Department of Plant Sciences, University of Cambridge, Cambridge, United Kingdom.
J Virol. 2024 Sep 17;98(9):e0099324. doi: 10.1128/jvi.00993-24. Epub 2024 Aug 20.
The cucumber mosaic virus (CMV) 2b protein is a potent counter-defense factor and symptom determinant that inhibits antiviral silencing by titrating short double-stranded RNAs. Expression of the CMV subgroup IA strain Fny-CMV 2b protein in transgenic plants disrupts microRNA-mediated cleavage of host mRNAs by binding Argonaute 1 (AGO1), leading to symptom-like phenotypes. This also triggers AGO2-mediated antiviral resistance and resistance to CMV's aphid vectors. However, in authentic viral infections, the Fny-CMV 1a protein modulates 2b-AGO1 interactions, inhibiting induction of AGO2-mediated virus resistance and aphid resistance. Contrastingly, 2b proteins encoded by the subgroup II strain LS-CMV and the recently discovered subgroup IA strain Ho-CMV induce no symptoms. Confocal laser scanning microscopy, bimolecular fluorescence complementation, and co-immunoprecipitation showed that Fny-CMV and Ho-CMV 2b proteins interact with Fny-CMV and LS-CMV 1a proteins, while the CMV-LS 2b protein cannot. However, Fny-CMV, Ho-CMV, and LS-CMV 2b proteins, all interacted with AGO1, but while AGO1-Fny2b complexes occurred in the nucleus and cytoplasm, corresponding AGO1-2b complexes for LS-CMV and Ho-CMV accumulated almost exclusively in nuclei. transcript accumulation was used to assess the inhibition of AGO1-mediated mRNA degradation. Fny-CMV 2b induced a fivefold increase in accumulation, but LS-CMV and Ho-CMV 2b proteins induced only twofold increases. Thus, these 2b proteins bind AGO1 but are less effective at inhibiting AGO1 activity. We conclude that the intracellular localization of 2b-AGO1 complexes influences the degree to which a 2b protein inhibits microRNA-mediated host mRNA degradation and that cytoplasmic AGO1 has the strongest influence on miRNA-mediated cellular mRNA turnover.
The cucumber mosaic virus (CMV) 2b protein was among the first discovered viral suppressors of RNA silencing. It has additional pro-viral functions through effects on plant defensive signaling pathways mediated by salicylic acid and jasmonic acid, the abscisic acid pathway and virus-induced drought resistance, and on host plant interactions with insect vectors. Many of these effects occur due to interaction with the important host RNA silencing component Argonaute 1 (AGO1). It was thought that only 2b proteins of "severe" CMV strains interacted with AGO1 and inhibited its microRNA-mediated "slicing" of cellular mRNAs and that the lack of interaction with AGO1 explained the moderate symptoms typically seen in plants infected with mild CMV strains. Our work overthrows this paradigm by showing that mild strain CMV 2b proteins can interact with AGO1, but their localization prevents them from interacting with AGO1 molecules present in the infected cell cytoplasm.
黄瓜花叶病毒（CMV）2b 蛋白是一种有效的反防御因子和症状决定因子，通过滴定短双链 RNA 来抑制抗病毒沉默。转 CMV 亚组 IA 株 Fny-CMV 2b 蛋白的表达通过结合 Argonaute 1（AGO1）破坏 microRNA 介导的宿主 mRNA 切割，导致类似症状的表型。这也触发了 AGO2 介导的抗病毒抗性和对 CMV 的蚜虫载体的抗性。然而，在真正的病毒感染中，Fny-CMV 1a 蛋白调节 2b-AGO1 相互作用，抑制 AGO2 介导的病毒抗性和蚜虫抗性的诱导。相比之下，由亚组 II 株 LS-CMV 和最近发现的亚组 IA 株 Ho-CMV 编码的 2b 蛋白不会引起症状。共焦激光扫描显微镜、双分子荧光互补和共免疫沉淀显示，Fny-CMV 和 Ho-CMV 2b 蛋白与 Fny-CMV 和 LS-CMV 1a 蛋白相互作用，而 CMV-LS 2b 蛋白不能。然而，Fny-CMV、Ho-CMV 和 LS-CMV 2b 蛋白均与 AGO1 相互作用，但 AGO1-Fny2b 复合物存在于细胞核和细胞质中，而相应的 AGO1-2b 复合物对于 LS-CMV 和 Ho-CMV 几乎仅在细胞核中积累。使用 积累来评估 AGO1 介导的 mRNA 降解的抑制。Fny-CMV 2b 诱导 积累增加了五倍，但 LS-CMV 和 Ho-CMV 2b 蛋白仅诱导两倍增加。因此，这些 2b 蛋白结合 AGO1，但在抑制 AGO1 活性方面效果较差。我们得出结论，2b-AGO1 复合物的细胞内定位影响 2b 蛋白抑制 microRNA 介导的宿主 mRNA 降解的程度，并且细胞质 AGO1 对 miRNA 介导的细胞 mRNA 周转具有最强的影响。
黄瓜花叶病毒（CMV）2b 蛋白是最早发现的抑制 RNA 沉默的病毒抑制剂之一。它通过影响水杨酸和茉莉酸、脱落酸途径和病毒诱导的干旱抗性介导的植物防御信号通路，以及影响宿主植物与昆虫载体的相互作用，具有额外的促病毒功能。这些作用中的许多是由于与重要的宿主 RNA 沉默成分 Argonaute 1（AGO1）相互作用而产生的。人们认为，只有“严重”CMV 株系的 2b 蛋白与 AGO1 相互作用并抑制其 microRNA 介导的细胞 mRNA 的“切割”，而与 AGO1 缺乏相互作用解释了感染轻度 CMV 株系的植物中通常看到的中度症状。我们的工作推翻了这一范例，表明轻度株系 CMV 2b 蛋白可以与 AGO1 相互作用，但它们的定位阻止了它们与感染细胞细胞质中存在的 AGO1 分子相互作用。
