RDUR, a lncRNA, Promotes Innate Antiviral Responses and Provides Feedback Control of NF-κB Activation

Abstract

Influenza A virus (IAV), a highly infectious respiratory pathogen, remains a major threat to global public health. Numerous long non-coding RNAs (lncRNAs) have been shown to be implicated in various cellular processes. Here, we identified a new lncRNA termed RIG-I-dependent IAV-upregulated noncoding RNA (RDUR), which was induced by infections with IAV and several other viruses. Both in vitro and in vivo studies revealed that robust expression of host RDUR induced by IAV was dependent on the RIG-I/NF-κB pathway. Overexpression of RDUR suppressed IAV replication and downregulation of RDUR promoted the virus replication. Deficiency of mouse RDUR increased virus production in lungs, body weight loss, acute organ damage and consequently reduced survival rates of mice, in response to IAV infection. RDUR impaired the viral replication by upregulating the expression of several vital antiviral molecules including interferons (IFNs) and interferon-stimulated genes (ISGs). Further study showed that RDUR interacted with ILF2 and ILF3 that were required for the efficient expression of some ISGs such as IFITM3 and MX1. On the other hand, we found that while NF-κB positively regulated the expression of RDUR, increased expression of RDUR, in turn, inactivated NF-κB through a negative feedback mechanism to suppress excessive inflammatory response to viral infection. Together, the results demonstrate that RDUR is an important lncRNA acting as a critical regulator of innate immunity against the viral infection.

Keywords: NF-κB; inflammation; influenza A virus; innate immunity; long non-coding RNA.

Figure 1

Human RDUR is identified as a new lncRNA induced by influenza A virus and several other viruses. (A) The differentially expressed lncRNAs in A549 cells infected with or without A/WSN/33 influenza virus (WSN) were analyzed by a cDNA microarray (http://www.ncbi.nlm.nih.gov/geo/; GEO access number GSE58741). Shown are representative differentially expressed lncRNAs. (B) A549 cells infected with WSN were collected at 16 h post-infection (hpi). The differential expressions of 6 selected lncRNAs were confirmed by RT-PCR. RDUR (LOC152225) is indicated by red rectangle. (C) Quantitative real-time PCR (qRT-PCR) was performed to examine the kinetics of RDUR expression in WSN infected A549 cells (n = 3; means ± SD). (D) Shown is the abundance of two isoforms of RDUR examined by qRT-PCR. The error bars represent the SD. Shown are representative results from three independent experiments. (E) RNA-FISH was performed to determine the localization of RDUR in WSN infected A549 cells. Shown were representative images from at least three independent experiments with similar results. (F) RDUR expression was examined by RT-PCR in indicated human cell lines infected with WSN (moi=1) for 16 h. Plotted are the average results from three independent experiments. Data are shown as means ± SD. **P < 0.01. (G) RDUR expression was examined by qRT-PCR in indicated cells infected with influenza viruses (PR8 and CA04), Sendai virus (SeV), Muscovy duck reovirus (MDRV), or herpes simplex virus type 1 (HSV-1). Plotted are the average results from three independent experiments. Data are shown as means ± SD. *P < 0.05, **P < 0.01.

Figure 2

Altering RDUR expression impacts IAV replication in A549 cells. (A) RDUR knockdown A549 cell lines were infected with WSN and the virus titers in the supernatants were measured by hemagglutinin (HA) assay at indicated time points. Luciferase shRNA (sh-Luc) served as a control. Shown are average results from three independent experiments. Data are shown as means ± SD. (B) RDUR knockdown A549 cells were infected with WSN for 16 h and the virus titers were measured by plaque assay. Plotted are the average levels from three independent experiments. Data are shown as means ± SD, **P < 0.01. (C) A549 cells overexpressing RDUR were infected by WSN and the virus titers at indicated time points were measured as described in (A). Data are shown as means ± SD. (D) A549 cells overexpressing RDUR were infected by WSN for 16 h and the virus titers were measured by plaque assay. Cells expressing empty vector (EV) were used as control. Plotted are the average results from three independent experiments. Data are shown as means ± SD, **P < 0.01. (E) RDUR knockdown A549 cells were infected by CA04 virus for 16 h and the titers were measured by plaque assay. Plotted are the average levels from three independent experiments. Data are shown as means ± SD. *P< 0.05, **P < 0.01. (F) RDUR overexpressed A549 cells were infected by CA04 virus and the virus titers were measured by HA assay at indicated time points. Data are shown as means ± SD.

