Honeysuckle-encoded atypical microRNA2911 directly targets influenza A viruses

Abstract

Influenza A viruses (IAVs), particularly H1N1, H5N1 and H7N9, pose a substantial threat to public health worldwide. Here, we report that MIR2911, a honeysuckle (HS)-encoded atypical microRNA, directly targets IAVs with a broad spectrum. MIR2911 is highly stable in HS decoction, and continuous drinking or gavage feeding of HS decoction leads to a significant elevation of the MIR2911 level in mouse peripheral blood and lung. Bioinformatics prediction and a luciferase reporter assay showed that MIR2911 could target various IAVs, including H1N1, H5N1 and H7N9. Synthetic MIR2911 significantly inhibited H1N1-encoded PB2 and NS1 protein expression, but did not affect mutants in which the MIR2911-binding nucleotide sequences were altered. Synthetic MIR2911, extracted RNA from HS decoction and HS decoction all significantly inhibited H1N1 viral replication and rescued viral infection-induced mouse weight loss, but did not affect infection with a mutant virus in which the MIR2911-binding nucleotide sequences of PB2 and NS1 were altered. Importantly, the inhibitory effect of HS decoction on viral replication was abolished by an anti-MIR2911 antagomir, indicating that the physiological concentration of MIR2911 in HS decoction could directly and sufficiently suppress H1N1 viral replication. MIR2911 also inhibited H5N1 and H7N9 viral replication in vitro and in vivo. Strikingly, administration of MIR2911 or HS decoction dramatically reduced mouse mortality caused by H5N1 infection. Our results demonstrate that MIR2911 is the first active component identified in Traditional Chinese Medicine to directly target various IAVs and may represent a novel type of natural product that effectively suppresses viral infection.

Figure 1

MIR2911 is highly enriched in HS decoction and is delivered into mouse lungs. (A, B) The levels (sequencing reads) of plant miRNAs in HS (A) and HS decoction (B) detected by Illumina sequencing. (C) The concentrations of MIR2911 in HS and HS decoction determined by RT-qPCR. (D) Northern blotting analysis showing MIR2911 expression in 10 g of HS or 10 ml of HS decoction (final concentration: ∼0.2 g HS/ml). (E) MIR2911 kinetics in mouse plasma after administration of 500 μl of HS decoction (MIR2911 concentration: 0.12 nM) by gavage (0 h, n = 30; other time point, n = 6). (F) MIR2911 kinetics in mouse lung tissue after administration of 500 μl of HS decoction (MIR2911 concentration: 0.12 nM) by gavage (n = 5). (G, H) The MIR2911 level in mouse plasma (G) and lung tissue (H) 3 days after drinking HS decoction (MIR2911 concentration: 0.06 nM) (n = 5). (I, J) The MIR2911 kinetics in mouse plasma (I) and lung tissue (J) after administration of 100 pmol synthetic MIR2911 in 100 μl PBS by gavage (n = 5). (K) Fluorescently labeled MIR2911 in mouse lung tissue 3 h after administration of 2 nmol synthetic, fluorescently labeled MIR2911 by gavage (representative images are shown). Note that labeled MIR2911 (green dots) rapidly accumulated in mouse lungs. ^*P < 0.05; ^***P < 0.001. All values are the mean ± SEM. P values were determined using Student's t-test.

Figure 2

MIR2911 targets various influenza viruses. (A) Prediction of the viral genome sequence targeted by MIR2911. (B) Validation of the predicted MIR2911 target sequence using a luciferase reporter assay (n = 3). (C) Western blot analysis of the PB2 and NS1 levels after transfecting HEK293T cells with the plasmids expressing PB2 or NS1, MIR2911 or ncRNA. (D) Western blot analysis of the mutant PB2 and NS1 levels after transfecting HEK293T cells with the plasmids expressing mutant PB2 or NS1, MIR2911 or ncRNA. (E, F) Anti-H1N1 effects of synthetic MIR2911 (E) and HS total RNA (F) in MDCK cells (MOI = 0.1, n = 6). (G, H) Effects of synthetic MIR2911 (G) and HS total RNA (H) on mutant H1N1 virus in MDCK cells (MOI = 0.1, n = 6). ^*P < 0.05; ^***P < 0.001. All values are the mean ± SEM. P values were determined using Student's t-test.

Figure 3

MIR2911 suppressed the replication of H1N1 influenza virus but not mutant virus in mice. The mice were treated with different reagents before being inoculated with H1N1 influenza viruses (A-C) or mutant H1N1 influenza virus (D-F). The body weight was recorded every day. On days 3, 5 and 7 post infection, the mice were sacrificed, and lung tissue was obtained to measure viral replication. (A, B) Body weight changes in mice subjected to synthetic MIR2911 (A) and HS decoction (B) treatment after inoculation with 10⁶ EID₅₀ of H1N1 influenza virus (n = 8). (C) Viral titers (EID₅₀) in the lungs of mice subjected to synthetic MIR2911 and HS decoction treatment at different times after inoculation with 10⁶ EID₅₀ of H1N1 influenza virus (n = 6). (D, E) Body weight changes in mice subjected to synthetic MIR2911 (D) and HS decoction (E) treatment after inoculation with 10⁶ EID₅₀ of mutant H1N1 influenza virus (n = 8). (F) Viral titers (EID₅₀) in the lungs of mice subjected to synthetic MIR2911 and HS decoction treatment at different times after inoculation with 10⁶ EID₅₀ of mutant H1N1 influenza virus (n = 6). ^*P < 0.05; ^**P < 0.01. All values are the mean ± SEM. P values were determined using Student's t-test by comparing to the virus alone group.

Figure 4

MIR2911 suppressed the replication of the H5N1 and H7N9 influenza viruses in mice. (A, B) The anti-H5N1 effects of synthetic MIR2911 (A) and HS total RNA (B) in MDCK cells (MOI = 0.1, n = 6). (C) Survival rates of mice subjected to synthetic MIR2911 and HS decoction treatment after intranasal inoculation with 10³ EID₅₀ of H5N1 influenza virus (n = 7-8). (D, E) Body weight changes in mice subjected to synthetic MIR2911 (D) and HS decoction (E) treatment after inoculation with 10³ EID₅₀ of H5N1 influenza virus (n = 7-8). (F) Viral titers (EID₅₀) in the lungs of mice subjected to synthetic MIR2911 and HS decoction treatment at different times after inoculation with 10³ EID₅₀ of H5N1 influenza virus (n = 6). (G, H) The anti-H7N9 effects of synthetic MIR2911 (G) and HS total RNA (H) in MDCK cells (MOI = 0.1, n = 6). (I, J) Body weight changes in mice subjected to synthetic MIR2911 (I) and HS decoction (J) treatment after inoculation with 10⁶ EID₅₀ of H7N9 influenza virus (n = 8). (K) Viral titers (EID₅₀) in the lungs of mice subjected to synthetic MIR2911 and HS decoction treatment at different times after inoculation with 10⁶ EID₅₀ of H7N9 influenza virus (n = 6). ^*P < 0.05. All values are the mean ± SEM. P values were determined using Student's t-test by comparing to the virus alone group.