LincRNA‐EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction
2022; Springer Nature; Volume: 23; Issue: 5 Linguagem: Inglês
10.15252/embr.202153937
ISSN1469-3178
AutoresJingfei Zhu, Shengchuan Chen, Liqiong Sun, Siying Liu, Xue Bai, Dapei Li, Fan Zhang, Zigang Qiao, Liang Li, Haiping Yao, Yu Xia, Ping Xu, Xiaohui Jiang, Zhengrong Chen, Yongdong Yan, Feng Ma,
Tópico(s)RNA Research and Splicing
ResumoArticle21 March 2022Open Access Transparent process LincRNA-EPS impairs host antiviral immunity by antagonizing viral RNA–PKR interaction Jingfei Zhu Jingfei Zhu CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Data curation, Investigation, Writing - original draft Search for more papers by this author Shengchuan Chen Shengchuan Chen CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Contribution: Investigation Search for more papers by this author Li-Qiong Sun Li-Qiong Sun Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing, China Contribution: Investigation Search for more papers by this author Siying Liu Siying Liu CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Data curation, Software, Investigation Search for more papers by this author Xue Bai Xue Bai CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Dapei Li Dapei Li CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Fan Zhang Fan Zhang CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Zigang Qiao Zigang Qiao CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Liang Li Liang Li CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Haiping Yao Haiping Yao CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Yu Xia Yu Xia Suzhou Center for Disease Control and Prevention, Suzhou, China Contribution: Resources Search for more papers by this author Ping Xu Ping Xu Department of Laboratory Medicine, The Fifth People's Hospital of Suzhou, Suzhou, China Contribution: Resources, Funding acquisition Search for more papers by this author Xiaohui Jiang Xiaohui Jiang Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China Contribution: Investigation Search for more papers by this author Zhengrong Chen Zhengrong Chen Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China Contribution: Resources Search for more papers by this author Yongdong Yan Corresponding Author Yongdong Yan [email protected] Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China Contribution: Conceptualization, Supervision Search for more papers by this author Feng Ma Corresponding Author Feng Ma [email protected] orcid.org/0000-0002-2975-0118 CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Jingfei Zhu Jingfei Zhu CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Data curation, Investigation, Writing - original draft Search for more papers by this author Shengchuan Chen Shengchuan Chen CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Contribution: Investigation Search for more papers by this author Li-Qiong Sun Li-Qiong Sun Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing, China Contribution: Investigation Search for more papers by this author Siying Liu Siying Liu CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Data curation, Software, Investigation Search for more papers by this author Xue Bai Xue Bai CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Dapei Li Dapei Li CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Fan Zhang Fan Zhang CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Zigang Qiao Zigang Qiao CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Liang Li Liang Li CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Haiping Yao Haiping Yao CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Contribution: Investigation Search for more papers by this author Yu Xia Yu Xia Suzhou Center for Disease Control and Prevention, Suzhou, China Contribution: Resources Search for more papers by this author Ping Xu Ping Xu Department of Laboratory Medicine, The Fifth People's Hospital of Suzhou, Suzhou, China Contribution: Resources, Funding acquisition Search for more papers by this author Xiaohui Jiang Xiaohui