Negative regulation of human antiviral RNAi by PACT

PACT 对人类抗病毒 RNAi 的负调控

基本信息

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
项目类别：
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

项目摘要

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.

项目概要由病毒产生的双链 RNA 加工而成的小干扰 RNA (siRNA) (dsRNA) 介导有效的抗病毒免疫，通常称为抗病毒 RNA 干扰 (RNAi)，真菌、植物和无脊椎动物的抗病毒RNAi通常是由切碎机蛋白启动的，切碎机蛋白会斩断病毒。 dsRNA 转化为 siRNA。Argonaut (AGO) 蛋白招募 dicer 产生的 siRNA 并将其用作序列指南用于识别和破坏具有匹配序列的病毒RNA转录本。目标病毒转录物的表达是由 AGO 蛋白的切片活性介导的。 (DRBP) 还可以通过促进 siRNA 的产生或加载到 AGO 中来促进抗病毒 RNAi 蛋白质。目前，RNAi是否在生理条件下介导哺乳动物的抗病毒免疫？尽管哺乳动物RNAi的抗病毒活性在某些情况下仍处于激烈争论中。未分化干细胞和新生小鼠在分化细胞和完全发育的小鼠的事实是，dicer 和 AGO 蛋白都是 siRNA 生产所必需的。在分化细胞中保持功能活性表明存在一种调节机制目前，如何积极抑制分化细胞中哺乳动物 RNAi 的抗病毒活性。由于关键的哺乳动物 RNAi 基因，这种调节机制的作用仍然未知。如那些编码 dicer 和 Ago2 的基因，是 miRNA 的生物发生或功能所必需的，调节重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，而不引起细胞生长停滞或致死。通过人类切丁机和 Ago2（具有切片机活性的人类 Ago 蛋白之一）的共同递送， PI 的实验室成功地在秀丽隐杆线虫中重建了人类抗病毒 RNAi。人类 TRBP 转基因存在时活性进一步增强，但似乎受到抑制 TRBP 和 PACT 都是 dsRNA 结合蛋白。先前的体外研究表明，TRBP 具有相似的结构域。促进 dicer 对 dsRNA 的加工 PACT 似乎抑制 dsRNA 的 dicer 加工。观察结果与我们的发现一起表明 TRBP 和 PACT 反向调节抗病毒作用哺乳动物 RNAi 和 PACT 的活性可能主导调节，导致抗病毒作用受到抑制在这里我们建议研究人类生理条件下的负调节。线虫中 PACT 的抗病毒 RNAi 拟议研究的结果可能不仅使我们能够深入了解哺乳动物抗病毒 RNAi 在分化过程中的调节机制细胞还促进了预防病毒感染的新治疗策略的开发。