Discovery of dapivirine, a nonnucleoside HIV-1 reverse transcriptase inhibitor, as a broad-spectrum antiviral against both influenza A and B viruses

Abstract

The emergence of multidrug-resistant influenza viruses poses a persistent threat to public health. The current prophylaxis and therapeutic interventions for influenza virus infection have limited efficacy due to the continuous antigenic drift and antigenic shift of influenza viruses. As part of our ongoing effort to develop the next generation of influenza antivirals with broad-spectrum antiviral activity and a high genetic barrier to drug resistance, in this study we report the discovery of dapivirine, an FDA-approved HIV nonnucleoside reverse transcriptase inhibitor, as a broad-spectrum antiviral against multiple strains of influenza A and B viruses with low micromolar efficacy. Mechanistic studies revealed that dapivirine inhibits the nuclear entry of viral ribonucleoproteins at the early stage of viral replication. As a result, viral RNA and protein synthesis were inhibited. Furthermore, dapivirine has a high in vitro genetic barrier to drug resistance, and its antiviral activity is synergistic with oseltamivir carboxylate. In summary, the in vitro antiviral results of dapivirine suggest it is a promising candidate for the development of the next generation of dual influenza and HIV antivirals.

Keywords: Broad-spectrum antiviral; Dapivirine; Drug resistance; Dual antiviral; Influenza virus.

Figure 1. Chemical structures and antiviral activity of the three HIV NNTIs

(A) Dapivirine; (B) Etavirine; (C) Rilpivirine;( D) EC₅₀ of the three NNTIs against A/California/07/2009 and B/Brisbane/60/2008 and CC₅₀ of the three NNTIs with a 48 h incubation time with MDCK cell line. The EC₅₀ and CC₅₀ values are the mean of two independent experiments ± standard deviation.

Figure 2. Antiviral activity of dapivirine against A/WSN/1933 (H1N1) and A/California/7/2009 (H1N1) with different cell lines at low and high MOI

(A) CC₅₀ of dapivirine against MDCK and A549 cell lines with a 48 h incubation time; The CC₅₀ values are the mean of two independent experiments ± standard deviation. (B) CC₅₀ of dapivirine against MDCK, HEK 293 or A549 cell lines with a 24 h incubation time. The concentration at 3 μM (Figure A) and 10 μM (Figure B) were indicated with arrows. The CC₅₀ values are the mean of two independent experiments ± standard deviation. (C) HEK293 or MDCK cells were infected with A/California/7/2009 (H1N1) at MOI of 0.01; 3 μM dapivirine or 1 μM oseltamivir carboxylate was added after viral infection. Viruses from supernatant were harvested 24 h p.i. and titers were determined by plaque assay. *** P < 0.001. The value of viral titer is the mean of two independent experiments ± standard deviation. (D) A549 cells were infected with A/WSN/1933 (H1N1) at MOI of 0.01; 3 μM dapivirine or 1 μM oseltamivir carboxylate was added after viral infection. Viruses from supernatant were harvested 24 h p.i. and the titers were determined by plaque assay. Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of viral titer is the mean of two independent experiments ± standard deviation. (E) A549 cells were infected with A/WSN/1933 (H1N1) at MOI of 0.01; DMSO (control) or 3 μM dapivirine was added after infection. Viruses from supernatant were harvested 2, 4, 6, 8, 12, 24, 48, 72, or 96 h post infection. Virus titers were determined by plaque assay.

Figure 3. Time-of-addition experiments

MDCK cells were infected with the A/WSN/33 (H1N1) virus at −2 h time point; viruses were first incubated at 4 °C for 1 h for attachment followed by 37 °C for 1 h for viral entry. At time point 0 h, cells were washed with PBS buffer and viruses were harvested at 12 h p.i. The titer of harvested virus was determined by plaque assay. Arrows indicate the period in which (A) 1 μM oseltamivir carboxylate or (B) 3 μM dapivirine was present. Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of viral titer is the mean of two independent experiments ± standard deviation.

Figure 4. Dapivirine inhibited the nuclear entry of vRNP complex at the early stage of viral replication

(A) Retention of influenza vRNPs in cytoplasm by dapivirine. A549 cells infected with the A/WSN/33 (H1N1) virus (MOI = 30) were treated with DMSO or 30 μM of dapivirine. After fixation at indicated times (2, 4, and 6 h p.i.), cells were stained with mouse anti-influenza A NP antibody, rabbit anti-M1, and DAPI to determine the viral NP, M1 protein, and nucleus, respectively. (B) CC₅₀ of dapivirine against A549 cell line with a 6 h incubation time. The concentration at 30 μM was indicated with an arrow. The CC₅₀ value is the mean of two independent experiments ± standard deviation. (C–F) Dapivirine reduced the levels of viral RNAs and proteins. A549 cells infected with the A/WSN/33 (H1N1) virus (MOI = 1) were treated with DMSO and 10 or 30 μM of dapivirine. At 6 h p.i., cells were harvested and total viral RNA was extracted and quantified by RT-qPCR (C and D). Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of RNA level is the mean of two independent experiments ± standard deviation. Viral protein levels were quantified by western blot (E) and immunofluorescence (F). (G–I) Mini-genome assay. A549 cells were transfected with a combination of plasmids for minigenome assay (see in Materials and methods). Dapivirine was added to the culture medium at 2 h post transfection. After 22 h incubation, cells were harvest and the activities of Firefly (G) and Renilla (H) luciferases were measured using the dual luciferease kit from Promega. Renilla was served as an internal control to normalize the transfection efficiency (I). Asterisks indicate statistically significant difference in comparison with the DMSO control (student’s t-test, *** P < 0.001). The value of luciferase signal is the mean of two independent experiments ± standard deviation.