Figure 3

IAV-induced robust expression of RDUR is RIG-I dependent both in vitro and in vivo. (A) A549 cells were transfected with indicated amount of WSN genomic RNA (VG-RNA) using Lipofectamine 2000. Effect of VG-RNA on the expression of RDUR was determined by qRT-PCR. (B) Different amounts of total RNA from A549 cells infected with (named as Viral RNA) or without (named as Cellular RNA) WSN virus were transfected into native A549 cells respectively using Lipofectamine 2000. Expression of RDUR in transfected A549 cells was examined by qRT-PCR. (C) Viral RNA from IAV-infected A549 cells was treated with/without alkaline phosphatase calf intestinal (CIAP), and was transfected into native A549 cells. Expression of RDUR in A549 cells was then examined by RT-PCR. (D) Different amounts of poly (I:C) were transfected into A549 cells and expression of RDUR in transfected A549 cells was examined by RT-PCR. (E) A549 cell line stably expressing shRNAs targeting RIG-I was generated. Then, the cells were infected with WSN for 16 h, and the expression of RDUR was examined by RT-PCR. (F) RIG-I knockout A549 cells were generated by utilizing CRISPR-Cas9. These cells and native A549 cells were infected with WSN for 16 h, and the expression of RDUR was examined by qRT-PCR. (G, H) RIG-I knockout mice were infected with WSN for 1 day, and the expression of RDUR in lungs was detected by RT-PCR (G) and qRT-PCR (H). Shown are representative data from three independent experiments. The error bars represent the SD, *P < 0.05, **P < 0.01.

Figure 4

IAV-induced RDUR expression is regulated by NF-κB but not IRF3 and IFNs. (A) sh-MAVS and sh-Luc (control) infected A549 cells were analyzed by qRT-PCR to determine the interference efficiency. (B) The RNA levels of RDUR in sh-MAVS and sh-Luc (control) A549 cells infected with or without WSN were determined by qRT-PCR. (C) The RNA levels of RDUR in A549 cells treated with or without Bay 11-7082 and infected with or without WSN were determined by qRT-PCR. (D, E) A549 cells were transfected with siRNAs targeting NF-κB (P65/NFKB3) or control siRNA for 36 h and infected with WSN for 16 h. The knockdown efficiency (D) and the RNA level of RDUR (E) were then detected by RT-PCR. (F, G) A549 cells were stimulated with TNF-α for 180 min, and the activation of NF-κB was confirmed by Western blotting (F) and the expression of RDUR was examined by qRT-PCR (G). (H, J) A549 cells were treated with different amounts of IFNs including IFN-β (H), IFN-γ (I) and IFN-λ1 (J) for 90 min. The expression levels of RDUR and ISGs (Mx1 or ISG15) in the cells were examined by qRT-PCR. (K) Shown is IRF3 (sh-IRF3) knockdown efficiency analyzed by qRT-PCR in A549 cells. (L) The RNA levels of RDUR in sh-IRF3 and sh-Luc A549 cells infected with or without WSN were determined by qRT-PCR. Shown are representative data from three independent experiments. Plotted are the average levels from three independent experiments. The error bars represent the SD, **P < 0.01, and ns represents no statistical significance.

Figure 5

mRDUR-deficient mice are more susceptible to IAV and PRV infections. (A) The expression of mRDUR in three mouse cell lines infected with or without WSN for 16 h was examined by qRT-PCR. The error bars represent the SD, **P < 0.01, *P < 0.05. (B) The mRDUR expression levels in the lungs of mice infected with or without WSN for 0-72 h were examined by RT-PCR. (C, D) Shown are the body weight change and survival rates of mRDUR knockout and control mice intranasally inoculated with WSN or Mock. Body weight was measured every day (8 mice for each Mock group and 14 mice for each WSN infection group). (E) Viral titers in the lungs of mRDUR knockout and control mice inoculated intranasally with WSN for 72 h were measured by plaque assay. Plotted are the average levels from three independent experiments. The error bars represent the SD, *P < 0.05. (F) Expression levels of mRDUR in different organs (lung, liver, kidney, heart, thymus and spleen) of mice infected with WSN or Mock were examined by qRT-PCR (n = 3; means ± SD; **P < 0.01). (G) Shown are representative micrographs of H&E stained lung (left) and spleen (right) sections of the mRDUR knockout and control mice infected with or without WSN. (H) WT and RUUR-/- mice were intramuscularly infected with PRV for 2 days, and shown is representative clinic-pathological phenotype from three independent experiments.

