近日，国际著名杂志The Plant Cell 在线刊登了中国科学院微生物研究所研究人员的最新研究成果“Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein”，文章中，研究者揭示了他们在病毒沉默抑制子研究方面获得的进展。
在植物和病毒相互作用的长期过程中，两者之间存在着防御、反防御的复杂斗争关系。寄主的RNA沉默机制和病毒编码沉默抑制子就是防御-反防御斗争的典型。由黄瓜花叶病毒（CMV）编码的基因沉默抑制子2b蛋白是早在1998年就被发现的抑制子之一，CMV 2b尽管是一个只有111个氨基酸的小蛋白，却具有非常复杂的生化特性和亚细胞定位特性，包括与RNA沉默途径效应复合物的关键因子AGO蛋白互作，与短/长的dsRNA结合，靶向细胞核和核仁；CMV 2b还具有复杂的抑制子活性，包括抑制转录后基因沉默（PTGS）和依赖RNA的DNA甲基化（RdDM），抑制沉默的细胞间传递，抑制AGO蛋白的剪切活性，抑制寄主的RDR6-依赖的抗病毒途径等。但是关于2b的这111个氨基酸究竟是如何决定的2b的这些生化特性，又是如何影响2b复杂的抑制子活性，其机制目前还研究得很不透彻。
在国家自然科学基金重点项目，国家重点基础研究发展计划（973计划）等项目资助下，中国科学院微生物研究所植物基因组学国家重点实验室郭惠珊课题组，通过分段克隆构建了CMV 2b蛋白的11个突变体，详细分析了2b各种突变体的亚细胞定位、RNA结合特性、与AGO间的互作、对PTGS和RdDM途径的抑制作用等。研究发现，2b的dsRNA的结合活性以及2b的核定位所需要的结构域与2b与AGO蛋白的互作所需要的结构域是分开的。他们鉴定出2b的核仁定位信号（NoLS）位于2b结合dsRNA的结构域（dsRBD）之间；2b和AGO蛋白的体外互作不需要dsRBD。有趣的是，SD2b与AGO蛋白在体内的互作需要这个有功能的NoLS，并且2b和AGO的体内互作改变2b和AGO的细胞核定位。通过体内抑制子活性检测发现，2b蛋白通过捕获siRNAs、长的dsRNAs前体dsRNA，而不是通过作用于AGO蛋白，来实现其抑制子活性。也就是说，体内抑制PTGS和RdDM都不依赖于2b-AGO的相互作用。（生物谷Bioon.com）
doi:10.1105/tpc.111.092718
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Suppression of Arabidopsis ARGONAUTE1-Mediated Slicing, Transgene-Induced RNA Silencing, and DNA Methylation by Distinct Domains of the Cucumber mosaic virus 2b Protein[W]
Cheng-Guo Duana,1, Yuan-Yuan Fanga,1, Bang-Jun Zhoua,b, Jian-Hua Zhaoa,b, Wei-Na Houa, Hui Zhua,b, Shou-Wei Dingc and Hui-Shan Guoa,2
Unique among the known plant and animal viral suppressors of RNA silencing, the 2b protein interacts directly with both small interfering RNA (siRNA) and ARGONAUTE1 (AGO1) and AGO4 proteins and is targeted to the nucleolus. However, it is largely unknown which regions of the 111-residue 2b protein determine these biochemical properties and how they contribute to its diverse silencing suppressor activities. Here, we identified a functional nucleolar localization signal encoded within the 61–amino acid N-terminal double-stranded RNA (dsRNA) binding domain (dsRBD) that exhibited high affinity for short and long dsRNA. However, physical interaction of 2b with AGOs required an essential 33-residue region C-terminal to the dsRBD and was sufficient to inhibit the in vitro AGO1 Slicer activity independently of its dsRNA binding activities. Furthermore, the direct 2b–AGO interaction was not essential for the 2b suppression of posttranscriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) in vivo. Lastly, we found that the 2b–AGO interactions in vivo also required the nucleolar targeting of 2b and had the potential to redistribute both the 2b and AGO proteins in nucleus. These findings together suggest that 2b may suppress PTGS and RdDM in vivo by binding and sequestering siRNA and the long dsRNA precursor in a process that is facilitated by its interactions with AGOs in the nucleolus.
