A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants

Abstract

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs.

Keywords: 4-hydroxyquinolinone; antiviral; nucleoprotein; oligomerization; resistance.

Figure 1

Effects of NUD-1 and naproxen on nucleoprotein (NP) oligomerization. (A) Purified recombinant NP was analyzed using 10% SDS-PAGE followed by Coomassie brilliant blue staining. (B) The migration of protein markers (thyroglobulin, 669 kDa; apoferritin, 443 kDa; β-amylase, 200 kDa) and NP mixed with yeast (0.05, 0.15, 0.45, 1.35, and 4 μM) was analyzed using blue native polyacrylamide gel electrophoresis (BN-PAGE). (C) NP (2.5 μM, equivalent to 2 μg) was mixed with RNA (0.15, 0.45, 1.35, and 4 μM) in the absence of any compound or in the presence of 100 μM NUD-1 or naproxen and incubated at room temperature overnight before analysis via BN-PAGE. The intensity of the smear at the top of the gel (enclosed by bracket) was quantified using ImageJ software. The relative band intensity in the presence of NUD-1 or naproxen was calculated in reference to that in the absence of a compound. Three independent experiments were performed, and representative data are shown. (D) The relative band intensities of high-molecular-weight and low-molecular-weight NP treated with 1.35 µM RNA (no compound, lane 4; NUD-1, lane 8; naproxen, lane 12), and NP treated with 4 µM RNA (no compound, lane 5; NUD-1, lane 9; naproxen, lane 13) were quantified from three independent experiments. The asterisk indicates p < 0.05.

Figure 1

Effects of NUD-1 and naproxen on nucleoprotein (NP) oligomerization. (A) Purified recombinant NP was analyzed using 10% SDS-PAGE followed by Coomassie brilliant blue staining. (B) The migration of protein markers (thyroglobulin, 669 kDa; apoferritin, 443 kDa; β-amylase, 200 kDa) and NP mixed with yeast (0.05, 0.15, 0.45, 1.35, and 4 μM) was analyzed using blue native polyacrylamide gel electrophoresis (BN-PAGE). (C) NP (2.5 μM, equivalent to 2 μg) was mixed with RNA (0.15, 0.45, 1.35, and 4 μM) in the absence of any compound or in the presence of 100 μM NUD-1 or naproxen and incubated at room temperature overnight before analysis via BN-PAGE. The intensity of the smear at the top of the gel (enclosed by bracket) was quantified using ImageJ software. The relative band intensity in the presence of NUD-1 or naproxen was calculated in reference to that in the absence of a compound. Three independent experiments were performed, and representative data are shown. (D) The relative band intensities of high-molecular-weight and low-molecular-weight NP treated with 1.35 µM RNA (no compound, lane 4; NUD-1, lane 8; naproxen, lane 12), and NP treated with 4 µM RNA (no compound, lane 5; NUD-1, lane 9; naproxen, lane 13) were quantified from three independent experiments. The asterisk indicates p < 0.05.

Figure 2

Inhibitory effects of NUD-1 on viral transcription activity. In this experiment, 293T cells were co-transfected with the viral protein expression plasmids (pCAGGS-PA-WSN, pCAGGS-PB1-WSN, pCAGGS-PB2-WSN, and pCAGGS-NP-WSN), the model viral genome expression plasmid (pPolI/NP(0)GFP(0)), and the control plasmid pDsRed2-monomer-N1. Two hours post-transfection, the cells were treated with DMSO, oseltamivir (10 μM), or NUD-1 (10 μM) and further incubated for 24 h. (A) Illustration of the miningenome reporter system. (B) Transcription inhibition was assessed using a fluorescence microscope. Representative data from two independent experiments are shown. Scale bar, 200 μm. (C) Transcription activity (%) was calculated as the ratio of the number of GFP- and DsRed-positive cells in the presence of oseltamivir or NUD-1 in reference to the ratio of GFP- and DsRed-positive cells in DMSO-treated cells. Means ± standard deviations from three different microscopy fields are shown. Statistical analysis was done in comparison to the DMSO control. The asterisk indicates p < 0.001.

Figure 3

Effect of NUD-1 on the expression of viral proteins. Madin–Darby canine kidney cells were infected with A/WSN/33 virus (multiplicity of infection = 1) in the presence of oseltamivir (100 μM), NUD-1 (12.5 μM), or DMSO. Nine hours post-infection, the cells were collected for Western blotting. (A) Viral protein expression was detected by Western blotting using anti-PA, anti-NP, anti-M1, anti-HA, and anti-actin antibodies. (B) The band intensity was quantified using ImageJ software. Relative band intensity (%) in the presence of NUD-1 and oseltamivir was calculated in reference to DMSO (control). The level of actin was used for normalization. The data represent the means and standard deviations of three independent experiments. Statistical analysis was performed in comparison to DMSO control. The asterisk indicates p < 0.01.

Figure 4

Subcellular localization of nucleoprotein (NP). Madin–Darby canine kidney cells were infected with the A/WSN/33 virus (multiplicity of infection = 5) in the presence of DMSO, oseltamivir (16 μM), or NUD-1 (16 μM) and incubated for 9 h. (A) The cells were fixed and stained with anti-NP antibody followed by Alexa Fluor 488-conjugated secondary antibody to determine the subcellular localization of NP. One representative result from three independent experiments is shown. Scale bar, 25 μm. (B) The percentage of cells exhibiting both nuclear and cytoplasmic localization and only nuclear localization of NP was calculated by counting two microscopy fields per experiment. The presented data are the mean and standard deviation of three independent experiments with statistical analysis in comparison to DMSO control. The asterisk indicates a p value of less than 0.05.