Conceived and designed the experiments: AMH ASF AF EGB GVD RH XL. Performed the experiments: AMH ASF AF EGB GVD RH. Analyzed the data: AMH ASF AF EGB GVD RH XL. Contributed reagents/materials/analysis tools: RH XL. Wrote the paper: AMH ASF AF EGB RH XL.
The authors have declared that no competing interests exist.
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.
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.
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.
Influenza viruses cause a highly contagious respiratory tract infection. The frequent mutations of influenza genes, particularly those encoding surface hemagglutinin (HA) and neuraminidase (NA) proteins, allow the virus to evade the host immune system. This gives rise to new infectious strains responsible for annual epidemics associated with significant morbidity and mortality
Vaccines, either inactivated or live attenuated viruses, offer the best protection against influenza infection by inducing neutralizing antibodies against HA and NA antigens of specific influenza strains
Presently, only two classes of antiviral agents have been developed and approved for prophylaxis and treatment of seasonal influenza infection
Aurintricarboxylic acid (ATA) is a polyaromatic carboxylic acid derivative
Madin-Darby canine kidney (MDCK) cells (ATCC: CCL-34) were obtained from the American Type Culture Collection (Manassas, VA, USA) and were grown in modified minimum essential medium (modified MEM) containing Earle's balanced salts and supplemented with 2 mM L-glutamine, 1.5 g/l sodium bicarbonate (pH 7.2), 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 10% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin (Invitrogen, Carlsbad, CA) in a humidified atmosphere of 5% CO2. All viruses were amplified and titrated in MDCK cells and stored at −80°C until use. Influenza A strains A/Puerto Rico/8/34 (H1N1) (hereafter referred to as PR8), A/New Caledonia/20/99 (H1N1) (hereafter referred to as NC), and A/New York/55/01 (H3N2) (hereafter referred to as NY) were kindly provided by Dr. Jim Robertson at the National Institute for Biological Standards and Control (Potters Bar, UK). The oseltamivir-resistant influenza virus A/WSN/33 with the substitution of H274Y was made by reverse genetics and provided by Dr. Guy Boivin (Laval University, Quebec City, QC, Canada) and is hereafter referred to as H274Y in this paper. The parental virus influenza A/WSN/33 (H1N1) virus (hereafter referred to as WSN) was obtained from Dr. Earl Brown at University of Ottawa, Ottawa, ON, Canada. Influenza B/Singapore/222/97 virus (hereafter referred to as B) was provided by Dr. Kathryn Wright (Biochemistry, Microbiology and Immunology Department, University of Ottawa, Canada). Confluent MDCK monolayers in 6 or 24-well plates were washed twice with PBS and incubated with viruses at a multiplicity of infection (MOI) of 0.001 in MEM for 2 h at 37°C. After viral adsorption, media was removed, cells were washed twice with PBS and incubated with post-adsorption medium [MEM with 2 mM L-glutamine and Earle's balanced salts supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin, 25 mM HEPES buffer (pH 7.2), 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 2 µg/ml L-1-(tosylamido-2-phenyl) ethyl chloromethyl ketone (TPCK)-treated trypsin (Invitrogen, Carlsbad, CA)]. Plates were incubated for 48 h at 37°C, 5% CO2.
Aurintricarboxylic acid (ATA), amantadine hydrochloride (AH), and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NAA) were from Sigma (St. Louis, MO, USA). Stock solutions of each drug were prepared immediately before use in dimethyl sulfoxide (DMSO) and filtered using a 0.22 nm filter.
