorylation were assessed using dual-luciferase reporter assays and Western blotting.Co-immunoprecipitation(Co-IP) combined with mass spectrometry was employed to screen for GTPVRF-interacting proteins and to identify the key domains involved in these interactions.Results GTPVRF was localized in the cytoplasm.GTPVRF significantly inhibited the transcriptional activity of NF-κB(p < 0.0001),and the inhibitory effect tended to increase with increasing GTPVRF expression levels.Both the KILA-N and RING finger domains played important roles in this inhibition.GTPVRF suppressed p-IκBα degradation and NF-κB-p65 nuclear translocation without affecting the phosphorylation levels of IKKα or IκBα.GTPVRF did not interact with the NF-κB pathway-related factors IKKα,IκBα,p-IκBα,or p65,but interacted with the mass spectrometry-screened proteins UBE2K,G3BP2,and CUL4B.UBE2K and CUL4B interacted primarily with the RING finger domain of GTPVRF,whereas GTPVRF was unable to interact with G3BP2 when either domain was deleted.Conclusion GTPVRF down-regulates the NF-κB signaling pathway by inhibiting p-IκBα degradation and NF-κB-p65 nuclear translocation.Both the KILA-N and RING finger domains are involved in this regulatory effect,and the interacting proteins UBE2K,G3BP2,and CUL4B may synergistically participate in this process.This study is the first to demonstrate the inhibitory effect of GTPVRF on the NF-κB signaling pathway and to preliminarily explore its underlying mechanism,thereby laying a foundation for elucidating the function of this class of proteins encoded by poxviruses.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.260085
中图分类号:S852.65
引用信息:
[1]冯秋媛,余翰维,杨云凤,等.山羊痘病毒RING finger蛋白对NF-κB信号通路的调控作用[J].病毒学报,2026,42(04):1128-1139.DOI:10.13242/j.cnki.bingduxuebao.260085.
基金信息:
国家自然科学基金项目（项目号：32260874）,题目：羊痘病毒RING finger蛋白对NF-κB通路的调控作用研究~~
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