Jiang Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China Contribution: Investigation Search for more papers by this author Zhengrong Chen Zhengrong Chen Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China Contribution: Resources Search for more papers by this author Yongdong Yan Corresponding Author Yongdong Yan [email protected] Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China Contribution: Conceptualization, Supervision Search for more papers by this author Feng Ma Corresponding Author Feng Ma [email protected] orcid.org/0000-0002-2975-0118 CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China Suzhou Institute of Systems Medicine, Suzhou, China Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Jingfei Zhu1,2,†, Shengchuan Chen1,2,3,†, Li-Qiong Sun4,†, Siying Liu1,2, Xue Bai1,2, Dapei Li1,2, Fan Zhang1,2, Zigang Qiao1,2, Liang Li1,2, Haiping Yao1,2, Yu Xia5, Ping Xu6, Xiaohui Jiang7, Zhengrong Chen7, Yongdong Yan *,7 and Feng Ma *,1,2,3 1CAMS Key Laboratory of Synthetic Biology Regulatory Elements, Institute of Systems Medicine, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China 2Suzhou Institute of Systems Medicine, Suzhou, China 3Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China 4Institute of Chinese Medicinal Materials, Nanjing Agricultural University, Nanjing, China 5Suzhou Center for Disease Control and Prevention, Suzhou, China 6Department of Laboratory Medicine, The Fifth People's Hospital of Suzhou, Suzhou, China 7Department of Pulmonary Medicine, Children's Hospital of Soochow University, Suzhou, China † These authors contributed equally to this work *Corresponding author. Tel: +86 512 8069 8303; E-mail: [email protected] *Corresponding author (lead contact). Tel: +86 512 6287 3679; Fax: +86 512 6287 3779; E-mail: [email protected] EMBO Reports (2022)23:e53937https://doi.org/10.15252/embr.202153937 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract LincRNA-EPS is an important regulator in inflammation. However, the role of lincRNA-EPS in the host response against viral infection is unexplored. Here, we show that lincRNA-EPS is downregulated in macrophages infected with different viruses including VSV, SeV, and HSV-1. Overexpression of lincRNA-EPS facilitates viral infection, while deficiency of lincRNA-EPS protects the host against viral infection in vitro and in vivo. LincRNA-EPS−/− macrophages show elevated expression of antiviral interferon-stimulated genes (ISGs) such as Mx1, Oas2, and Ifit2 at both basal and inducible levels. However, IFN-β, the key upstream inducer of these ISGs, is downregulated in lincRNA-EPS−/− macrophages compared with control cells. RNA pulldown and mass spectrometry results indicate that lincRNA-EPS binds to PKR and antagonizes the viral RNA–PKR interaction. PKR activates STAT1 and induces antiviral ISGs independent of IFN-I induction. LincRNA-EPS inhibits PKR-STAT1-ISGs signaling and thus facilitates viral infection. Our study outlines an alternative antiviral pathway, with downregulation of lincRNA-EPS promoting the induction of PKR-STAT1-dependent ISGs, and reveals a potential therapeutic target for viral infectious diseases. Synopsis The lncRNA lincRNA-EPS binds to PKR in the cytoplasm of macrophages, antagonizes viral RNA-PKR interaction, and thus impairs PKR-STAT1-dependent host antiviral immunity. LincRNA-EPS is downregulated by host antiviral immunity Knockout of lincRNA-EPS protects host against viral infection LincRNA-EPS antagonizes viral RNA-PKR interaction LincRNA-EPS impairs PKR-STAT1-dependent induction of antiviral ISGs Introduction The innate immune system is the first line of defense against pathogenic microbes including numerous life-threatening viruses. During viral infection, viral RNA or DNA are recognized by pattern recognition receptors (PRRs) to initiate complex signal transduction pathways, which ultimately leads to the induction of type I interferon (IFN-I) and proinflammatory cytokines (Akira et al, 2006; Goubau et al, 2013). Retinoic acid-inducible gene I (RIG-I) is one of the key cytosolic RNA sensors that recognize viral RNA from