Neutral red is a vital dye that is incorporated into the vacuoles of viable cells, can be detected spectrophotometrically and is used to determine cell viability. The in vitro efficacy and toxicity of antiviral compounds have previously been determined by measuring the uptake of neutral red
Total RNA was extracted from cells 48 h following infection with influenza PR8 and treatment with ATA using an RNeasy mini kit (Qiagen Inc. Valencia, CA). Extracted RNA was treated with DNAse I (Ambion Inc., Streetsville, Ontario, Canada) and single step reverse transcription-PCR was performed using a Titan One Tube RT-PCR kit (Roche Applied Science, 68298 Mannheim, Germany) according to manufacturer's instructions. Influenza PR8 nucleoprotein RNA was amplified from 200 ng RNA in a total reaction volume of 50 µl using the forward and reverse primers
Extracellular influenza A antigens were detected in cell culture supernatants using a commercial ELISA kit (Takara Bio Inc., Otsu, Shiga, Japan). Culture media were removed 48 h after cells were infected with virus and treated with ATA, AH or both. Supernatants were clarified by centrifugation at 5000×g for 5 min. Samples, positive control and standards diluted in diluent/lysis buffer were added to individual wells (100 µl/well), and incubated at 37°C for 1 h with immobilized monoclonal antibody directed against influenza A nucleoprotein (NP), then were washed three times with PBS containing 0.1% Tween-20. Samples were incubated with biotinylated rabbit polyclonal anti-influenza virus antibodies for 1 h at 37°C. Following three washes, streptavidin-o-phenylenediamine dihydrochloride conjugates were added to wells and the plate incubated for 30 min at 37°C. Wells were washed four times and incubated with the substrate solution consisting of hydrogen peroxide and tetramethylbenzidine at room temperature for 15 min. The reaction was stopped by the addition of 1 N H2SO4 prior to absorbance reading at 450 nm using a Synergy™ 2 Multi-Mode Microplate Reader. Viral abundance was calculated as hemagglutination (HA) units from a standard curve using a positive control with known HA content. One HA unit is equal to the quantity of virus required to completely aggregate erythrocytes in an HA assay (100 µl of 0.25% v/v). The commercial ELISA kit has certain limitations as it employs the anti-nucleoprotein as the capture antibodies coated on the plates and anti-influenza viral proteins (total influenza viral proteins including hemagglutinins) as the detecting antibodies in solution for sandwich ELISA. Because hemagglutinins vary from strain to strain in terms of amino acid sequences or the ratio of viral proteins, one cannot directly compare the HA units between two different strains. Therefore, we present the results as percentage of HA units from treated infected samples relative to that from untreated infected control for the same strain.
Confluent cells in 6 well plates were washed twice with PBS and inoculated with 1 ml MEM containing PR8 for 2 h at 37 °C. Inoculums were aspirated, cells were washed twice with PBS and treated with 1% DMSO, 100 µg/ml ATA, 100 µg/ml AH or 100 µg/ml NAA for 48 h at 37°C. Cells were scraped off wells and collected by centrifugation at 1600×g for 5 min. Supernatants were transferred to a new tube, then cells were lysed by 2 cycles of freezing and thawing. Supernatants and lysates were subjected to plaque assay as described previously
PR8, NC, NY, WSN, H274Y and influenza B viruses, at a concentration of ∼100 plaque forming units (pfu), were incubated with ATA at 37°C for 30 min. 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 as described previously
Confluent cells in 6 well plates were inoculated with PR8 virus for 2 h at 37°C, then treated with DMSO, 100 µg/ml ATA, 100 µg/ml AH in post-adsorption medium for 24 h at 37°C. Cells were scraped off wells and centrifuged at 1600×g for 5 min. Medium was discarded and cells were incubated with ice-cold fixative (2.5% glutaraldehyde in 0.2 M cacodylate buffer, pH 7.4) for 50 min, with gentle agitation. Cells were pelleted by centrifugation at 20,000×g for 2 min at room temperature. Cell pellets were re-suspended in 0.5 ml fixative then rinsed in 0.5 M cacodylate buffer twice for 10 min and post-fixed with 2% osmium tetroxide for 2 h. The fixed cells were washed with water twice for 10 min, dehydrated with increasing concentrations of ethanol from 50 to 100% and embedded in spurr resin. Thin (70–80 nm) sections were cut on an ultramicrotome and counter stained with uranyl acetate and lead citrate. The sections were viewed and photographed on a JEOL 1010 transmission electron microscope.
The NA-Star® Influenza Neuraminidase Inhibitor Resistance Detection Kit (Applied Biosystems, Foster City, CA, USA) was used to measure the inhibition of NA activity as described previously
All data were expressed as means ± standard deviation. NA inhibition assay and IC50 calculations were determined using nonlinear curve fit in GraphPad Prism version 5. Statistical analysis was conducted using either one-way or two-way ANOVA when appropriate. To adjust for multiple comparisons, Bonferroni comparison post test was used. P value of <0.05 was regarded as statistically significant.
We and others have reported that ATA can inhibit certain RNA viruses; therefore, we postulated that ATA may also inhibit the replication of influenza viruses. To evaluate the ability of ATA to protect MDCK cells from influenza infection, cells inoculated with different influenza A viral strains were incubated in the presence or absence of ATA (50 or 100 µg/ml) for 2 days. Microscopic examination revealed a drastic reduction of influenza-induced CPE following treatment with ATA (data not shown). The viability of MDCK cells infected with influenza A viruses and incubated in the presence or absence of ATA was then assayed by incorporation of neutral red dye. Infected cells treated with ATA had increased neutral red uptake (
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.
(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).
