Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity
Cucumber Mosaic Virus 2b Protein Subcellular Targets and Interactions: Their Significance to RNA Silencing Suppressor Activity
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DOI：
10.1094/mpmi-23-3-0294
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发表时间：
2010-03-01
影响因子：
3.5
通讯作者：
Canto, Tomas
中科院分区：
文献类型：
--
作者：
Gonzalez, Inmaculada;Martinez, Llucia;Canto, Tomas
黄瓜花叶病毒（CMV）2b蛋白的RNA沉默抑制剂活性已被不同地归因于其核靶向、其与Argonaute 1（AGO 1）的相互作用和抑制、或其在体外结合小RNA的能力。此外，番茄无精病毒的2b直系同源物在体外形成聚集体并结合RNA。我们进一步研究了CMV 2b蛋白沉默抑制剂活性与其亚细胞分布、体内蛋白-蛋白相互作用以及与小干扰RNA的体外相互作用之间的关系。为了做到这一点，我们用荧光标记物标记了蛋白质，并表明它保留了抑制活性。我们发现2b蛋白存在于核仁中，并且它在体内与AGO 1和AGO 4相互作用。使用电池的突变体，我们表明，假定的核定位信号和磷酸化基序的2b蛋白不需要自我相互作用或与AGO蛋白的相互作用。这些相互作用或核仁靶向的发生都不足以提供局部沉默抑制活性。相反，2b蛋白结合小RNA的能力似乎是沉默抑制子功能不可或缺的。
The RNA silencing suppressor activity of the 2b protein of Cucumber mosaic virus (CMV) has been variously attributed to its nuclear targeting, its interaction with and inhibition of Argonaute 1 (AGO1), or its ability to bind small RNAs in vitro. In addition, the 2b ortholog of Tomato aspermy virus forms aggregates and binds RNAs in vitro. We have further studied the relationships between CMV 2b protein silencing suppressor activity and its subcellular distribution, protein-protein interactions in vivo, and interactions with small interfering RNAs in vitro. To do this, we tagged the protein with fluorescent markers and showed that it retained suppressor activity. We showed that the 2b protein is present in the nucleolus and that it self-interacts and interacts with AGO1 and AGO4 in vivo. Using a battery of mutants, we showed that the putative nuclear localization signals and phosphorylation motif of the 2b protein are not required for self-interaction or for interaction with AGO proteins. The occurrence of neither of these interactions or of nucleolar targeting was sufficient to provide local silencing-suppression activity. In contrast, the ability of the 2b protein to bind small RNAs appears to be indispensable for silencing suppressor function.