Eckenfelder Agathe, Ségéral Emmanuel, Pinzón Natalia, Ulveling Damien, Amadori Céline, Charpentier Marine, Nidelet Sabine, Concordet Jean-Paul, Zagury Jean-François, Paillart Jean-Christophe, Berlioz-Torrent Clarisse, Seitz Hervé, Emiliani Stéphane, Gallois-Montbrun Sarah
INSERM, U1016, Institut Cochin, Paris 75014, France.
CNRS, UMR8104, Paris 75014, France.
Nucleic Acids Res. 2017 Apr 20;45(7):4158-4173. doi: 10.1093/nar/gkw1289.
Argonaute (Ago) proteins associate with microRNAs (miRNAs) to form the core of the RNA-induced silencing complex (RISC) that mediates post-transcriptional gene silencing of target mRNAs. As key players in anti-viral defense, Ago proteins are thought to have the ability to interact with human immunodeficiency virus type 1 (HIV-1) RNA. However, the role of this interaction in regulating HIV-1 replication has been debated. Here, we used high throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP) to explore the interaction between Ago2 and HIV-1 RNA in infected cells. By only considering reads of 50 nucleotides length in our analysis, we identified more than 30 distinct binding sites for Ago2 along the viral RNA genome. Using reporter assays, we found four binding sites, located near splice donor sites, capable of repressing Luciferase gene expression in an Ago-dependent manner. Furthermore, inhibition of Ago1 and Ago2 levels in cells expressing HIV-1 led to an increase of viral multiply spliced transcripts and to a strong reduction in the extracellular CAp24 level. Depletion of Dicer did not affect these activities. Our results highlight a new role of Ago proteins in the control of multiply spliced HIV-1 transcript levels and viral production, independently of the miRNA pathway.
AGO蛋白与微小RNA（miRNA）结合，形成RNA诱导沉默复合体（RISC）的核心，该复合体介导靶mRNA的转录后基因沉默。作为抗病毒防御的关键参与者，AGO蛋白被认为具有与人免疫缺陷病毒1型（HIV-1）RNA相互作用的能力。然而，这种相互作用在调节HIV-1复制中的作用一直存在争议。在这里，我们使用交联免疫沉淀法分离RNA的高通量测序（HITS-CLIP）来探索感染细胞中AGO2与HIV-1 RNA之间的相互作用。在我们的分析中，仅考虑长度为50个核苷酸的读数，我们在病毒RNA基因组上鉴定出30多个不同的AGO2结合位点。使用报告基因检测，我们发现四个位于剪接供体位点附近的结合位点，能够以AGO依赖的方式抑制荧光素酶基因表达。此外，在表达HIV-1的细胞中抑制AGO1和AGO2水平会导致病毒多重剪接转录本增加，细胞外CAp24水平大幅降低。Dicer的缺失不影响这些活性。我们的结果突出了AGO蛋白在控制HIV-1多重剪接转录本水平和病毒产生方面的新作用，这一作用独立于miRNA途径。
