Negative regulation of human antiviral RNAi by PACT

PACT 对人类抗病毒 RNAi 的负调控

• 批准号: 10667112
• 负责人: Rui Lu
• 金额: $ 7.5万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-23 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10667112
• 关键词: 2019-nCoV; Affinity; Antiviral Agents; Binding; Biogenesis; Caenorhabditis elegans; Cell Differentiation process; Cell division; Cell physiology; DNA Sequence Alteration; Defect; Development; Double-Stranded RNA; FBXW7 gene; Gene Expression; Genes; Genome; Human; Human Activities; In Vitro; Infection; Infection prevention; Invertebrates; Mammals; Mediating; MicroRNAs; Molecular; Mus; NCOA6 gene; Nematoda; Newborn Infant; Physiological; Plants; Process; Production; Proteins; RNA Interference; RNA Viruses; Regulation; Research; Small Interfering RNA; Structure; Therapeutic; Transcript; Transgenes; Viral; Viral Load result; Viral Physiology; Virus; Virus Diseases; Virus Replication; Work; antiviral immunity; cell growth; ds RNA-Binding Proteins; fungus; improved; in vivo; insight; knockout gene; novel therapeutic intervention; reconstitution; recruit; stem cells; treatment strategy; viral RNA

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.

项目摘要 由病毒产生的双链RNA处理的小干扰RNA（siRNA） （DSRNA）介导潜在的抗病毒免疫组织化学，通常称为抗病毒RNA干扰（RNAI） 真菌，植物和无脊椎动物。抗病毒RNAi通常是由dicer蛋白引发的，这些蛋白会切碎病毒 dsrnas进入sirnas。 Argonaut（AGO）蛋白质招募DICER生产的siRNA，并将其用作 鉴定和破坏具有匹配序列的病毒RNA转录本的序列指南。破坏 靶病毒转录物的含量是由AGO蛋白的切片活性介导的。 dsRNA结合蛋白 （DRBP）还通过支持siRNA的产生或加载到AGO来促进抗病毒RNAi 蛋白质。 目前，RNAi是否在生理学下介导哺乳动物的抗病毒免疫史地 条件仍在炎热的辩论中。尽管哺乳动物RNAi的抗病毒活性在活性 非差异的干细胞和新生小鼠在分化细胞和 完全发育的小鼠。 siRNA产生所需的dicer和AGO蛋白的事实 在分化细胞中保持功能活跃，表明有一种调节机制 积极抑制分化细胞中哺乳动物RNAi的抗病毒活性。目前，如何 这种监管机制的工作仍然很大程度上是未知的。由于关键的哺乳动物RNAi基因，因此 作为编码DICER和AGO2的人，需要miRNA的生物发生或功能，这 调节必需的细胞分化和分裂，不可能淘汰这些基因来研究 哺乳动物RNAi的抗病毒功能，而不会引起细胞生长停滞或致死性。 由人dicer和Ago2的共同传递，Ago2是一个具有切片活性的人类AGO蛋白， PI的实验室成功地重新建立了秀丽隐杆线虫中的人类抗病毒RNAi。重要的是，抗病毒 在存在人类TRBP转换的情况下，活动进一步增强，但似乎被抑制 在人类公立转变的情况下。 TRBP和PACT都是DSRNA结合蛋白 共享类似的域结构。以前的体外研究表明，trbp 通过DICER PACT促进DSRNA的加工似乎抑制了DSRNA的DICER处理。这些 观察结果与我们的发现表明TRBP和PACT相反调节抗病毒 哺乳动物RNAi和Pact的活性可能主导调节，导致抗病毒毒 生理条件下的活动。在这里，我们建议研究人类的负面调节 秀丽隐杆线虫中的抗病毒RNAi。拟议研究的发现可能不仅使我们能够 洞察哺乳动物抗病毒RNAi在分化中调节的机制 细胞但还促进了预防病毒感染的新型治疗策略的发展。