Negative regulation of human antiviral RNAi by PACT

项目总结 由病毒产生的双链RNA加工的小干扰RNA (DsRNA)介导强大的抗病毒免疫，通常被称为抗病毒RNA干扰(RNAi)，在 真菌、植物和无脊椎动物。抗病毒RNAi通常是由切割病毒的DICER蛋白启动的 DsRNA转化为siRNA。ArgAert(AGO)蛋白招募DICER产生的siRNA并将其用作 识别和销毁具有匹配序列的病毒RNA转录本的序列指南。这场毁灭 靶病毒转录本的一部分是由AGO蛋白的切片器活性介导的。双链RNA结合蛋白 (DRBPs)还通过促进siRNA的产生或装载到AgO中而有助于抗病毒RNAi 蛋白质。 目前，RNAi是否在生理状态下介导哺乳动物的抗病毒免疫 条件仍在激烈的辩论中。尽管哺乳动物RNAi的抗病毒活性在 未分化的干细胞和新生小鼠，在分化的细胞和 发育完全的小鼠。DICER和AGO蛋白都是生产siRNA所需的事实 在分化的细胞中保持功能活跃表明存在一种调节机制， 在分化细胞中主动抑制哺乳动物RNAi的抗病毒活性。目前，如何 这种监管机制的作用在很大程度上仍不为人所知。由于哺乳动物的关键RNAi基因，如 因为编码DICER和Ago2的那些是miRNAs的生物发生或功能所必需的，这 调节必要的细胞分化和分裂，不可能敲除这些基因来研究 哺乳动物RNAi的抗病毒功能，而不会导致细胞生长停滞或致死。 通过人类DICER和Ago2的联合传递，Ago2是一种具有切片器活性的人类AGO蛋白， PI的实验室成功地在线虫体内重建了人类抗病毒RNAi。重要的是，抗病毒药物 在人类TRBP转基因的存在下，活性进一步增强，但似乎受到抑制 在人类契约转基因存在的情况下。TRBP和PACT都是dsRNA结合蛋白 共享相似的域结构。之前的体外研究已经证明，尽管TRBP 通过DICER PACT促进dsRNA的加工似乎抑制了dsRNA的DICER加工。这些 结合我们的发现，观察结果表明，TRBP和PACT相反地调节抗病毒 哺乳动物RNAi和PACT的活性可能主导调节，导致抑制抗病毒作用 生理条件下的活动。在这里，我们建议研究人类的负性调节 在线虫中通过PACT进行抗病毒RNAi。拟议中的研究结果可能不仅使我们能够 深入了解哺乳动物抗病毒RNAi在分化过程中的调控机制 这不仅有助于开发预防病毒感染的新型治疗策略，而且还有助于开发新的治疗策略。

PROJECT SUMMARY Small interfering RNAs (siRNAs) processed from virus-produced double-stranded RNA (dsRNA) mediate potent antiviral immunity, often termed as antiviral RNA interference (RNAi), in fungi, plants and invertebrates. Antiviral RNAi is often initiated by dicer proteins which chop viral dsRNAs into siRNAs. Argonaut (AGO) proteins recruit dicer-produced siRNAs and use them as sequence guide to identify and destroy viral RNA transcripts with matching sequence. The destruction of target viral transcripts is mediated by the slicer activity of AGO proteins. dsRNA binding proteins (DRBPs) also contribute to antiviral RNAi by facilitating siRNA production or loading into AGO proteins. Currently, whether RNAi mediates antiviral immunity in mammals under physiological conditions is still under hot debate. Although the antiviral activity of mammalian RNAi is active in non-differentiated stem cells and newborn mice it becomes undetectable in differentiated cells and fully developed mice. The fact that both dicer and the AGO protein required for siRNA production remain functionally active in differentiated cells suggest that there is a regulatory mechanism that actively suppresses the antiviral activity of mammalian RNAi in differentiated cells. Currently, how this regulatory mechanism works remains largely unknown. Since key mammalian RNAi genes, such as those encoding dicer and Ago2, are required for the biogenesis or function of miRNAs, which regulate essential cell differentiation and division, it is impossible to knock out these genes to study the antiviral function of mammalian RNAi without causing cell growth arrest or lethality. By co-delivery of human dicer and Ago2, one of the human Ago proteins with slicer activity, the PI’s lab successfully reconstituted human antiviral RNAi in C. elegans. Importantly, the antiviral activity is further enhanced in the presence of a human TRBP transgene but appears to be suppressed in the presence of a human PACT transgene. Both TRBP and PACT are dsRNA-binding proteins sharing similar domain structure. Previous in vitro studies have demonstrated that whereas TRBP facilitates the processing of dsRNA by dicer PACT seems to inhibit dicer processing of dsRNA. These observations together with our finding suggest that TRBP and PACT conversely regulate the antiviral activity of mammalian RNAi and PACT may dominate the regulation, leading to suppressed antiviral activity under physiological conditions. Here we propose to study the negative regulation of human antiviral RNAi by PACT in C. elegans. Findings from the proposed research may not only allow us to gain insight into the mechanism by which mammalian antiviral RNAi is regulated in differentiated cells but also facilitate the development of novel therapeutic strategies for viral infection prevention.