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. 2015 Dec 25;8(1):6.
doi: 10.3390/v8010006.

Quercetin as an Antiviral Agent Inhibits Influenza A Virus (IAV) Entry

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Free PMC article

Quercetin as an Antiviral Agent Inhibits Influenza A Virus (IAV) Entry

Wenjiao Wu et al. Viruses. .
Free PMC article

Abstract

Influenza A viruses (IAVs) cause seasonal pandemics and epidemics with high morbidity and mortality, which calls for effective anti-IAV agents. The glycoprotein hemagglutinin of influenza virus plays a crucial role in the initial stage of virus infection, making it a potential target for anti-influenza therapeutics development. Here we found that quercetin inhibited influenza infection with a wide spectrum of strains, including A/Puerto Rico/8/34 (H1N1), A/FM-1/47/1 (H1N1), and A/Aichi/2/68 (H3N2) with half maximal inhibitory concentration (IC50) of 7.756 ± 1.097, 6.225 ± 0.467, and 2.738 ± 1.931 μg/mL, respectively. Mechanism studies identified that quercetin showed interaction with the HA2 subunit. Moreover, quercetin could inhibit the entry of the H5N1 virus using the pseudovirus-based drug screening system. This study indicates that quercetin showing inhibitory activity in the early stage of influenza infection provides a future therapeutic option to develop effective, safe and affordable natural products for the treatment and prophylaxis of IAV infections.

Keywords: entry inhibitor; hemagglutinin; influenza A virus; quercetin.

