Aurintricarboxylic acid is a potent inhibitor of influenza A and B virus neuraminidases

Abstract

Background: Influenza viruses cause serious infections that can be prevented or treated using vaccines or antiviral agents, respectively. While vaccines are effective, they have a number of limitations, and influenza strains resistant to currently available anti-influenza drugs are increasingly isolated. This necessitates the exploration of novel anti-influenza therapies.

Methodology/principal findings: We investigated the potential of aurintricarboxylic acid (ATA), a potent inhibitor of nucleic acid processing enzymes, to protect Madin-Darby canine kidney cells from influenza infection. We found, by neutral red assay, that ATA was protective, and by RT-PCR and ELISA, respectively, confirmed that ATA reduced viral replication and release. Furthermore, while pre-treating cells with ATA failed to inhibit viral replication, pre-incubation of virus with ATA effectively reduced viral titers, suggesting that ATA may elicit its inhibitory effects by directly interacting with the virus. Electron microscopy revealed that ATA induced viral aggregation at the cell surface, prompting us to determine if ATA could inhibit neuraminidase. ATA was found to compromise the activities of virus-derived and recombinant neuraminidase. Moreover, an oseltamivir-resistant H1N1 strain with H274Y was also found to be sensitive to ATA. Finally, we observed additive protective value when infected cells were simultaneously treated with ATA and amantadine hydrochloride, an anti-influenza drug that inhibits M2-ion channels of influenza A virus.

Conclusions/significance: Collectively, these data suggest that ATA is a potent anti-influenza agent by directly inhibiting the neuraminidase and could be a more effective antiviral compound when used in combination with amantadine hydrochloride.

Figure 1. ATA protects MDCK cells from infection with influenza A strains PR8, NY or NC.

MDCK cells infected with influenza A viruses (MOI 0.001) were treated with ATA for 48 h. Cell viability was assessed by neutral red assay. The columns represent the means of triplicates and error bars represent standard deviations. * = corrected p-value <0.05.

Figure 2. Determination of the selective index of ATA in MDCK cells.

(A) Cytotoxicity of ATA in MDCK cells. MDCK cells were treated with ATA for 48 h at the indicated ATA concentrations (µg/ml). Cell viability was determined by neutral red assay by measuring the absorbance at 540 nm. Samples were tested in quadruplicate and showed as means and standard deviations (error bars). (B) Inhibition of influenza A PR8 infection in MDCK cells by ATA is concentration-dependent. MDCK cells were infected with influenza A PR8 virus (MOI 0.001) and treated with ATA at increasing concentrations for 48 h. Cell viability was determined by neutral red assay by measuring the absorbance at 540 nm. Samples were tested in duplicate and showed as means and standard deviations (error bars).

Figure 3. ATA reduces the level of influenza NP RNA detectable in MDCK cells.

MDCK cells were infected with influenza A PR8 virus and treated with the indicated concentrations of ATA for 24 h. Total RNA was extracted and reverse-transcription PCR was performed to determine NP and β-actin RNA levels.

Figure 4. ATA reduces viral release from infected MDCK cells into the media.

MDCK cells were inoculated with influenza A viruses (MOI 0.001), then treated with ATA for 48 h. Media were collected and released viruses were quantified by ELISA. The columns represent the means of triplicates and error bars represent standard deviations. * = corrected p-value <0.05.

Figure 5. ATA and AH protect MDCK cells from influenza A infection.

Uninfected MDCK cells, or MDCK cells infected with influenza A strains were treated with either ATA alone, AH alone, or in combinations at concentrations indicated. (A) MDCK cells infected with influenza A PR8 virus; (B) MDCK cells infected with influenza A NC virus; (C) MDCK cells infected with influenza A NY virus. Protection of cells from infection was determined by NR dye uptake. Cell viability was determined by measuring the absorbance at 540 nm. The columns represent the means of triplicates and error bars represent standard deviations. * = corrected p-value <0.05. Not all statistically significant differences are shown.

Figure 6. ATA and AH decrease the abundance of viruses released by infected MDCK cells.

Viruses in culture supernatants were detected by ELISA 48 h following treatment with ATA, AH or both compounds. (A) MDCK cells infected with influenza A PR8 virus; (B) MDCK cells infected with influenza A NC virus; (C) MDCK cells infected with influenza A NY virus. The columns represent the means of 6 replicates and error bars represent standard deviations. * = corrected p-value <0.05. Not all statistically significant differences are shown.

Figure 7. ATA inactivates influenza A and B viruses.

Influenza PR8, NC, NY and B viruses were pre-incubated with ATA (0, 50, 100 µg/ml) for 30 min, then the virus-ATA mixture was transferred to confluent cell monolayers in 6-well plates, incubated at 37°C for 2 h and subjected to plaque assay.

Figure 8. Influenza PR8 virus forms aggregates upon ATA treatment.

MDCK cells were infected with influenza A PR8 virus and exposed to DMSO, AH or ATA, then processed for electron microscopy. (A and B) Low and high magnification view, respectively, of MDCK cells infected with influenza A PR8 virus in the presence of DMSO only. (C and D) Low and high magnification view, respectively, of MDCK cells infected with influenza A PR8 virus in the presence of AH. (E and F) Low and high magnification view, respectively, of MDCK cells infected with influenza A PR8 virus in the presence of ATA.

Figure 9. ATA inhibits NA enzymatic activity.

ATA inhibits enzymatic activity of NA (A) derived from PR8, NC, NY and B viruses, (B) recombinant N1 and N4 proteins. Viruses or recombinant proteins were incubated with increasing concentrations of ATA and NA enzymatic activity was determined by a chemiluminescent assay. Samples were tested in quadruplicates and presented as means and standard deviations (error bars).

Figure 10. ATA inhibits H274Y oseltamivir-resistant virus.

(A) ATA inactivates influenza WSN and H274Y viruses. Influenza WSN and H274Y viruses were pre-incubated with ATA (0, 50, 100 µg/ml) for 30 min, then the virus-ATA mixture was transferred to confluent cell monolayers in 6-well plates, incubated at 37°C for 2 h and subjected to plaque assay. (B) ATA inhibits enzymatic activity of NA derived from WSN and H274Y viruses. WSN (•) and H274Y (▪) viruses were incubated with increasing concentrations of ATA and NA enzymatic activity was determined by a chemiluminescent assay. Samples were tested in quadruplicates and presented as means and standard deviations (error bars).