A novel intronic circular RNA antagonizes influenza virus by absorbing a miRNA that degrades CREBBP and accelerating IFN-ß production

Virus-host interactions are complicated processes, and multiple cellular proteins have been reported to promote or inhibit viral replication through different mechanisms. Recent progress has implicated circular RNAs (circRNA) in cancer biology and progression; however, the role of circRNAs in viral infection remains largely unclear. Here, we detected 11,620 circRNAs in A549 cells and found that 411 of them were differentially expressed in influenza virus-infected A549 cells. We characterized a novel intronic circRNA, AIVR, that was upregulated in influenza virus-infected A549 cells, and found that silencing of AIVR significantly promoted influenza virus replication in A549 cells. We further found that AIVR predominantly localizes in the cytoplasm and works as a microRNA (miRNA) sponge. One of the miRNAs absorbed by AIVR binds the mRNA of CREBBP, which is an important component of the large nucleoprotein complex IFN-ß enhanceosome that accelerates IFN-ß production. AIVR-overexpression significantly increased the mRNA and protein levels of INF-ß in the influenza virus-infected A549 cells. Therefore, the upregulation of AIVR is a cellular antiviral strategy, with AIVR exerting its antiviral effect by absorbing miRNA and promoting the expression of CREBBP to facilitate IFN-ß production. Our study provides new insights into the roles of circRNAs in the cellular innate antiviral response. Overall design: To investigate the differential expression of circRNAs in influenza virus-infected cells, human alveolar epithelial cells (A549) were mock-infected or infected with an H9N2 avian influenza virus, A/chicken/Jiangsu/C4258/12 (H9N2) (H9N2 virus). At 12 h post-infection (p.i.), total RNA was extracted from the cells, and the circRNAs, miRNAs, and mRNAs were deep-sequenced and computationally analyzed.

病毒与宿主的相互作用是一类复杂的生物学过程，已有多项研究报道，多种宿主细胞蛋白可通过不同机制调控病毒复制，发挥促病毒或抗病毒作用。近年来的研究进展表明环状RNA（circular RNA, circRNA）参与了肿瘤的发生与发展进程，但circRNA在病毒感染过程中的具体作用仍未得到充分阐明。本研究在A549细胞中检测到11620条circRNA，其中411条在流感病毒感染的A549细胞中呈现差异表达。我们鉴定出一条新型内含子来源circRNA——AIVR，其在流感病毒感染的A549细胞中表达上调；实验发现，沉默AIVR可显著促进流感病毒在A549细胞中的复制。进一步研究表明，AIVR主要定位于细胞质，并作为microRNA（miRNA）海绵发挥功能。AIVR所吸附的其中一条miRNA可靶向结合CREBBP的mRNA；CREBBP是介导干扰素β（IFN-β）产生的大型核蛋白复合物IFN-β增强体的关键组成部分。过表达AIVR可显著提升流感病毒感染的A549细胞中IFN-β的mRNA及蛋白表达水平。综上，AIVR的表达上调是一种宿主细胞抗病毒策略：AIVR通过吸附miRNA、促进CREBBP的表达以加速IFN-β的产生，从而发挥抗病毒作用。本研究为阐明circRNA在宿主细胞天然抗病毒应答中的功能提供了全新视角。

实验设计：为探究流感病毒感染细胞后circRNA的差异表达情况，本研究将人肺泡上皮细胞A549分为两组，分别进行空白对照感染（mock感染）以及H9N2亚型禽流感病毒A/chicken/Jiangsu/C4258/12 (H9N2)（简称H9N2病毒）感染。于感染后12小时（h.p.i.）收集细胞并提取总RNA，随后对circRNA、miRNA及mRNA进行深度测序与生物信息学分析。