RETRACTED: IAV Antagonizes Host Innate Immunity by Weakening the LncRNA-LRIR2-Mediated Antiviral Functions

Abstract

A growing number of studies have shown that long non-coding RNAs (lncRNAs) are implicated in many biological processes, including the regulation of innate immunity and IAV replication. In addition, IAV has been found to be able to hijack lncRNAs and thus antagonize host innate immunity. Nonetheless, whether IAV can antagonize host innate immunity by weakening the antiviral functions mediated by lncRNAs is unknown. In this study, we found that LncRNA-ENST00000491430 regulates IAV replication and named it LRIR2. Interestingly, we found that the expression of LRIR2 was suppressed during IAV infection. Importantly, LRIR2 overexpression inhibited IAV replication, suggesting that LRIR2 plays an antiviral role during IAV infection. Mechanistically, we demonstrated that LRIR2 inhibits the transcription and replication of the IAV genome. In addition, the antiviral function of LRIR2 is mainly dependent on the stem-loop structures of 1-118 nt and 575-683 nt. Taken together, IAV could antagonize host innate immunity by weakening the LncRNA-LRIR2-mediated antiviral functions. Our study provides novel perspectives into viral strategies to antagonize host innate immunity. It lays a theoretical foundation for the design of novel anti-IAV drugs that target host lncRNAs or the antagonism effect.

Keywords: antagonism; antiviral drug targets; antiviral functions; influenza A virus; innate immunity; lncRNA.

Figure 1

LRIR2 expression is inhibited during IAV infection. (A) A heat map showing the 22 selected lncRNAs with markedly different expressions. ENST00000491430 is highlighted with a red border. (B) Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine the different expressions of ENST00000491430 in A549 cells infected with or without WSN. (C,D) qRT-PCR and RT-PCR were performed to detect the differential expressions of LRIR2 in A549 cells infected with or without WSN. (E,F) A549 cells were infected with different multiplicities of infection (MOI) of WSN for 24 h. The expression level of LRIR2 was examined by qRT-PCR and RT-PCR. Data represent means ± standard deviations (n  =  3; **, p < 0.01).

Figure 2

Biological properties of LRIR2. (A,B) A549 cells were treated with IFN-β at the indicated concentrations for 24 h. The expression levels of LRIR2 and Mx1 (a known ISG) in the cells were detected by qRT-PCR. Data represent means ± standard deviations (n  =  3; **, p < 0.01). NS stands for no significant difference. (C) A549 cells were mock-infected or infected with WSN at an MOI of 1 for 12 h. The RNA levels of LRIR2, cytoplasmic control (GAPDH mRNA), and nuclear control (U6 RNA) were examined by qRT-PCR in the cytoplasmic and nuclear fractions from A549 cells. The total RNA was used as an input control. Data are shown as % input (means ± SEM; n = 3). (D) The secondary structure analysis of LRIR2 was predicted with RNAfold.

Figure 3

LRIR2 suppresses IAV replication. (A) A549 cells were transfected with pcDNA3.1/pcDNA3.1-LRIR2, followed by infection with WSN (MOI = 1). The cell culture supernatants were harvested at the indicated times to determine viral growth curves. (B) A549 cells seeded in 6-well plates were transfected with si-NC/si-LRIR2, followed by infection with WSN (MOI = 1). The cell culture supernatants were harvested at the indicated times to determine viral growth curves. Data represent means ± standard deviations (n  =  3; **, p < 0.01).

Figure 4

Antiviral functions of LRIR2 are independent of type I interferon-mediated antiviral immune response. (A–F) qRT-PCR was conducted to validate the expression of IFIT1, IFIT3, IFITM3, ISG15, Mx1, and OASL between LRIR2-overexpressing and control cells at 24 hpi. NS stands for no significant difference.

Figure 5

LRIR2 inhibits the transcription and replication of the IAV genome. (A–F) A549 cells were transfected with pcDNA3.1/pcDNA3.1-LRIR2 (A–D) or si-NC/si-LRIR2 (E,F), followed by infection with WSN (MOI = 1) for 24 h. Total RNA was extracted, and qRT-PCR was conducted to examine the mRNA, vRNA, and cRNA levels of several viral genes. Data represent means ± standard deviations (n  =  3; **, p < 0.01).

Figure 6

The antiviral functions of LRIR2 mainly depend on the stem-loop structures of 1–118 nt and 575–683 nt. (A) Schematic diagram of the truncated mutants of LRIR2. (B,C) Secondary structure predictions of LRIR2 and the LRIR2 mutants were performed using RNAfold. The mutation locations were labeled with orange circles. (D) A549 cells expressing LRIR2 or its mutants were infected with WSN, and the virus titers in culture supernatants were detected by plaque assays. (E) A549 cells expressing LRIR2 or its mutants were infected with WSN, and the NP mRNA level was determined by qRT-PCR. Data represent means ± standard deviations (n  =  3; **, p < 0.01). NS stands for no significant difference.