Bafilomycin A1 and U18666A Efficiently Impair ZIKV Infection

Abstract

Zika virus (ZIKV) is a highly transmissive virus that belongs to the Flaviviridae family, which comprises several other pathogens that threaten human health. This re-emerging virus gained attention during the outbreak in Brazil in 2016, where a considerable number of microcephaly cases in newborns was associated with ZIKV infection during pregnancy. Lacking a preventive vaccine or antiviral drugs, efforts have been made to better understand the viral life cycle. In light of this, the relevance of the endosomal-lysosomal compartment for the ZIKV life cycle was investigated. A549 and SH-SY5Y cells were infected with either the African strain (associated with mild symptoms) or the French Polynesia strain (associated with neurological complications). For both strains, the V-ATPase inhibitor, bafilomycin A1, efficiently inhibited ZIKV entry and prevented the spread of the infection by interfering with viral maturation. Additionally, affecting cholesterol metabolism and transport with the drug U18666A, which inactivates late endosomes and lysosomes, impairs the viral life cycle. The data presented show a clear antiviral effect of two compounds that target the same compartments in different ways. This highlights the relevance of the endosomal-lysosomal compartment for the viral life cycle that should be considered as a target for antivirals.

Keywords: U18666A; Zika virus; antiviral effect; bafilomycin A1; endosomal–lysosomal compartment; viral life cycle.

Figure 1

Bafilomycin A1 prevents the establishment of ZIKV infection in A549 cells (A) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre-infection treatment). Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. Infected untreated cells were used as control; (B) Quantification of the amount of extracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre-infection treatment). Quantification was performed by RT-qPCR and infected untreated cells served as control; (C) Quantification of the number of intracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre-infection treatment). Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). Infected untreated cells were used as control; (D) Quantification of the number of extracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre-infection treatment). Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). Infected untreated cells served as control; (E) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre-infection treatment). Cells were fixed with ethanol:acetone (1:1) and nuclei were visualized with DAPI (blue) and the ZIKV envelope protein with a specific antibody (green). Pictures were taken with the 16× objective. * p ≤ 0.05; **** p ≤ 0.0001.

Figure 2

Bafilomycin A1 severely diminishes ZIKV infection in A549 cells. (A) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (post-infection treatment) during the indicated times. Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. Infected untreated cells were used as control; (B) Quantification of the amount of extracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (post-infection treatment) during the indicated times. Quantification was performed by RT-qPCR and infected untreated cells served as control; (C,D) Quantification of the number of intracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia (C) or the Uganda (D) strain and treated with 10 nM bafilomycin A1 (post-infection treatment) during the indicated times. Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). Infected untreated cells were used as control; (E,F) Quantification of the number of extracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia (E) or the Uganda (F) strain and treated with 10 nM bafilomycin A1 (post-infection treatment) during the indicated times. Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). Infected untreated cells served as control; (G) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (post-infection treatment) during the indicated times. Cells were fixed with ethanol:acetone (1:1) and nuclei were visualized with DAPI (blue) and the ZIKV envelope protein with a specific antibody (green). Pictures were taken with the 16× objective; (H) A549 cells were treated with 10 nM bafilomycin A1 during the indicated times and cell viability was quantified by PrestoBlue assay. Cells treated with 2% Triton X-100 (TX-100) served as positive control. Untreated cells were used for normalization. ns = not significant p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 3

Bafilomycin A1 treatment of ZIKV-infected A549 cells affects the infectiveness of the supernatant. (A) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 50 nM bafilomycin A1 (24 h treatment). Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. DMSO was included as vehicle control and infected untreated cells were used as control; (B) Quantification of the amount of extracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 50 nM bafilomycin A1 (24 h treatment). Quantification was performed by RT-qPCR. DMSO was used as vehicle control and infected untreated cells served as control; (C) Quantification of the number of intracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 50 nM bafilomycin A1 (24 h treatment). Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). DMSO was used and vehicle control and infected untreated cells served as control; (D) Quantification of the number of extracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 50 nM bafilomycin A1 (24 h treatment). Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). DMSO was used as vehicle control and infected untreated cells served as control; (E) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (post-infection treatment). The supernatants (used in Section 3.2) and cell lysates were analyzed after 24 hpi by Western blot with a specific antibody against ZIKV-prM/pr peptide. ß-Actin was used as loading control; (F) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 50 nM bafilomycin A1 (post-infection treatment) for 24 h. The supernatants and cell lysates were analyzed after 72 hpi by Western blot with a specific antibody against ZIKV-prM/pr peptide. β-Actin served as loading control; (G) A549 cells were treated with 50 nM bafilomycin A1 during 24 h and cell viability was quantified by PrestoBlue assay. Cells treated with DMSO 1:200 and 2% Triton X-100 (TX-100) served as vehicle and positive control, respectively. Untreated cells were used for normalization. ns = not significant p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 4