To examine the prophylactic potential of ATA against influenza A infection, MDCK cells were exposed to ATA 24 h prior to viral infection. The cell viability was then assessed by measuring neutral red uptake. We found that pre-exposing MDCK cells to ATA 24 h prior to infection did not protect cells from virus-induced CPE (data not shown). Collectively, these data suggest that ATA is a potent anti-influenza agent with relatively low toxicity in tissue culture, as demonstrated by the SI value of 88.8.
To examine whether the protective effect of ATA is due to inhibition of viral replication, the level of influenza nucleoprotein RNA isolated from PR8-infected MDCK cells was determined by reverse-transcription PCR. Cells infected with PR8 had low levels of β -actin RNA (
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.
To determine if ATA treatment also reduces the level of influenza viruses released into the medium, supernatants from ATA-treated infected cell cultures were subjected to the analyses of viral proteins by ELISA. As shown in
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.
Since an ELISA measures both infectious and non-infectious particles, we determined whether ATA treatment specifically reduces the abundance of infectious particles. Cells were infected with PR8, then exposed to ATA or other previously established anti-influenza agents, AH, an M2 blocker or NAA, a neuraminidase inhibitor. Virus titres, of both the cell lysate and supernatant fraction, were determined by plaque assay. ATA treatment significantly reduced both the cell-associated and extracellular virus yields when compared to either AH or NAA (
Virus yield (PFU/ml) | ||
Treatment | Cells | Medium |
No treatment | 1×107 | 1×107 |
DMSO | 1×107 | 1×107 |
ATA | 5×104 | 1.3×104 |
AH | 4.7×105 | 3×105 |
NAA | 6.2×106 | 4.7×106 |
Confluent MDCK cells in 6 well plates were infected with 0.001 MOI of influenza A PR8 virus and incubated for 48 h with 100 µg/ml of AH, ATA, NAA or DMSO alone. Culture media and cell lysates were collected and viral titers were determined by plaque assay.
Upon demonstrating that ATA protects MDCK cells from influenza infection by reducing viral replication and release, we sought to investigate the mechanism underlying the anti-influenza activities of ATA. The protection of influenza-infected MDCK cells should be enhanced by simultaneous treatment with two antivirals acting via different mechanisms. Therefore, MDCK cells infected with influenza were treated concomitantly with ATA and an agent with a known antiviral mechanism. As shown in
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.
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.
To begin to elucidate how ATA protects cells from influenza infection, we investigated whether ATA elicits its inhibitory actions directly on the virus. In previous experiments, the consequences of ATA were examined after viral infection. To determine if ATA inhibits influenza viruses directly, MDCK cells were infected with viruses that had been pre-incubated with ATA, and then subjected to plaque assay. As shown in
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.
To gain insight into how ATA directly inhibits influenza virus, MDCK cells infected with PR8 and treated with ATA or AH were examined by election microscopy. Compared to untreated PR8-infected cells, or those incubated with AH, ATA treatment was found to induce viral aggregation on the cell surface (
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.
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).
Next we tested the effect of ATA on a common oseltamivir-resistant viral strain, i.e. WSN (H1N1) virus with the substitution at H274Y, in comparison with the parental wild-type WSN H1N1 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).
The influenza virus is highly contagious and results in significant morbidity and mortality
Antiviral drugs currently in use (such as the M2 blockers amantadine and rimantidine and NA inhibitors oseltamivir and zanamivir) can reduce the duration of flu symptoms. However, a single mutation can render influenza viruses resistant to antiviral drugs such as M2 blockers or NA inhibitors
Although ATA has been reported to have antiviral activity against human immunodeficiency virus
During the preparation of this manuscript, we noted that Hung et al.
Since inhibition of influenza by ATA and AH is mediated by two distinct mechanisms, it is not surprising that we observed additive effects upon simultaneous treatment with both compounds. Recently the Advisory Committee on Immunization Practices (ACIP) recommended against the use of amantadine or rimantidine to treat influenza infection
The toxicity of ATA will need to be evaluated further in animals. In this study, we showed that ATA is associated with relatively low toxicity in tissue cultures, with the SI being around 88.8. Although in vivo toxicity studies of ATA are rather limited, previous research in hamsters has shown that infusion of ATA was well tolerated in a dose of up to 1 mg/kg/hour for 2 weeks
In short, ATA is an NA inhibitor that may prove to be a valuable inclusion to the current arsenal of anti-influenza agents. The data presented here provide compelling evidence to further study the anti-influenza potential of ATA in animal models.
We thank Drs Michael Rosu-Myles and Shiv Prasad (Health Canada) for providing constructive comments on the manuscript. We also wish to thank Caroline Gravel, Monika Tocchi for technical assistance.