Triethylamine inhibits influenza A virus infection and growth via mechanisms independent of viral neuraminidase and RNA-dependent RNA polymerase

Abstract

Triethylamine (Et3N) is a proton (H⁺) acceptor that is widely used in various industrial organic synthesis processes, including the production of pharmaceuticals, agrochemicals, and polymers. Inhalation of high Et3N concentrations can damage human respiratory tract and lungs. Given the compound's known reactivity and membrane-penetrating properties, we hypothesized that non-toxic concentrations of Et₃N may exert modulatory effects on virus-host interactions in epithelial cells. We thus investigated the anti-influenza activity of Et3N and found that it enhanced the viability of influenza A H1N1 and H3N2 virus-infected Madin-Darby canine kidney (MDCK) cells. Non-cytotoxic Et3N concentrations reduced the number of infected cells and suppressed influenza A virus nucleoprotein expression as well as viral gene and antiviral host gene upregulation in infected MDCK cells. Selectivity index values of Et₃N against influenza A virus infection, ranging from approximately 10 to over 50. These findings indicated that Et3N inhibited influenza A H1N1 and H3N2 viral infections. Additionally, Et3N suppressed influenza A H1N1 and H3N2 virus titers in the infected MDCK cell culture supernatant, suggesting that it inhibited viral growth in infected cells. This implies that Et3N may suppress influenza A virus release and/or replication by targeting viral or host cell factors. However, Et3N did not inhibit influenza A viral neuraminidase or RNA-dependent RNA polymerase activity, which are involved in viral release and replication, respectively. These results suggest that Et3N targets other viral proteins or host cell factors essential for influenza A virus growth. Our findings demonstrate that Et3N exerts anti-influenza activity, providing new insights into the development of antiviral agents based on Et3N skeleton.

Fig 1. Effects of Et₃N on survival in influenza A virus-infected MCDK cells.

(A) Chemical structure of Et₃N. (B) The cytotoxicity of Et₃N in MDCK cells using the WST-8 assay. Data represent the mean ± SEM (n = 8) of two independent experiments. (C) The effects of Et₃N on influenza A H1N1 (A/PR/8/34 or AWSN/33) and H3N2 (A/Aichi/2/68) virus-infected MDCK cell viability using NB staining. Data are representative of four independent experiments, with results consistently reproducible across these experiments.

Fig 2. Immunofluorescence analysis of the inhibitory effect of Et₃N on influenza A virus infection.

(A) IF staining for influenza NP in Et₃N-treated MDCK cells infected with influenza A H1N1 (A/PR/8/34 and A/WSN/33) or H3N2 (A/Aichi/2/68) viruses. The white scale bars in each image represent 100 µm. (B–D) The percentage of NP-positive cells that calculated relative to the total number of DAPI-stained nuclei. A/PR/8/34 (B), A/WSN/33 (C), or A/Aichi/2/68 (D). Data are expressed as the mean ± SEM (n = 6) from two independent experiments. **P < 0.01 and ***P < 0.001 indicate statistical significance compared to the negative control (Water), as determined by one-way ANOVA followed by Dunnett’s post-hoc test. The results were consistently reproducible across experiments.

Fig 3. Inhibitory effects of Et₃N on the expressions of influenza A viral NP and genes in virus-infected MDCK cells.

(A–C) WB analysis of influenza A viral NP expression in MDCK cells treated with Et₃N and infected with influenza A viruses. Water (control) = 1. A/PR/8/34 (B), A/WSN/33 (C), or A/Aichi/2/68 (D). Data are presented as the mean ± SEM (n = 6) of two independent experiments. **P < 0.01 and ***P < 0.001 indicate statistical significance compared to the negative control (Water), as determined by one-way ANOVA followed by Dunnett’s post-hoc test. (D–J) The gene expression levels of viral NP (D), NS1 (E), PA (F), PB1 (G), PB2 (H), M1 (I), and M2 (J) in MDCK cells treated with Et₃N and infected with A/PR/8/34 determined by RT-qPCR. Virus-infected Water-treated cells = 1. Data are presented as the mean ± SEM (n = 6) of two independent experiments. ***P < 0.001 indicates statistical significance compared to virus-infected Water-treated cells, as determined by one-way ANOVA followed by Tukey’s post-hoc test. Results were consistently reproducible across experiments.

Fig 4. Inhibitory effects of Et₃N on the upregulation of antiviral host genes in influenza A virus-infected MDCK cells.

The expression levels of canine Ifn-β (A) and Mx1 (B) determined by RT-qPCR. A/PR/8/34-infected Water-treated cells = 1. Data are presented as the mean ± SEM (n = 6) of two independent experiments. ***P < 0.001 indicates statistical significance for the indicated comparisons, as determined by one-way ANOVA followed by Tukey’s post-hoc test. Results were consistently reproducible in this experiment.

Fig 5. Inhibitory effect of Et3N on influenza A viral growth.

(A) Schematic representation of the virus pre-infection experiment. (B–D) Influenza viral titers of culture supernatant in influenza A viruses-infected MDCK cells treated with Et3N at 48 h post-infection. A/PR/8/34 (B), A/WSN/33 (C), or A/Aichi/2/68 (D) viruses. Data are presented as the mean ± SEM (n = 6) of two independent experiments. ***P < 0.001 indicates statistical significance compared to the negative control (Water), as determined by one-way ANOVA followed by Dunnett’s post-hoc test. Results were consistently reproducible in this experiment.

Fig 6. Et₃N did not inhibit NA and RdRp activities.

(A–D) NAI assay using influenza A viral particles. without virus (−) (A). A/PR/8/34 (B), A/WSN/33 (C), or A/Aichi/2/68 (D). The fluorescence intensity in wells was measured at 365 nm excitation wavelength and 445 nm emission wavelength at 0, 3, 6, and 24 h. Data are presented as the mean ± SEM (n = 6) of two independent experiments. ***P < 0.001 indicates statistical significance for the indicated comparisons, as determined by one-way ANOVA followed by Tukey’s post-hoc test. (E) A minigenome assay for influenza viral RdRp activity. Firefly_LUC and Renilla_LUC activities in the transfected MDCK cells were measured after 24 h. The RLU ratio of Firefly_LUC to Renilla_LUC was calculated. Data are presented as the mean ± SEM (n = 6) of two independent experiments. ***P < 0.001 indicates statistical significance compared to Water treatment, as determined by one-way ANOVA followed by Tukey’s post-hoc test. Results were consistently reproducible in these experiments.