invaded viruses (Yoneyama et al, 2004). Viral RNA-triggered activation of RIG-I results in the phosphorylation of TBK1 and IRF3, which activates transcription factors including NF-κB, AP-1, and IRF3/7 to induce proinflammatory cytokines and IFN-I (Honda et al, 2006). IFN-I including IFN-α and IFN-β further trigger the phosphorylation of transcription factors STAT1 and STAT2 via the JAK-STAT pathway to induce multiple IFN-stimulated genes (ISGs) such as MX1, OAS2, ISG15, and IFIT2, which synergistically inhibit viral infection by targeting almost all the steps of viral life cycles (Sadler & Williams, 2008; Schneider et al, 2014). However, overproduction of IFN-I and hyperactivation of IFN-α/β receptor (IFNAR) downstream signaling lead to autoimmune diseases including systemic lupus erythematosus (SLE) and Aicardi-Goutières syndrome (AGS) (Chaussabel et al, 2008; Crow & Manel, 2015). The innate immune signaling cascades during viral infection are precisely controlled by various negative feedback pathways, which protect the host by efficiently clearing invaded pathogens but avoiding autoimmunity (Wang et al, 2017b; Vierbuchen & Fitzgerald, 2021). In addition to RIG-I, PKR is well-known as a nucleic acids receptor of viral dsRNA produced from replication or transcription intermediates of a wide range of virus families such as negative-strand RNA viruses VSV and Sendai virus (SeV) (Stojdl et al, 2000; Dauber & Wolff, 2009), positive-strand RNA virus Hepatitis C virus (Targett-Adams et al, 2008), and DNA virus Herpes simplex virus type 1 (HSV-1) (Jacquemont & Roizman, 1975). PKR is autophosphorylated and activated following sensing viral dsRNA and then phosphorylates eukaryotic translation initiation factor 2 on its α subunit (eIF2α) to inhibit translation initiation of viral proteins (Dalet et al, 2015). Cellular non-coding RNAs, such as the inverted Alu repeats (IRAlus) elements located in the 3′-untranslated regions (3′-UTR) and mitochondrial RNAs (mtRNAs) formed intermolecular dsRNA, also activate PKR through direct interaction to regulate cellular proliferation or metabolism (Kim et al, 2014, 2018). In addition to be activated by the cellular RNA, PKR is suppressed during binding with the cytoplasmic circular RNAs (circRNAs) that tend to form 16–26 bp imperfect RNA duplexes, and viral infection relieves this inhibition following circRNAs degradation by RNase L to activate PKR activity (Liu et al, 2019). However, it is unclear whether any linear long non-coding RNAs (lncRNAs) regulate PKR-dependent antiviral immunity. PKR is also required for the activation of MAPK and IKK complex, as well as the transcriptional activities of IRF1 and STAT1 (Wong et al, 1997; Garcia et al, 2006; Gal-Ben-Ari et al, 2018). As an IFN-I inducible gene, PKR directly associates with STAT1 via the PKR dsRNA-binding domain (Tanaka & Samuel, 1994). PKR is essential for the phosphorylation of STAT1 on Ser727 and Tyr701 under the response to IFN-γ and LPS (Ramana et al, 2000; Lee et al, 2005; Karehed et al, 2007). LncRNA GRASLND acts to inhibit IFN-γ signaling by binding PKR and in turn inhibiting STAT1 activity during chondrogenesis (Huynh et al, 2020). However, it is unexplored whether any PKR-interacted lncRNAs contribute to the regulation of the IFN-I-JAK-STAT pathway during host innate immunity against viral infection. The long intergenic noncoding RNA lincRNA-EPS was initially reported to inhibit apoptosis during erythroid cell differentiation in part through repressing the expression of the proapoptotic gene Pycard (Hu et al, 2011). During inflammatory responses, lincRNA-EPS is tightly regulated in macrophages to control the expression of immune response genes (IRGs) at the transcription level by interacting with hnRNPL (Atianand et al, 2016). The deficiency of lincRNA-EPS enhances inflammatory response and leads to death in the endotoxin-shock mouse model while protecting the host from Listeria monocytogenes infection (Atianand et al, 2016; Agliano et al, 2019). Furthermore, knockdown of lincRNA-EPS promoted autophagy in Bacillus Calmette-Guérin (BCG)-infected