Plant Protection Institute, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary.
Institute for Veterinary Medical Research, Centre for Agricultural Research, Hungarian Academy of Sciences, Budapest, Hungary.
Sci Rep. 2017 Oct 18;7(1):13444. doi: 10.1038/s41598-017-13870-7.
The 2b protein of Cucumber mosaic virus has a role in nearly all steps of the viral cycle including cell-to-cell movement, symptom induction and suppression of antiviral RNA silencing. Previous studies demonstrated the presence of 2b protein in the nucleus and in cytoplasm as well. Phosphorylation site of 2b protein is conserved in all CMV isolates, including proposed constitute motifs for casein kinase II and cyclin-dependent kinase 2. To discern the impact of 2b protein phosphorylation, we created eight different mutants to mimic the non-phosporylated (serine to alanine) as well as the phosphorylated state (serine to aspartic acid) of the protein. We compared these mutants to the wild-type (Rs-CMV) virus in terms of symptom induction, gene silencing suppressor activity as well as in cellular localization. Here, in this study we confirmed the phosphorylation of 2b protein in vivo, both in infected N. benthamiana and in infiltrated patches. Mutants containing aspartic acid in the phosphorylation site accumulated only in the cytoplasm indicating that phosphorylated 2b protein could not enter the nucleus. We identified a conserved dual phosphorylation switch in CMV 2b protein, which equilibrates the shuttling of the 2b protein between the nucleus and the cytoplasm, and regulates the suppressor activity of the 2b protein.
黄瓜花叶病毒 2b 蛋白在病毒周期的几乎所有步骤中都发挥作用，包括细胞间运动、症状诱导和抗病毒 RNA 沉默抑制。先前的研究还表明 2b 蛋白存在于细胞核和细胞质中。黄瓜花叶病毒所有分离株的 2b 蛋白磷酸化位点都保守，包括推测的酪蛋白激酶 II 和细胞周期蛋白依赖性激酶 2 的构成基序。为了探究 2b 蛋白磷酸化的影响，我们创建了八个不同的突变体来模拟非磷酸化（丝氨酸突变为丙氨酸）和磷酸化状态（丝氨酸突变为天冬氨酸）的蛋白。我们将这些突变体与野生型（Rs-CMV）病毒进行比较，从症状诱导、基因沉默抑制活性和细胞定位等方面进行比较。在本研究中，我们证实了 2b 蛋白在体内被磷酸化，无论是在感染的 N. benthamiana 中还是在渗透斑点中。含有磷酸化位点天冬氨酸的突变体仅在细胞质中积累，表明磷酸化的 2b 蛋白不能进入细胞核。我们鉴定出黄瓜花叶病毒 2b 蛋白中存在一个保守的双磷酸化开关，它平衡了 2b 蛋白在细胞核和细胞质之间的穿梭，并调节 2b 蛋白的抑制活性。
