Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs.
Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs.
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DOI：
10.1371/journal.ppat.1000996
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发表时间：
2010-07-15
期刊：
影响因子：
6.7
通讯作者：
Burgyán J
中科院分区：
文献类型：
--
作者：
Giner A;Lakatos L;García-Chapa M;López-Moya JJ;Burgyán J
RNA沉默是一种进化保守的序列特异性基因失活系统，在高等植物和昆虫中也起抗病毒机制的作用。为了克服抗病毒RNA沉默，病毒表达沉默抑制蛋白。这些病毒蛋白可以靶向沉默机制中的一个或多个关键点。在这里，我们发现在甘薯轻度斑疹病毒（SPMMV， Ipomovirus属，potyvirus科的类型成员）中，沉默抑制因子的作用由P1蛋白（所有已知的potyvirus中最大的丝氨酸蛋白酶）发挥，尽管在其基因组中存在HC-Pro蛋白，该蛋白在potyvirus中充当抑制因子。通过体内研究，我们已经证明SPMMV P1抑制si/ mirna编程的RISC活性。正如我们通过免疫沉淀所显示的，RISC活性的抑制是通过将P1结合到成熟的高分子量RISC上发生的。我们的研究结果表明，P1靶向RISC的催化单元Argonaute1 (AGO1)，并且抑制/结合活性位于P1的n端一半。在这一区域发现了三个与后生动物和植物中保守的ago结合线性肽基序相似的WG/GW基序。定点突变证明这三个基序对于结合和抑制AGO1功能都是绝对必需的。与目前分析的其他病毒沉默抑制子不同，P1抑制现有的和新形成的含有RISC复合物的AGO1。因此，P1代表了一种新的RNA沉默抑制机制。P1介导的沉默抑制的分子基础的发现可能有助于更好地了解含有RISC的多蛋白的功能和组装。RNA沉默是一种进化保守的序列特异性基因失活系统，在高等植物和昆虫中也是一种主要的抗病毒机制。病毒rna被dicer样蛋白加工成小干扰（si） rna，从而触发rna诱导的沉默复合体（RISC）组装。然后装载siRNA的RISC灭活同源病毒RNA。然而，病毒沉默抑制因子进化为对抗RNA沉默，靶向沉默机制中的一个或多个关键点。本研究表明，在甘薯轻度斑斑病毒中，沉默抑制因子的作用由P1蛋白发挥，它通过靶向Argonaute 1 （AGO1）抑制si/ mirna负载的RISC起作用。我们通过免疫沉淀和体外结合实验证实，P1与装载AGO1的小RNA之间的相互作用是特异性和直接的。抑制活性定位于P1的n端部分，其中包含三个WG/GW基序，类似于后生动物和植物中保守的ago结合线性肽基序。定点突变证明这三个基序在结合和抑制AGO1功能中都是必不可少的。P1蛋白是迄今为止发现的唯一抑制活性RISC的沉默抑制因子，这是首次证明WG/GW蛋白对RNA沉默具有负面影响。
RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as an antiviral mechanism in higher plants and insects. To overcome antiviral RNA silencing, viruses express silencing-suppressor proteins. These viral proteins can target one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus (SPMMV, type member of the Ipomovirus genus, family Potyviridae), the role of silencing suppressor is played by the P1 protein (the largest serine protease among all known potyvirids) despite the presence in its genome of an HC-Pro protein, which, in potyviruses, acts as the suppressor. Using in vivo studies we have demonstrated that SPMMV P1 inhibits si/miRNA-programmed RISC activity. Inhibition of RISC activity occurs by binding P1 to mature high molecular weight RISC, as we have shown by immunoprecipitation. Our results revealed that P1 targets Argonaute1 (AGO1), the catalytic unit of RISC, and that suppressor/binding activities are localized at the N-terminal half of P1. In this region three WG/GW motifs were found resembling the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are absolutely required for both binding and suppression of AGO1 function. In contrast to other viral silencing suppressors analyzed so far P1 inhibits both existing and de novo formed AGO1 containing RISC complexes. Thus P1 represents a novel RNA silencing suppressor mechanism. The discovery of the molecular bases of P1 mediated silencing suppression may help to get better insight into the function and assembly of the poorly explored multiprotein containing RISC. RNA silencing is an evolutionarily conserved sequence-specific gene-inactivation system that also functions as a major antiviral mechanism in higher plants and insects. Viral RNAs are processed by Dicer-like proteins into small interfering (si) RNAs, which trigger the RNA-induced silencing complex (RISC) assembly. Then siRNA loaded RISC inactivates cognate viral RNA. However, viral silencing suppressors evolved to counteract with RNA silencing targeting one or more key points in the silencing machinery. Here we show that in Sweet potato mild mottle virus, the role of silencing suppressor is played by P1 protein and it works by inhibiting si/miRNA-loaded RISC through targeting Argonaute 1 (AGO1). We confirmed using immunoprecipitation and in vitro binding assays that the interaction between P1 and small RNA loaded AGO1 is specific and direct. The suppression activity mapped to the N-terminal part of P1 containing three WG/GW motifs that resemble the AGO-binding linear peptide motif conserved in metazoans and plants. Site-directed mutagenesis proved that these three motifs are essential for both binding and suppression of AGO1 function. P1 protein is the only silencing suppressor identified so far that inhibits active RISC and this is the first demonstration of a WG/GW protein having negative effect on RNA silencing.
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DOI：
10.1073/pnas.95.22.13079
发表时间：
1998-10-27
影响因子：
11.1
作者：
Anandalakshmi, R;Pruss, GJ;Vance, VB
通讯作者：
Vance, VB
影响因子：
11.4
作者：
Bouche, Nicolas;Lauressergues, Dominique;Vaucheret, Herve
通讯作者：
Vaucheret, Herve
影响因子：
11.6
作者：
Diaz-Pendon, Juan A.;Li, Feng;Ding, Shou-Wei
通讯作者：
Ding, Shou-Wei
影响因子：
2.7
作者：
Adams, MJ;Antoniw, JF;Fauquet, CM
通讯作者：
Fauquet, CM
影响因子：
64.5
作者：
Buhler, Marc;Verdel, André;Moazed, Danesh
通讯作者：
Moazed, Danesh