Long noncoding RNA AVAN promotes antiviral innate immunity by interacting with TRIM25 and enhancing the transcription of FOXO3a

Chengcai Lai; Lihui Liu; Qinghua Liu; Sijie Cheng; Keyu Wang; Lingna Zhao; Min Xia; Cheng Wang; Hongjing Gu; Yueqiang Duan; Zhongpeng Zhao; Lili Zhang; Ziyang Liu; Jianjun Luo; Jianxun Song; Penghui Yang; Runsheng Chen; Xiliang Wang

proliferation, genomic imprinting, and stem cell self-renewal (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) . lncRNAs are also 48 reportedly involved in innate and adaptive immune responses, such as immune cell 49 proliferation and cytokine production (11) (12) (13) (14) . Previous studies have shown that lncRNAs 50 exert their roles in regulating chromatin accessibility, mRNA stability and protein activity by 51 interacting with chromatin DNA, mRNAs or proteins (15-18). With the discovery and 52 characterization of an increasing number of lncRNAs, several mechanisms of these lncRNAs 53 in virus-host interactions have been elucidated (19, 20) , however, many others remain 54 uncharacterized. recovery-stage counterparts (FC>2, p<0.05) and subjected them to a cluster analysis ( Fig 1B) . 100 To investigate the association between lncRNAs and IAV infection, in silico analysis 101 identified 26 novel lncRNAs candidates with notably aberrant expression (Fig 1C) . To 102 confirm the expression of these lncRNAs, we separated the neutrophils and monocytes from 103 healthy volunteers and infected the cells with influenza virus A/Beijing/501/2009 (abbreviated 104 as IAV-BJ501 or BJ501) for 12 h and found that XLOC_040025 (AVAN) was most 105 significantly up-regulated after IAV-BJ501 challenge (Fig 1D, E) . We also infected human 106 alveolar epithelial cells (A549) with IAV-BJ501 and measured the expression of the 26 lncRNAs and found that AVAN was most strongly up-regulated after IAV-BJ501 infection (Fig 108 1F ). 109 110 AVAN is preferentially up-regulated following virus infection 111 We then performed qRT-PCR to quantify AVAN expression in the RNA-Seq samples and 112 found that the results were consistent with the RNA-Seq data ( Fig S2A) . Thus, AVAN was 113 chosen for subsequent investigation. To further confirm the expression of AVAN in 114 IAV-infected patients, we collected blood samples from 63 additional IAV-positive patients 115 and measured the expression of AVAN, which was significantly up-regulated in the neutrophils 116 of these patients (Fig 2A; Table S1 ). Similarly, AVAN was up-regulated in A549 cells after 117 infection with multiple sub-strains of IAV ( Fig 2B) and several other RNA viruses, including 118 Sendai virus (SeV) and respiratory syncytial virus (RSV) ， but not the DNA virus, adenovirus 119 (ADV) (Fig S2B) . In addition, Poly (I:C), IFN-β also could significantly stimulate the 120 expression of AVAN ( Fig S2B) . Moreover, AVAN expression was significantly up-regulated 121 upon IAV-BJ501 challenge in a time-and dose-dependent manner in A549 cells (Fig 2C, D) . 122 Absolute copy number measurement showed that AVAN was expressed at relatively low 123 levels without infection, with 11 or 14 transcript copies in per neutrophil or A549 cell 124 respectively ( Figure S2 ). Additionally, AVAN expression was also significantly up-regulated in 125 a time-and dose-dependent manner in the human promyelocytic leukemia cell line (HL60), 126 which is often a substitute for neutrophils, upon IAV-BJ501 challenge (Fig S2B, C) . 127 Subcellular fractionation followed by qRT-PCR in IAV-BJ501-infected A549 cells revealed labeling-probes was weaken (Fig S2E) . AVAN exhibits no protein-coding potential according 135 to ORF finder (33) and the coding potential calculator (34) (Fig S2F) . Only one transcript 136 variant (approximately 500 nt) of AVAN was found in A549 cells, and it was found to be 137 up-regulated upon IAV-BJ501 infection usi