Figures

Figure 1
Figure 1
(A) The chemical structure of quercetin; (B) Validation of the protection of MDCK cells from influenza A/Puerto Rico/8/34 (H1N1) infection by quercetin. Original magnification, 40 ×. (C,D) The inhibitory effect of quercetin on the influenza A/Puerto Rico/8/34 (H1N1) HA mRNA expression in MDCK cells and A549 cells were detected by quantitative real-time PCR at 24 h post-infection. ** p < 0.05.
Figure 1
Figure 1
(A) The chemical structure of quercetin; (B) Validation of the protection of MDCK cells from influenza A/Puerto Rico/8/34 (H1N1) infection by quercetin. Original magnification, 40 ×. (C,D) The inhibitory effect of quercetin on the influenza A/Puerto Rico/8/34 (H1N1) HA mRNA expression in MDCK cells and A549 cells were detected by quantitative real-time PCR at 24 h post-infection. ** p < 0.05.
Figure 2
Figure 2
Quercetin inhibited vRNP localization in the nucleus. MDCK cells were infected with virus in the presence of quercetin at the concentration of 100, 50 and 25 µg/mL. Viral NP protein was detected with NP-specific monoclonal antibody and Alexa 488-conjugated goat anti-mouse secondary antibody (green); the nuclei were counterstained with DAPI (blue). Original magnification, 40×.
Figure 3
Figure 3
Quercetin performed the inhibitory activity in the initial stage of influenza virus infection. (A) The cells were infected with influenza A/Puerto Rico/8/34, and the virus-infected cells were then treated with quercetin in 0–2 h, 2–5 h, 5–8 h, 8–10 h and 0–10 h time intervals respectively. At 10 h post-infection, the viral HA protein was detected by Western blotting; (B) The cells were infected with influenza A/Puerto Rico/8/34, and the cells were then treated with quercetin in 0–2 h, 2–5 h, 5–8 h, 8–10 h and 0–10 h time intervals respectively. The viral HA mRNA was detected by quantitative real-time PCR at 10 h post-infection; (C) Serially diluted compound was added 1 h before virus infection (−1 h p.i.) or 1 h after virus infection (1 h p.i.). The extent of virus infection in the cell was determined at 48 h post-infection using MTT assay; (D) Different modes of treatment, namely co-treatment, pre-treatment of cells and pre-treatment of virus, were conducted to clarify whether the quercetin targets the host cell or the influenza virus. The protection of quercetin to the virus-infected cell was detected at 48 h post-infection by MTT assay.
Figure 3
Figure 3
Quercetin performed the inhibitory activity in the initial stage of influenza virus infection. (A) The cells were infected with influenza A/Puerto Rico/8/34, and the virus-infected cells were then treated with quercetin in 0–2 h, 2–5 h, 5–8 h, 8–10 h and 0–10 h time intervals respectively. At 10 h post-infection, the viral HA protein was detected by Western blotting; (B) The cells were infected with influenza A/Puerto Rico/8/34, and the cells were then treated with quercetin in 0–2 h, 2–5 h, 5–8 h, 8–10 h and 0–10 h time intervals respectively. The viral HA mRNA was detected by quantitative real-time PCR at 10 h post-infection; (C) Serially diluted compound was added 1 h before virus infection (−1 h p.i.) or 1 h after virus infection (1 h p.i.). The extent of virus infection in the cell was determined at 48 h post-infection using MTT assay; (D) Different modes of treatment, namely co-treatment, pre-treatment of cells and pre-treatment of virus, were conducted to clarify whether the quercetin targets the host cell or the influenza virus. The protection of quercetin to the virus-infected cell was detected at 48 h post-infection by MTT assay.
Figure 4
Figure 4
The interaction between the influenza HA and quercetin was analyzed with PlexArray HT systems. (A) The binding curve of quercetin with HA protein in SPR assay; (B) The binding curve of curcumin with HA protein in SPR assay. The compounds were immobilized on a sensor chip, and the recombinant influenza HA protein was injected as analytes at various concentrations dissolved in a running buffer at a flow rate of 3 μL/s. The contact time and dissociation time were 300 s respectively. The chip platform was regenerated with gly-HCl (pH 2.0) and washed with the running buffer. Curcumin was used as a positive control in this assay. The affinity constant KD is the ratio of dissociation constant Kd with association constant Ka, KD = Kd/Ka.
Figure 5
Figure 5
Protein-binding assays in biological liquids using microscale thermophoresis (MST). (A) The binding curve of quercetin with NT647 labeled recombinant influenza HA protein; (B) The binding curve of CL-385319 with NT647 labeled recombinant influenza HA protein. NT647 (NanoTemper Technologies) labeled recombinant influenza HA protein was mixed with two-fold diluted compounds in the same volume as that of the final concentrations. MST experiments were performed on a Monolith NT.115 system (NanoTemper Technologies) using 100% LED and 20% IR-laser power. The compound CL-385319 was used as the positive control. Kd means the dissociation constant.
Figure 5
Figure 5
Protein-binding assays in biological liquids using microscale thermophoresis (MST). (A) The binding curve of quercetin with NT647 labeled recombinant influenza HA protein; (B) The binding curve of CL-385319 with NT647 labeled recombinant influenza HA protein. NT647 (NanoTemper Technologies) labeled recombinant influenza HA protein was mixed with two-fold diluted compounds in the same volume as that of the final concentrations. MST experiments were performed on a Monolith NT.115 system (NanoTemper Technologies) using 100% LED and 20% IR-laser power. The compound CL-385319 was used as the positive control. Kd means the dissociation constant.
Figure 6
Figure 6
(A) Hemagglutinin inhibition assay for quercetin. 4HAU influenza A/Puerto Rico/8/34 (H1N1) virus was incubated with serally diluted quercetin at room temperature for 30 min. 0.5% cRBCs was added, and the virus then incubated at room temperature for 1 h. Note the inhibition on hemagglutination of quercetin; (B) Hemolysis inhibition assay of quercetin against the influenza A/Puerto Rico/8/34 (H1N1) virus strain. Quercetin diluted in PBS was mixed with an equal volume of the influenza virus A/Puerto Rico/8/34 (H1N1) in a 96-deep well plate. After incubating at room temperature for 30 min, 2% chicken erythrocytes pre-warmed at 37 °C were added. The mixture was incubated at 37 °C for another 30 min. To trigger hemolysis, sodium acetate (0.5 M; pH 5.0) was added and incubated at 37 °C for 15 min. Plate was centrifuged at the end of incubation at 3000 rpm for 5 min, and the supernatants were transferred to another flat-bottom 96-well plate. The OD540 was read on a microtiter plate reader.
Figure 7
Figure 7
The inhibitory activity of quercetin on H5N1 pseudovirus infection. (A) Infectivities of a series of H5N1 pseudoviruses, named A/Anhui/1/2005A, A/Xinjiang/1/2006, A/HongKong/156/1997, A/Qinghai/59/2005, A/Thailand/Kan353/2004, and A/VietNam/1194/2004—Env-pseudovirus and cells-only (mock) were used as the negative control; (B) The inhibitory activity of quercetin against the infection of different H5N1 subtype pseudoviruses and VSV-G pseudovirus on MDCK cells.
Figure 7
Figure 7
The inhibitory activity of quercetin on H5N1 pseudovirus infection. (A) Infectivities of a series of H5N1 pseudoviruses, named A/Anhui/1/2005A, A/Xinjiang/1/2006, A/HongKong/156/1997, A/Qinghai/59/2005, A/Thailand/Kan353/2004, and A/VietNam/1194/2004—Env-pseudovirus and cells-only (mock) were used as the negative control; (B) The inhibitory activity of quercetin against the infection of different H5N1 subtype pseudoviruses and VSV-G pseudovirus on MDCK cells.

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