Furin inhibitor I diminishes ZIKV infectivity in A549 cells. (A) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 10 µM furin inhibitor I (24 h treatment). Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. DMSO was included as vehicle control and infected untreated cells were used as control; (B) Quantification of the amount of extracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 10 µM furin inhibitor I (24 h treatment). Quantification was performed by RT-qPCR. DMSO was used as vehicle control and infected untreated cells served as control; (C) Quantification of the number of intracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 10 µM furin inhibitor I (24 h treatment). Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). DMSO was used and vehicle control and infected untreated cells served as control; (D) Quantification of the number of extracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia or the Uganda strain for 72 h and treated with 10 µM furin inhibitor I (24 h treatment). Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). DMSO was used as vehicle control and infected untreated cells served as control; (E) A549 cells were treated with different concentrations of furin inhibitor I during 24 h and cell viability was quantified by PrestoBlue assay. Cells treated with DMSO 1:314 and 2% Triton X-100 (TX-100) served as vehicle control for the chosen concentration and positive control, respectively. Untreated cells were used for normalization. ns = not significant p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 5

Autophagy modulation in ZIKV-infected cells. (A–C) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre- and post-infection treatment) during 24 (A), 48 (B), and 72 (C) hpi. Cell lysates of the indicated times were analyzed by Western blot with specific antibodies against the ZIKV NS1, p62, LC3, and β-Actin. The latest was included as loading control; (D,E) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with either 5 mM 3-MA (D) or 100 nM rapamycin (E) (post-infection treatment) during the indicated times. Cells were fixed with ethanol:acetone (1:1) and analyzed by confocal laser scanning microscopy. Nuclei were visualized with a DAPI (blue) and the ZIKV envelope protein (green) and p62 (red) with specific antibodies.

Figure 5

Autophagy modulation in ZIKV-infected cells. (A–C) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 10 nM bafilomycin A1 (pre- and post-infection treatment) during 24 (A), 48 (B), and 72 (C) hpi. Cell lysates of the indicated times were analyzed by Western blot with specific antibodies against the ZIKV NS1, p62, LC3, and β-Actin. The latest was included as loading control; (D,E) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with either 5 mM 3-MA (D) or 100 nM rapamycin (E) (post-infection treatment) during the indicated times. Cells were fixed with ethanol:acetone (1:1) and analyzed by confocal laser scanning microscopy. Nuclei were visualized with a DAPI (blue) and the ZIKV envelope protein (green) and p62 (red) with specific antibodies.

Figure 6

U18666A decreases ZIKV infection in A549 cells. (A) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain and treated with 2 µg/mL U18666A (post-infection treatment) during the indicated times. Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. Infected untreated cells were used as control; (B) Quantification of the amount of extracellular ZIKV RNA of A549 cells infected with either the French Polynesia or the Uganda strain and treated with 2 µg/mL U18666A (post-infection treatment) during the indicated times. Quantification was performed by RT-qPCR and infected untreated cells served as control; (C,D) Quantification of the number of intracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia (C) or the Uganda (D) strain and treated with 2 µg/mL U18666A (post-infection treatment) during the indicated times. Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). Infected untreated cells were used as control; (E,F) Quantification of the number of extracellular infectious viral particles (VPs) from A549 cells infected with either the French Polynesia (E) or the Uganda (F) strain and treated with 2 µg/mL U18666A (post-infection treatment) during the indicated times. Quantification was performed in Vero cells by plaque assay and the number of infectious viral particles is expressed in plaques forming units per mL (pfu/mL). Infected untreated cells served as control; (G) A549 cells were treated with 2 µg/mL U18666A during the indicated times and cell viability was quantified by PrestoBlue assay. Cells treated with 2% Triton X-100 (TX-100) served as positive control. Untreated cells were used for normalization. ns = not significant p > 0.05; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.