Figure 6

RDUR regulates the expression of several critical antiviral genes in vitro and in vivo. (A) The levels of IFN-β, IFITM3, Mx1 and ISG15 were examined by qRT-PCR in A549 cells expressing shRNAs specifically targeting RDUR or luciferase (control) after infection with or without WSN. Plotted are the average levels from three independent experiments. Data are shown as means ± SD. **P < 0.01. (B) A549 cell lines stably expressing specific shRNAs targeting RDUR and luciferase (control) were infected with or without WSN and harvested at 16 hpi, followed by Western blotting with the indicated antibodies. Shown are representative data from three independent experiments with similar results. (C) The mRNA levels of IFN-β, IFITM3, Mx1, ISG15 and OAS2 in RDUR over-expression and empty vector (EV) control A549 cells infected with or without WSN were determined by qRT-PCR. Plotted are the average levels from three independent experiments. Data are shown as means ± SD. *P < 0.05, **P < 0.01. (D, E) The protein levels of IFN-β were determined by ELISA in the lungs of mRDUR knockout or WT mice infected with WSN or seasonal IAV (3 mice each group). Plotted are the average levels from three independent experiments. Data are shown as means ± SD. *P < 0.05, **P < 0.01. (F) RDUR knockdown and control A549 cells were infected with or without WSN, and Western blotting was performed to detect indicated proteins. (G) RDUR overexpression and EV control A549 cells were infected with or without WSN and examined by Western blotting with the indicated antibodies. (H) A549 cell lines stably expressing specific shRNAs targeting RDUR and luciferase (control) were infected with or without WSN and examined by Western blotting with the indicated antibodies. (I, J) mRDUR knockout and control mice were infected with WSN or CA04 influenza virus. Western blotting was performed by using indicated antibodies. Shown are representative immunoblots from three independent experiments.

Figure 7

RDUR deficiency induces NF-κB activation and aggravates inflammatory response after IAV infection in vivo. (A) A549 cell lines stably expressing specific shRNAs targeting RDUR and luciferase (control) were infected with or without WSN and harvested at 16 hpi., followed by Western blotting with the indicated antibodies. (B) Shown are the protein levels in the lungs of mRDUR knockout and WT mice infected with WSN or Mock, detected by Western blotting with the indicated antibodies. (C–E) The mRNA levels of IL-6 (C), IL-1β (D) and TNF-α (E) in the lungs of mRDUR knockout and WT mice infected with WSN or Mock were determined by qRT-PCR. Plotted are the average results from three independent experiments. Data are shown as means ± SD. **P < 0.01. (F, G) The Protein levels of IL-1β (F) and IL-6 (G) in the lungs of mRDUR knockout and WT mice infected with WSN and seasonal IAV or Mock were determined by qRT-PCR (3 mice per group). Plotted are the average results from three independent experiments. Data are shown as means ± SD. **P < 0.01. (H–J) The mRNA levels of IL-6 (H), IL-1β (I) and TNF-α (J) in RDUR overexpression and EV control A549 cells infected with WSN or Mock were determined by qRT-PCR. Plotted are the average results from three independent experiments. Data are shown as means ± SD. **P < 0.01.

Figure 8

RDUR interacts with ILF2 and ILF3 RNA binding proteins. (A, B) RIP assay was performed using ILF2-specific antibody (A) or ILF3-specific antibody (B) or normal IgG control and quantitative real-time PCR was performed to detect RDUR in A549 cells infected with IAV for 16 h. (C) ILF3 knockdown A549 cells were infected with IAV for 14 h and the virus titers were measured by plaque assay. Plotted are the average levels from three independent experiments. Data are shown as means ± SD, **P < 0.01. (D) ILF3 knockdown A549 cell lines were infected with PR8 and the virus titers in the supernatants were measured by HA assay at indicated time points. Shown are average results from three independent experiments. Data are shown as means ± SD. (E) A549 cell lines stably expressing specific shRNAs targeting ILF3 and luciferase (control) were infected with or without IAV and harvested at 14 hpi., followed by Western blotting with the indicated antibodies. (F) A549 cells overexpressing RDUR were lysed and Western blotting was performed to detect indicated protein levels. (G) The ILF2 and ILF3 level in the lungs of WT and mRDUR knockout mice were detected by Western blotting.