RAW264.7 macrophages by activating the JNK/MAPK pathway, and the downregulation of lincRNA-EPS was shown in active pulmonary tuberculosis (PTB) patients (Ke et al, 2020). Our previous study has described that lincRNA-EPS alleviates severe acute pancreatitis by suppressing HMGB1-triggered inflammation in pancreatic macrophages (Chen et al, 2021). Although lincRNA-EPS has been well identified as a key immunoregulatory lncRNA that restrains inflammatory responses, the role of lincRNA-EPS in antiviral immunity was not yet studied. In this study, we have found that the expression of lincRNA-EPS is also precisely controlled during host antiviral immunity in IFN-I- and NF-κB-dependent manners. Downregulation of lincRNA-EPS protects the host against viral infection in vitro and in vivo. LincRNA-EPS binds to PKR and thus negatively regulates PKR-STAT1-dependent host antiviral immunity by antagonizing the interaction between viral RNA and PKR. Our study has indicated that lincRNA-EPS plays an important role in modulating host innate antiviral immunity. Results Downregulation of lincRNA-EPS by host antiviral immunity To check the impact of viral infection on lincRNA-EPS expression, mouse bone marrow-derived macrophages (BMDMs) were infected with several viruses including RNA viruses VSV and SeV, and DNA virus HSV-1. All three viruses dramatically suppressed the expression of lincRNA-EPS (Fig 1A). Transfection of viral RNA mimics polyI:C and viral DNA mimics polydA:dT also led to the downregulation of lincRNA-EPS expression in BMDMs (Fig 1B). To further confirm whether host antiviral immunity regulates lincRNA-EPS expression, BMDMs were treated with IFN-α and IFN-β, the key cytokines which are always induced during viral infection. Both IFN-Is significantly suppressed lincRNA-EPS expression at the very early stage (2 h post stimulation) and at the concentration as low as 20 U/ml (Fig 1C–F). Consistently, higher expression of lincRNA-EPS was detected in the Ifnar1−/− BMDMs than the WT BMDMs during cells were transfected with polyI:C or infected with SeV (Fig 1G and H). However, lincRNA-EPS expression was still downregulated in the polyI:C-transfected and SeV-infected Ifnar1−/− BMDMs (Fig 1G and H). In addition, there were no differences of lincRNA-EPS expression between Ifnar1−/− and WT BMDMs when the cells were infected with WSN and VSV (Fig EV1A), which suggest an IFN-I-independent pathway also contributed to suppress lincRNA-EPS expression during viral infection. Hence, we used inhibitors to specifically target NF-κB, p38, ERK, and JNK signaling pathways (Fig EV1B), which are also activated during viral infection. Inhibition of NF-κB rather than the MAPK pathways significantly reversed the downregulation of lincRNA-EPS triggered by VSV infection (Figs 1I and EV1C). Figure 1. Downregulation of lincRNA-EPS by host antiviral immunity A. RT–qPCR analysis of lincRNA-EPS transcripts in the BMDMs infected with VSV (MOI 1), SeV (MOI 1), or HSV-1 (MOI 5) for indicated time points. B. RT–qPCR analysis of lincRNA-EPS transcripts in the BMDMs transfected with 1 μg/ml low molecular weight (LMW), high molecular weight (HMW) polyI:C, and polydA:dT for 6 h. C, D. RT–qPCR analysis of lincRNA-EPS transcripts in the BMDMs stimulated with 500 U/ml IFN-α for different time points (C) or different concentrations for 2 h (D). E, F. RT–qPCR analysis of lincRNA-EPS transcripts in the BMDMs stimulated with 500 U/ml IFN-β for different time points (E) or different concentrations for 2 h (F). G, H. RT–qPCR analysis of lincRNA-EPS transcripts in the WT and Ifnar1−/− BMDMs transfected with 1 μg/ml polyI:C (LMW) for 6 h (G) and infected with SeV (MOI 1) for indicated time points (H). I. BMDMs were pretreated with NF-κB inhibitor BAY11-7082 (1 μM) for 1 h and infected with VSV (MOI 1) for 6 h. The percentage of lincRNA-EPS transcripts downregulation in the VSV-infected group compared with the Mock group was calculated. J. Cell nucleus and cytoplasm were separated from untreated (Mock) and VSV-infected iBMMs, RNA was extracted for RT–qPCR analysis and compared with Mock group. Data information: Data of (A-J) are shown as the mean ± s.d. from three independent experiments. *P < 0.05 and **P < 0.01 by unpaired Student's t-test. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. The signaling pathways modulating lincRNA-EPS expression RT–qPCR analysis of lincRNA-EPS transcripts in the WT and Ifnar1−/− BMDMs infected with WSN (MOI 1) and VSV (MOI 1) for 6 h. BMDMs were pretreated with NF-κB inhibitor BAY11-7082 (1 μM), p38 MAPK inhibitor SB203580 (5 μM), ERK inhibitor PD98059 (50 μM) and JNK inhibitor SP600125 (20 μM) for 1 h prior and infected with VSV (MOI 1) for 6 h. The phosphorylation and total proteins were detected by Western blot. GAPDH and α-tubulin were shown as loading control. The percentage of lincRNA-EPS transcripts downregulated in the VSV-infected group compared with Mock group were calculated. Copy-number analysis of lincRNA-EPS transcripts in several cell types by RT–qPCR. Standard curve was generated using in vitro transcribed RNA molecule of lincRNA-EPS as template. Data information: Data of (A, C) are shown as the mean ± s.d. from three independent experiments, **P < 0.01 and ns, not significant by unpaired Student's t-test. Data of (B, D) are representative results from three independent experiments. Download figure Download PowerPoint We further checked the cellular localization and abundance of lincRNA-EPS in macrophages. Similar to the lncRNA Neat1 which mainly localizes in the nucleus (Clemson et al, 2009), about 71% of lincRNA-EPS were detected in the nucleus. A more robust reduction of cytoplasmic lincRNA-EPS was observed than the nuclear lincRNA-EPS, although both nuclear and cytoplasmic lincRNA-EPS were significantly suppressed during VSV infection (Fig 1J). About 63, 23, and 8 copies per cell of lincRNA-EPS were detected in the RAW264.7, immortalized BMDMs (iBMMs), and BMDMs, respectively (Fig EV1D), indicating higher abundance of lincRNA-EPS in the macrophage cell lines than the primary macrophages. Taken together, lincRNA-EPS was downregulated during host immunity against viral infection, in IFN-I- and NF-κB-dependent manners. LincRNA-EPS facilitates viral infection in macrophages Next, we sought to investigate the function of lincRNA-EPS during host innate immunity against viral infection by using macrophage cell lines. We stably overexpressed lincRNA-EPS in iBMMs (Fig 2A). More VSV, SeV, and HSV-1 infections were detected in the lincRNA-EPS-overexpressed iBMMs than the control cells (Fig 2B–D), which suggested that lincRNA-EPS broadly facilitated viral infection. Meanwhile, we used a pair of sgRNAs to efficiently knock down the lincRNA-EPS expression in RAW264.7 cells, a macrophage cell line that is susceptible to multiple viruses and expresses high level lincRNA-EPS (Fig 2E). Less VSV-GFP-infected cells were observed in the lincRNA-EPS knockdown cells than the control cells (Fig 2F). Consistently, less VSV titer, fewer SeV and HSV-1 viral genes were detected in the lincRNA-EPS knockdown cells than the control cells (Fig 2G–I). These results indicated that lincRNA-EPS facilitated viral infection in macrophages. Figure 2. LincRNA-EPS facilitates viral infection in macrophages A. RT–qPCR analysis of lincRNA-EPS transcripts in the lincRNA-EPS-stably-overexpressed iBMMs (lincRNA-EPS) and corresponding control cells (EV). B. LincRNA-EPS iBMMs were infected with VSV (MOI 1) for 8 h, VSV titers were measured by plaque assay. C, D. The EV and lincRNA-EPS iBMMs were infected with SeV (MOI 1) (C) or HSV-1 (MOI 5) (D) for 12 h. The viral RNA was measured by RT–qPCR. E. The transcripts level of lincRNA-EPS in control (sgCtrl) and lincRNA-EPS knockdown (sglincRNA-EPS) RAW264.7 cells were measured by RT–qPCR. F, G. sgCtrl and sglincRNA-EPS RAW264.7 cells were infected with VSV-GFP (MOI 0.1) for 6 h or infected with VSV (MOI 0.1) for 8 h. The fluorescence of GFP was checked by microscope (F) and the viral titer of VSV from the cell supernatant was measured by plaque assay (G). H, I. sgCtrl and sglincRNA-EPS RAW264.7 cells were infected with SeV (MOI 1) for 8 h (H) and HSV-1 (MOI 5) for 12 h (I). The viral RNA was measured by RT–qPCR. Data information: Data of (A–E) and (G–I) are shown as the mean ± s.d. from three independent experiments. *P < 0.05 and **P < 0.01 by unpaired Student's t-test. Data of (F) are representative images from three independent experiments, scale bar, 100 μm. Download figure Download PowerPoint Knockout of lincRNA-EPS enhances host antiviral ability To further confirm the function of lincRNA-EPS in facilitating viral infection, we immortalized the lincRNA-EPS−/− and WT BMDMs (Fig 3A). Much less GFP-positive cells were observed in the VSV-GFP-infected lincRNA-EPS−/− iBMMs than the WT cells (Fig 3B). Plaque assay results also showed that lincRNA-EPS−/− iBMMs were more resistant to VSV than the WT iBMMs (Fig 3C). Similarly, as the phenotypes observed in the lincRNA-EPS knockdown RAW264.7 cells, less SeV and HSV-1 viral genes were detected in the lincRNA-EPS−/− iBMMs than the WT cells (Fig 3D and E). However, rescued expression of lincRNA-EPS in the lincRNA-EPS−/− iBMMs facilitated VSV and SeV infection (Fig EV2A–C), which indicated that knockout of lincRNA-EPS rather than the off-target effects of the sgRNAs regulated the host susceptibility to viral infection. Next, we isolated the primary peritoneal macrophages (PMs) to further validate the function of lincRNA-EPS. LincRNA-EPS−/− PMs showed more resistant to the VSV than the WT cells (Fig 3F). In addition to the in vitro experiments, we further challenged the WT and lincRNA-EPS−/− mice with VSV to investigate the function of lincRNA-EPS in vivo. The lincRNA-EPS−/− mice exhibited a much higher survival rate than the WT group during the mice infected with a lethal dose of VSV intravenously (Fig 3G). Consistently, alleviated liver and lung injuries including fewer inflammatory cells infiltration, less liver fibrotic septa, less alveolar wall thickening, and less alveolar cavity atrophy were observed in the lincRNA-EPS−/− mice than the WT group during the mice infected with a sublethal dose of VSV (Fig 3H). Moreover, less viral load in serum, livers, and lungs was detected in the VSV-infected lincRNA-EPS−/− mice comparing to the WT mice (Fig 3I). Interestingly, lower serum IFN-β was detected in the VSV-infected lincRNA-EPS−/− mice than in the WT group (Fig 3J). Together, these results demonstrated that lincRNA-EPS facilitated viral infection such as VSV infection in vitro and in vivo, likely in an IFN-I-independent manner. Figure 3. Knockout of lincRNA-EPS enhances host antiviral ability A, B. WT and lincRNA-EPS−/− iBMMs were infected with VSV-GFP (MOI 0.1) for 6 h, and the lincRNA-EPS transcripts were measured by RT–qPCR (A), and the fluorescence of GFP were checked by microscope (B). C. Cell supernatant from VSV-infected WT and lincRNA-EPS−/− iBMMs (MOI 0.1, 8 h) were collected and the viral titer were measured by plaque assay. D, E. WT and lincRNA-EPS−/− iBMMs were infected with SeV (MOI 1) for 8 h (D) or HSV-1 (MOI 5) for 12 h (E). The viral RNA was measured by RT–qPCR. F. Peritoneal macrophages isolated from WT and lincRNA-EPS−/− mice were infected with VSV (MOI 1) for 10 h, and the viral titer were measured by TCID50 assay. G. Eight weeks female lincRNA-EPS−/− mice (n = 12) and WT littermates (n = 12) were injected (i.v.) with VSV (lethal dose, 1 × 108 pfu/g), and the survival situation was monitored for 120 h. H–J. Eight weeks female lincRNA-EPS−/− mice (n = 5) and WT littermates (n = 5) were injected (i.v.) with VSV (sub-lethal dose, 6 × 107 pfu/g) for 12 h, and negative control groups were injected with PBS (n = 3). Pathological section of liver and lung were harvested by H&E staining (H). Serum, liver, and lung from the VSV-infected mice were collected. The viral load of serum and tissue homogenate were measured by TCID50 assay (I), and the serum IFN-β protein level were checked by ELISA (J). Data information: Data of (A, C–F) are shown as the mean ± s.d. from three independent experiments, data of (I, J) are shown as the mean ± s.d. of a typical representative result f
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