Figure 7

U18666A affects the amount and the intracellular localization of ZIKV proteins in A549 cells. (A–D) A549 cells were infected with either the French Polynesia (A,C) or the Uganda (B,D) strain and treated with 2 µg/mL of U18666A (post-infection treatment) during the indicated times. Cells were fixed with 4% formaldehyde and analyzed by confocal laser scanning microscopy. Nuclei were visualized with a lamin A-specific antibody (blue), the ZIKV envelope protein with a specific antibody (green), p62 with a specific antibody (red) and free cholesterol with the filipin complex dye (cyan); Pictures were taken with either the 16× (A,B) or 100× objective (C,D); (E) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 2 µg/mL of U18666A (post-infection treatment). Cell lysates of the indicated times were analyzed by Western blot with specific antibodies against the ZIKV NS1, p62, LAMP2, and β-Actin. The latest was included as loading control; (F) Transmission electron microscopy images of U18666A-treated cells.

Figure 7

U18666A affects the amount and the intracellular localization of ZIKV proteins in A549 cells. (A–D) A549 cells were infected with either the French Polynesia (A,C) or the Uganda (B,D) strain and treated with 2 µg/mL of U18666A (post-infection treatment) during the indicated times. Cells were fixed with 4% formaldehyde and analyzed by confocal laser scanning microscopy. Nuclei were visualized with a lamin A-specific antibody (blue), the ZIKV envelope protein with a specific antibody (green), p62 with a specific antibody (red) and free cholesterol with the filipin complex dye (cyan); Pictures were taken with either the 16× (A,B) or 100× objective (C,D); (E) A549 cells were infected with either the French Polynesia or the Uganda strain and treated with 2 µg/mL of U18666A (post-infection treatment). Cell lysates of the indicated times were analyzed by Western blot with specific antibodies against the ZIKV NS1, p62, LAMP2, and β-Actin. The latest was included as loading control; (F) Transmission electron microscopy images of U18666A-treated cells.

Figure 8

U18666A affects ZIKV replication in A549 cells. (A) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with an MOI = 0.1 after 16 hpi with either the French Polynesia or the Uganda strain and pre-treated with 2 µg/mL U18666A (pre-infection treatment) for 24 h. Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. Infected untreated cells were used as control; (B) Quantification of the amount of intracellular ZIKV RNA of A549 cells infected with an MOI = 1 after 16 hpi with either the French Polynesia or the Uganda strain and pre-treated with 2 µg/mL U18666A (pre-infection treatment) for 24 h. Quantification was performed by RT-qPCR and the amount of ZIKV RNA was normalized to the amount of the RPL27 transcripts. Infected untreated cells were used as control; (C) A549 cells were infected with an MOI = 1 with either the French Polynesia or the Uganda strain and pre-treated with 2 µg/mL U18666A (pre-infection treatment) for 24 h. Cells were fixed with 4% formaldehyde after 16 hpi and analyzed by confocal laser scanning microscopy. Nuclei were visualized with a lamin A-specific antibody (blue) and the ZIKV envelope protein with a specific antibody (green). Pictures were taken with the 40× objective. (D) Quantification of the number of ZIKV-positive cells from Figure 8C. Quantification is referent to at least 100 cells from each condition; (E) Luciferase reported gene assay of ZIKV-replicating A549 cells after 24, 48 and 72 h treatment with 2 µg/mL U18666A; (F,G) Western blot analysis of A549 cells that were infected with an MOI = 0.1 (F) and MOI = 1 (G) with either the French Polynesia or the Uganda strain and pre-treated with 2 µg/mL U18666A (pre-infection treatment) for 24 h. Infection was detected by ZIKV E and NS1 specific antibodies. ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001.