Repurposing Papaverine as an Antiviral Agent against Influenza Viruses and Paramyxoviruses

Abstract

Influenza viruses are highly infectious and are the leading cause of human respiratory diseases and may trigger severe epidemics and occasional pandemics. Although antiviral drugs against influenza viruses have been developed, there is an urgent need to design new strategies to develop influenza virus inhibitors due to the increasing resistance of viruses toward currently available drugs. In this study, we examined the antiviral activity of natural compounds against the following influenza virus strains: A/WSN/33 (H1N1), A/Udorn/72 (H3N2), and B/Lee/40. Papaverine (a nonnarcotic alkaloid that has been used for the treatment of heart disease, impotency, and psychosis) was found to be an effective inhibitor of multiple strains of influenza virus. Kinetic studies demonstrated that papaverine inhibited influenza virus infection at a late stage in the virus life cycle. An alteration in influenza virus morphology and viral ribonucleoprotein (vRNP) localization was observed as an effect of papaverine treatment. Papaverine is a well-known phosphodiesterase inhibitor and also modifies the mitogen-activated protein kinase (MAPK) pathway by downregulating the phosphorylation of MEK and extracellular signal-regulated kinase (ERK). Thus, the modulation of host cell signaling pathways by papaverine may be associated with the nuclear retention of vRNPs and the reduction of influenza virus titers. Interestingly, papaverine also inhibited paramyxoviruses parainfluenza virus 5 (PIV5), human parainfluenza virus 3 (HPIV3), and respiratory syncytial virus (RSV) infections. We propose that papaverine can be a potential candidate to be used as an antiviral agent against a broad range of influenza viruses and paramyxoviruses.IMPORTANCE Influenza viruses are important human pathogens that are the causative agents of epidemics and pandemics. Despite the availability of an annual vaccine, a large number of cases occur every year globally. Here, we report that papaverine, a vasodilator, shows inhibitory action against various strains of influenza virus as well as the paramyxoviruses PIV5, HPIV3, and RSV. A significant effect of papaverine on the influenza virus morphology was observed. Papaverine treatment of influenza-virus-infected cells resulted in the inhibition of virus at a later time in the virus life cycle through the suppression of nuclear export of vRNP and also interfered with the host cellular cAMP and MEK/ERK cascade pathways. This study explores the use of papaverine as an effective inhibitor of both influenza viruses as well as paramyxoviruses.

Keywords: ERK; MAPK; MEK; cAMP; influenza virus; inhibitors; nuclear export; papaverine; paramyxovirus; phosphodiesterase; vRNP.

FIG 1

(A) Antiviral effect of compounds (50 μM each) on influenza virus strains A/WSN/33 (H1N1) (blue), A/Udorn/72 (H3N2) (salmon), and B/Lee/40 (green). Data represent the average value of triplicate experiments with the standard deviation shown as error bars. (B) Molecular structure of papaverine (PAP) (drawn by ChemDraw [PerkinElmer Informatics]) and its effect on the growth of different influenza virus strains in a dose-dependent manner. (C) Virus growth in the presence of the indicated concentrations of papaverine was measured by plaque assay. The data represent one representative experiment of three replicates. The asterisk indicates statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (D) Confocal microscopy analysis was performed with the cells infected with influenza virus and treated with various papaverine concentrations. The cells were fixed and stained with influenza A-specific M2 antibody or influenza B-specific HN antibody followed by Alexa Fluor 488 conjugated host-specific secondary antibody treatment. Virus is shown in green color, and DAPI staining is in blue.

FIG 2

TOA and TOE assays were performed to identify the stage in the A/WSN/33 (H1N1) virus life cycle that is impacted by papaverine. (A) Schematic representation of the assays in which papaverine was added 0, 2, 4, 8, 12, or 16 hpi (top) or eliminated 0, 2, 4, 5, 6, or 8 hpi from the culture medium (bottom). For the pretreatment analysis, cells were treated with papaverine 4 h before virus infection, and culture medium was replaced with fresh medium without papaverine immediately after virus infection. Virus was harvested 24 hpi, and plaque assays were performed to calculate the virus titer. (B and C) The results of plaque assays are shown as the percentage of virus titer compared to that of the DMSO control. The pretreatment analysis bar is shown in black. The data denotes the mean ± standard deviation from triplicate experiments. The asterisk indicates statistical significance (*, P < 0.05; **, P < 0.01).

FIG 3

A/WSN/33 virus was incubated with either DMSO or papaverine for 1 h at 37°C. (A) The neuraminidase activity of the virus treated with DMSO or papaverine is displayed. The fluorescence intensity values were normalized with the control reaction. (B) The hemagglutination of RBCs by the virus with and without papaverine is shown. (C) Virus was grown with DMSO or papaverine at indicated concentrations, and semiquantitative RT-PCR was performed using the isolated RNA. The amplified products were collected at 34, 36, 38, and 40 cycles for each sample and analyzed by agarose gel electrophoresis. (D) Electron micrographs of negatively stained A/WSN/33 virus grown in the presence of DMSO (top) or papaverine (50 μM) (bottom) are displayed. (E) The length of 280 particles was measured from each sample and the distribution of the length of the particles is shown.

FIG 4

Papaverine interferes with the host cAMP and MEK/ERK signaling pathways and also inhibits viral nuclear export. (A) pERK, ERK, pMEK, MEK, NP, and actin levels are shown in HEK293T cells. The cells were infected with A/WSN/33 virus and treated with papaverine or DMSO. At 18 hpi, the cells were stimulated for an hour, lysed, and analyzed by Western blotting. The bands were quantified and the graph shows the mean protein levels calculated for triplicate experiments. The asterisk indicates statistical significance (*, P < 0.05). (B) Cells were infected with A/WSN/33 virus, papaverine treated, fixed at 8 hpi, and visualized with a confocal microscope. NP is rendered in green and the stained nuclei in blue. The nuclear accumulation of the vRNPs in papaverine-treated cells is shown by white arrows. (C) The level of PDE4D is shown in HEK293T cells infected with A/WSN/33 virus and treated with papaverine at the indicated concentrations. The blot shown here is the representative from the triplicate experiments, and the graph denotes the average of three independent experiments. (D) Confocal images of PF transfected HEK293T cells are displayed which were further infected with virus. The red color is the fluorescence signal from the PF reporter plasmid that proportionally corresponds to the cAMP level in the cells. The M2 protein of the A/WSN/33 virus is shown by green fluorescence, and the DAPI-stained nuclei is shown in blue. An increase in the fluorescent intensity of PF and a decrease in that of WSN M2 was observed with the increasing concentrations of papaverine.

FIG 5

Antiviral effect of papaverine on paramyxoviruses PIV5, RSV, and HPIV3 and rhabdovirus VSV. (A) The percentage of virus titer with increasing concentration of papaverine. The graphs represent the mean values along with the standard deviation of triplicate experiments. The asterisk indicates statistical significance (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (B) Cells grown on coverslips were infected with PIV5-GFP or VSV-GFP and treated with papaverine or DMSO. The fluorescence signal was observed using a confocal microscope. The cells infected with viruses are rendered in green and DAPI in blue. CV-1 and HEp2 cells infected with HPIV3 and RSV, respectively, and treated with papaverine were stained with Hema-3 reagent, and the cytopathic effect was observed under a light microscope.