Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b's efficient suppression of RNA silencing.
Importin/exportin-mediated nucleocytoplasmic shuttling of cucumber mosaic virus 2b protein is required for 2b's efficient suppression of RNA silencing.
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DOI：
10.1371/journal.ppat.1010267
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发表时间：
2022-01
期刊：
影响因子：
6.7
通讯作者：
Masuta C
中科院分区：
文献类型：
--
作者：
Kim H;Shimura H;Sueda K;Masuta C
黄瓜花叶病毒（CMV）的2b蛋白（2b）是一种RNA沉默抑制因子（RSS），是CMV的主要致病决定因子。2b定位于细胞核和细胞质中，其核输入由两个核定位信号（NLS）决定;预计载体蛋白（输入蛋白[IMPα]）参与2b的核转运。细胞质2bs通过结合小RNA和AGO蛋白在抑制RNA沉默中发挥作用。一个假定的核输出信号（内斯）基序也被发现在2b，但尚未被证明功能。在这里，我们确定了一个富含亮氨酸的基序在2b的C-末端的一半作为内斯。然后，我们发现NES缺陷的2b在细胞核中大量积累并失去了RSS活性，这表明从细胞核输出的2b可以发挥RSS的作用。虽然两个丝氨酸残基（S40和S42）被磷酸化，我们还发现，一个额外的磷酸化位点（S28）单独可以影响2b的核定位和RSS活性。S28位的丙氨酸取代削弱了IMPα介导的2b的核/核仁定位，与野生型2b相比，RSS活性甚至更强。在亚细胞分级分离试验中，在细胞核中检测到磷酸化的2bs，并且比较野生型2b和内斯突变体之间的核磷酸化-2b的积累水平，显示细胞核中磷酸化的内斯突变体的水平大大降低，这表明2bs在细胞核中被去磷酸化，并且可能以非磷酸化的形式易位到细胞质中。这些结果表明，2b操纵其核质转运，就好像它在RNA沉默途径中追踪其靶标，小RNA和AGO一样。我们推断2b的有效RSS活性是由磷酸化和去磷酸化的平衡维持的，磷酸化和去磷酸化与输入/输出蛋白介导的细胞核和细胞质之间的穿梭有关。RNA沉默是一种由小RNA（small RNA，sRNA）介导的序列特异性机制，是植物抗病毒的主要反应。Argonaute 1（AGO 1）是一种RNA沉默效应蛋白，与sRNA结合以降解病毒RNA或沉默细胞质中涉及生物胁迫的基因;这些功能通过其在细胞核和细胞质之间的易位来促进。为了在宿主RNA沉默中存活，黄瓜花叶病毒（CMV）编码RNA沉默抑制子（RSS），2b蛋白（2b）。2b通过结合细胞核和细胞质中的sRNA或AGO在抑制RNA沉默中起多种作用。然而，一些先前的报告表明，2b发挥其RSS活动时，主要是在细胞质中。在这里，我们发现，在位置28的丝氨酸残基是磷酸化的，其磷酸化负调控RSS活性的2b通过调节2b的核/核仁定位。此外，我们还发现2bs在细胞核中被脱磷酸化，并以输出蛋白依赖的方式输出到细胞质中。由于2b在细胞核和细胞质之间的转运类似于AGO，2b似乎在细胞中起作用，就好像它在操纵其核质转运以追踪RNA沉默途径中的靶标。
The 2b protein (2b) of cucumber mosaic virus (CMV), an RNA-silencing suppressor (RSS), is a major pathogenicity determinant of CMV. 2b is localized in the nucleus and cytoplasm, and its nuclear import is determined by two nuclear localization signals (NLSs); a carrier protein (importin [IMPα]) is predicted to be involved in 2b’s nuclear transport. Cytoplasmic 2bs play a role in suppression of RNA silencing by binding to small RNAs and AGO proteins. A putative nuclear export signal (NES) motif was also found in 2b, but has not been proved to function. Here, we identified a leucine-rich motif in 2b’s C-terminal half as an NES. We then showed that NES-deficient 2b accumulated abundantly in the nucleus and lost its RSS activity, suggesting that 2b exported from the nucleus can play a role as an RSS. Although two serine residues (S40 and S42) were previously found to be phosphorylated, we also found that an additional phosphorylation site (S28) alone can affect 2b’s nuclear localization and RSS activity. Alanine substitution at S28 impaired the IMPα-mediated nuclear/nucleolar localization of 2b, and RSS activity was even stronger compared to wild-type 2b. In a subcellular fractionation assay, phosphorylated 2bs were detected in the nucleus, and comparison of the accumulation levels of nuclear phospho-2b between wild-type 2b and the NES mutant showed a greatly reduced level of the phosphorylated NES mutant in the nucleus, suggesting that 2bs are dephosphorylated in the nucleus and may be translocated to the cytoplasm in a nonphosphorylated form. These results suggest that 2b manipulates its nucleocytoplasmic transport as if it tracks down its targets, small RNAs and AGOs, in the RNA silencing pathway. We infer that 2b’s efficient RSS activity is maintained by a balance of phosphorylation and dephosphorylation, which are coupled to importin/exportin-mediated shuttling between the nucleus and cytoplasm. RNA silencing, a small RNA (sRNA)-mediated sequence-specific mechanism, is a primary antiviral response of host plants against viral infection. Argonaute 1 (AGO1), an effector protein of RNA silencing, associates with sRNAs to degrade viral RNAs or silence the genes involved in biotic stresses in cytoplasm; such functions are promoted by its translocation between the nucleus and cytoplasm. To survive host RNA silencing, cucumber mosaic virus (CMV) encodes an RNA silencing suppressor (RSS), 2b protein (2b). 2b plays multiple roles in suppressing RNA silencing by binding to either sRNAs or AGOs, in the nucleus and the cytoplasm. However, several previous reports suggest that 2b exerts its RSS activity mainly when it is in the cytoplasm. Here, we found that the serine residue at position 28 is phosphorylated, and its phosphorylation negatively regulated the RSS activity of 2b by regulating 2b’s nuclear/nucleolar localization. Furthermore, we also found that 2bs were dephosphorylated in the nucleus and exported to the cytoplasm in an exportin-dependent manner. Because 2b’s transport between the nucleus and cytoplasm is similar to that of AGOs, 2b seems to work in cells as if it is manipulating its nucleocytoplasmic trafficking to track down its targets in the RNA silencing pathway.
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