Stimulation of alpha2-adrenergic receptors impairs influenza virus infection

Abstract

Influenza A viruses cause seasonal epidemics and occasional pandemics. The emergence of viruses resistant to neuraminidase (NA) inhibitors and M2 ion channel inhibitors underlines the need for alternate anti-influenza drugs with novel mechanisms of action. Here, we report the discovery of a host factor as a potential target of anti-influenza drugs. By using cell-based virus replication screening of a chemical library and several additional assays, we identified clonidine as a new anti-influenza agent in vitro. We found that clonidine, which is an agonist of the alpha2-adrenergic receptor (α2-AR), has an inhibitory effect on the replication of various influenza virus strains. α2-AR is a Gi-type G protein-coupled receptor that reduces intracellular cyclic AMP (cAMP) levels. In-depth analysis showed that stimulation of α2-ARs leads to impairment of influenza virus replication and that α2-AR agonists inhibit the virus assembly step, likely via a cAMP-mediated pathway. Although clonidine administration did not reduce lung virus titers or prevent body weight loss, it did suppress lung edema and improve survival in a murine lethal infection model. Clonidine may thus protect against lung damage caused by influenza virus infection. Our results identify α2-AR-mediated signaling as a key pathway to exploit in the development of anti-influenza agents.

Figure 1

Screening for novel influenza virus replication inhibitors. (A) Effect of screened compounds on influenza virus replication. AX4/PB2 cells were treated with the indicated compound (1 µM each) and subjected to a virus replication assay with Rluc. Each compound was tested in duplicate assay wells. (B) Effect of screened compounds on influenza vRNA transcription/replication activity. 293vRNP-Puro cells were cultured with the indicated compound (10 µM each) in the presence of puromycin, and vRNA transcription/replication activity was assessed by cell viability. Each compound was tested in duplicate assay wells. (C) Reproducibility of virus replication inhibition and cytotoxicity of the identified compounds. AX4/PB2 cells treated with various concentrations of the indicated compounds were subjected to a virus replication assay with Rluc and a cell viability assay. Each experiment includes data from duplicate assay wells. (D) Effect of clonidine on influenza virus replication. AX4/PB2 cells were treated with clonidine before virus infection and subjected to a virus replication assay with Rluc. Data are shown as means ± SEM of three independent experiments. (E) Effect of clonidine on NA activity. WSN/PB2-Rluc virus were mixed with the indicated compounds (zanamivir, 3 nM; clonidine, 10 µM), and the NA activity of the viruses was measured with the NA-Star kit. Data are shown as means ± SEM of five independent experiments.

Figure 2

Inhibitory effect of clonidine on influenza virus replication. (A,B) Effect of clonidine on influenza virus replication. AX4/PB2 cells were treated with the indicated concentration of clonidine 1 hour before viral infection. In the presence of clonidine, the AX4/PB2 cells were infected with WSN/PB2-Rluc virus. After incubation in the presence of clonidine, virus titers were determined by using plaque assays (A), and NA levels in the supernatant were measured with the NA-Star kit (B). (C) Effect of clonidine on the replication kinetics of influenza viruses. AX4/PB2 cells were treated with 1 µM clonidine (Red) or DMSO (Black), and infected with WSN/PB2-Rluc virus at an MOI of 0.025. The amount of virus was determined at the indicated time points by measuring the NA levels in the supernatant. Data are shown as means ± SD of triplicate assay wells (A and C) or means ± SEM of three independent experiments (B). *P < 0.005.

Figure 3

Broad-spectrum anti-influenza activity of clonidine. MDCK cells were infected with influenza A/WSN/33 (H1N1), A/California/04/2009 (H1N1pdm), A/Yokohama/UT-K4A/2011 (H3N2), and B/Yokohama/UT-K1A/2011 (Victoria lineage) at an MOI of 0.0001. The remaining viruses were removed after 1 h of the infection, and the infected cells were incubated further in the presence of clonidine. The virus titer in the supernatant was determined at the indicated time points by means of plaque assays. Data are shown as means ± SD of triplicate assay wells. *P < 0.05.

Figure 4

Stimulation of α2-ARs inhibits a step of the virus life cycle after protein synthesis. (A) Effect of α2-AR agonists on influenza virus replication. AX4/PB2 cells were treated with dexmedetomidine (Dex), guanfacine (Gua), rilmenidine (Ril), tizanidine (Tiz), or xyladine (Xyl), and subjected to a virus replication assay with Rluc. Data are shown as means ± SEM of three independent experiments. (B) Effect of α2-AR antagonists on clonidine-induced inhibition of virus replication. AX4/PB2 cells were treated with atipamezole (Ati), yohimbine (Yoh), or BRL-44408 (BRL) in the presence of 1 µM clonidine, and subjected to a virus replication assay with Rluc. Data are shown as means ± SEM of three independent experiments. (C) Effect of clonidine on viral protein expression. AX4/PB2 cells were infected with WSN/PB2-Rluc virus at an MOI of 1 and the remaining viruses were removed. After 6 h of treatment with 100 µM favipiravir (Fav), 1 µM zanamivir (Zan), or 10 µM clonidine (Clo), viral protein expression levels were assessed on the basis of Rluc expression levels. Data are shown as means ± SEM of three independent experiments.

Figure 5

Efficacy of clonidine against H1N1 influenza viruses in mice. Five mice (A and B) and three mice (C) per group were intranasally infected with 370,000 pfu of mouse-adapted pandemic A/California/04/2009. The infected mice were given clonidine orally at the indicated doses once daily for 5 days beginning 2 h pi. Survival (A) and body weight (B) were monitored daily for 16 days. The survival rate was determined by death or a cut-off of 35% lost body weight. *P < 0.05. (B) Data are shown as means of surviving mice. (C) Lung virus titers on days 3 and 6 pi were determined by using plaque assays. Data are shown as means ± SD of three mice.