与烟草曲茎病毒V2蛋白互作的本氏烟SKP1.1蛋白负调控病毒侵染

Nicotiana benthamiana SKP1.1 interacts with V2, of tobacco curly shoot virus and inhibits virus infection

中文关键词:双生病毒  烟草曲茎病毒  V2蛋白  沉默抑制子活性  SKP1.1蛋白  泛素化

keywords:geminivirus  tobacco curly shoot virus  V2 protein  RNA silencing suppressor activity  SKP1.1 protein  ubiquitination

中文摘要:为明确烟草曲茎病毒（tobacco curly shoot virus，TbCSV）V2蛋白在TbCSV侵染寄主过程中发挥的作用，基于前期以TbCSV V2蛋白为诱饵筛选到的与之互作的本氏烟Nicotiana benthamiana泛素连接酶E3复合体SKP1/CUL1/F-box（SCF）组分NbSKP1.1蛋白（GenBank登录号为AKQ53343.1），采用双分子荧光互补、荧光素酶互补成像和免疫共沉淀试验来验证V2蛋白与NbSKP1.1蛋白的互作，并通过实时荧光定量PCR技术检验NbSKP1.1基因的表达情况，采用激光共聚焦显微镜观察NbSKP1.1的亚细胞定位，通过根癌农杆菌Agrobacterium tumefaciens介导的蛋白瞬时表达系统和Western blot试验分析NbSKP1.1对V2蛋白的沉默抑制子活性和蛋白稳定性的影响。结果显示：TbCSV侵染可以显著上调NbSKP1.1基因的表达，通过烟草脆裂病毒（tobacco rattle virus，TRV）介导NbSKP1下调表达导致TbCSV侵染症状加重，病毒积累量显著上调，表明NbSKP1.1受到TbCSV侵染的诱导进而抑制病毒侵染。NbSKP1.1蛋白分布于细胞质及细胞核中，且TbCSV侵染或与V2蛋白共表达不影响其亚细胞定位。V2蛋白与NbSKP1.1蛋白瞬时共表达试验表明NbSKP1.1减弱了V2蛋白的沉默抑制子活性，但并不能直接导致V2蛋白降解，暗示NbSKP1.1对V2蛋白沉默抑制子活性的削弱可能是其抑制TbCSV侵染的一种机制。表明泛素化途径的寄主因子可以通过直接靶向TbCSV编码的病毒蛋白，干扰病毒蛋白的功能并抑制病毒侵染。

Abstract:To investigate the role of the tobacco curly shoot virus (TbCSV) V2 protein in infection, V2 was used to identify its interacting partner, SKP1.1 (GenBank number: AKQ53343.1), a component of the SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex in Nicotiana benthamianai. The interaction between V2 and NbSKP1.1 was confirmed through bimolecular fluorescence complementation, luciferase complementation imaging, and co-immunoprecipitation assays. The expression level of NbSKP1.1 was further examined by RT-qPCR, and its subcellular localization was determined using confocal laser scanning microscopy. In addition, the effects of NbSKP1.1 expression on the RNA silencing suppressor activity and protein stability of V2 were determined with Agrobacterium tumefaciens-mediated transient expression and western blot assays. The results showed that TbCSV infection significantly upregulated NbSKP1.1 expression. Silencing of NbSKP1 using tobacco rattle virus (TRV)-mediated RNA interference exacerbated TbCSV symptoms and increased viral accumulation, indicating that NbSKP1.1 was induced by TbCSV infection and played a role in restricting viral replication. Subcellular localization studies revealed that NbSKP1.1 was distributed in both the cytoplasm and the nucleus, and its localization was not altered by either TbCSV infection or co-expression with the V2 protein. In transient co-expression assays, NbSKP1.1 reduced the silencing suppressor activity of the V2 protein without causing its degradation, suggesting that the suppression of TbCSV infection by NbSKP1.1 may involve the attenuation of V2’s silencing suppressor activity. This study provides evidence that host factors in the ubiquitination pathway can target viral proteins encoded by TbCSV, disrupting their function and inhibiting viral infection.