College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou, China.
J Virol. 2014 May;88(10):5228-41. doi: 10.1128/JVI.00284-14. Epub 2014 Mar 5.
The Cucumber Mosaic Virus (CMV) 2b protein is an RNA-silencing suppressor that plays roles in CMV accumulation and virulence. The 2b proteins of subgroup IA CMV strains partition between the nucleus and cytoplasm, but the biological significance of this is uncertain. We fused an additional nuclear localization signal (NLS) to the 2b protein of subgroup IA strain Fny-CMV to create 2b-NLS and tested its effects on subcellular distribution, silencing, and virulence. The additional NLS enhanced 2b protein nuclear and nucleolar accumulation, but nuclear and nucleolar enrichment correlated with markedly diminished silencing suppressor activity in patch assays and abolished 2b protein-mediated disruption of microRNA activity in transgenic Arabidopsis. Nucleus/nucleolus-localized 2b protein possesses at least some ability to inhibit antiviral silencing, but this was not sufficient to prevent recovery from disease in younger, developing leaves in Arabidopsis. However, enhanced nuclear and nucleolar accumulation of 2b increased virulence and accelerated symptom appearance in older leaves. Experiments with Arabidopsis lines carrying mutant Dicer-like alleles demonstrated that compromised suppressor activity explained the diminished ability of 2b-NLS to enhance virus accumulation. Remarkably, the increased virulence that 2b-NLS engendered was unrelated to effects on microRNA- or short interfering RNA-regulated host functions. Thus, although nucleus- and nucleolus-localized 2b protein is less efficient at silencing suppression than cytoplasm-localized 2b, it enhances CMV virulence. We propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.
In this work, the main finding is that nucleus/nucleolus-localized 2b protein is strongly associated with CMV virulence, which is independent of its effect on small RNA pathways. Moreover, this work supports the contention that the silencing suppressor activity of CMV 2b protein is predominantly exerted by that portion of the 2b protein residing in the cytoplasm. Thus, we propose that partitioning of the 2b protein between the cytoplasmic and nuclear/nucleolar compartments allows CMV to regulate the balance between virus accumulation and damage to the host, presumably to maximize the benefit for the virus.
黄瓜花叶病毒（CMV）2b 蛋白是一种 RNA 沉默抑制子，在 CMV 积累和毒力中起作用。亚组 IA CMV 株的 2b 蛋白在核和细胞质之间分配，但这一生物学意义尚不确定。我们将亚组 IA 株 Fny-CMV 的 2b 蛋白融合了一个额外的核定位信号（NLS），以创建 2b-NLS，并测试其对亚细胞分布、沉默和毒力的影响。额外的 NLS 增强了 2b 蛋白的核和核仁积累，但核和核仁的富集与明显降低的斑点测定中的沉默抑制活性相关，并消除了 2b 蛋白介导的转基因拟南芥中 microRNA 活性的破坏。核/核仁定位的 2b 蛋白至少具有抑制抗病毒沉默的能力，但这不足以防止年轻、发育中的拟南芥叶片中的疾病恢复。然而，2b 蛋白的核和核仁积累增强增加了病毒的毒力并加速了老叶的症状出现。携带突变 Dicer-like 等位基因的拟南芥系的实验表明，抑制活性的降低解释了 2b-NLS 增强病毒积累能力的降低。值得注意的是，2b-NLS 引起的毒力增加与 microRNA 或短干扰 RNA 调节的宿主功能无关。因此，尽管核定位的 2b 蛋白比细胞质定位的 2b 蛋白在沉默抑制方面效率更低，但它增强了 CMV 的毒力。我们提出，2b 蛋白在细胞质和核/核仁隔室之间的分配允许 CMV 调节病毒积累和宿主损伤之间的平衡，可能是为了使病毒的利益最大化。
在这项工作中，主要发现是核/核仁定位的 2b 蛋白与 CMV 的毒力密切相关，这与其对小 RNA 途径的影响无关。此外，这项工作支持这样的观点，即 CMV 2b 蛋白的沉默抑制活性主要由该蛋白在细胞质中的部分发挥。因此，我们提出，2b 蛋白在细胞质和核/核仁隔室之间的分配允许 CMV 调节病毒积累和宿主损伤之间的平衡，可能是为了使病毒的利益最大化。
