State Key Laboratory of Plant Genomics and National Center for Plant Gene Research (Beijing), Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China.
J Virol. 2011 Dec;85(24):13384-97. doi: 10.1128/JVI.05806-11. Epub 2011 Oct 12.
RNA silencing provides protection against RNA viruses by targeting both the helper virus and its satellite RNA (satRNA). Virus-derived small interfering RNAs (vsiRNAs) bound with Argonaute (AGO) proteins are presumed participants in the silencing process. Here, we show that a vsiRNA targeted to virus RNAs triggers the host RNA-dependent RNA polymerase 6 (RDR6)-mediated degradation of viral RNAs. We confirmed that satRNA-derived small interfering RNAs (satsiRNAs) could be associated with different AGO proteins in planta. The most frequently cloned satsiRNA, satsiR-12, was predicted to imperfectly match to Cucumber mosaic virus (CMV) RNAs in the upstream area of the 3' untranslated region (3' UTR). Moreover, an artificial satsiR-12 (asatsiR-12) mediated cleavage of a green fluorescent protein (GFP) sensor construct harboring the satsiR-12 target site. asatsiR-12 also mediated reduction of viral RNAs in 2b-deficient CMV (CMVΔ2b)-infected Nicotiana benthamiana. The reduction was not observed in CMVΔ2b-infected RDR6i plants, in which RDR6 was silenced. Following infection with 2b-containing CMV, the reduction in viral RNAs was not observed in plants of either genotype, indicating that the asatsiR-12-mediated reduction of viral RNAs in the presence of RDR6 was inhibited by the 2b protein. Our results suggest that satsiR-12 targeting the 3' UTR of CMV RNAs triggered RDR6-dependent antiviral silencing. Competition experiments with wild-type CMV RNAs and anti-satsiR-12 mutant RNA1 in the presence of 2b and satRNA demonstrate the inhibitory effect of the 2b protein on the satsiR-12-related degradation of CMV RNAs, revealing a substantial suppressor function of the 2b protein in native CMV infection. Our data provide evidence for the important biological functions of satsiRNAs in homeostatic interactions among the host, virus, and satRNA in the final outcome of viral infection.
RNA 沉默通过靶向辅助病毒及其卫星 RNA（satRNA）为 RNA 病毒提供保护。病毒衍生的小干扰 RNA（vsiRNA）与 Argonaute（AGO）蛋白结合，被认为是沉默过程的参与者。在这里，我们表明靶向病毒 RNA 的 vsiRNA 触发了宿主 RNA 依赖性 RNA 聚合酶 6（RDR6）介导的病毒 RNA 降解。我们证实 satRNA 衍生的小干扰 RNA（satsiRNAs）可以在植物体内与不同的 AGO 蛋白相关联。最常克隆的 satsiRNA，satsiR-12，被预测与黄瓜花叶病毒（CMV）RNAs 在 3'非翻译区（3'UTR）上游区域不完全匹配。此外，人工 satsiR-12（asatsiR-12）介导对含有 satsiR-12 靶位点的绿色荧光蛋白（GFP）传感器构建体的切割。asatsiR-12 还介导了 2b 缺失 CMV（CMVΔ2b）感染的 Nicotiana benthamiana 中病毒 RNA 的减少。在 RDR6 沉默的 CMVΔ2b 感染的 RDR6i 植物中未观察到这种减少。在用含有 2b 的 CMV 感染后，在两种基因型的植物中均未观察到病毒 RNA 的减少，表明在存在 RDR6 的情况下，asatsiR-12 介导的病毒 RNA 的减少被 2b 蛋白抑制。我们的结果表明，靶向 CMV RNA 3'UTR 的 satsiR-12 触发了 RDR6 依赖性抗病毒沉默。在 2b 和 satRNA 的存在下，用野生型 CMV RNA 和抗 satsiR-12 突变 RNA1 进行竞争实验，证明了 2b 蛋白对 satsiR-12 相关的 CMV RNA 降解的抑制作用，揭示了 2b 蛋白在天然 CMV 感染中的重要抑制功能。我们的数据为 satsiRNAs 在病毒感染的最终结果中宿主、病毒和 satRNA 之间的动态相互作用中的重要生物学功能提供了证据。
