Pharmacological Agent GW4869 Inhibits Tick-Borne Langat Virus Replication to Affect Extracellular Vesicles Secretion

Abstract

GW4869, a cell-permeable, selective inhibitor of neutral sphingomyelinase is a pharmacological agent that blocks the production and release of extracellular vesicles (EVs). Our previous studies have shown that GW4869 inhibits flaviviral loads in tick, mosquito and mammalian cells, including murine cortical neurons. Yet the mechanism(s) of GW4869 inhibitor upon viral infections were not addressed. In the current study, we focused on how GW4869 interferes with Langat Virus (LGTV, a tick-borne flavivirus) replication in ISE6 tick cells. First, we found that GW4869 is neither cytotoxic at tested doses of 50, 100, and 150 µM in tick cells, nor does it directly bind to the free LGTV present in cell culture supernatants. When tick cells were treated with GW4869, followed by infection with viral stock at dilutions of 10^-2, 10^-3, 10^-4 (the infectious dose determination by viral dilution assay), it affected LGTV replication in tick cells. A reduction in viral burden was noted in GW4869-treated tick cells, which constituted more than half the amount of decrease when compared to the mock control. Next, GW4869 treatment not only resulted in decreased LGTV transcript levels in tick cells and EVs derived from these infected cells, but also revealed diminished EVs concentrations. Enhanced IsSMase transcripts in the LGTV-infected group was noted upon GW4869 treatment, thus suggesting a host response to perhaps inhibit virus replication. In addition, GW4869 treatment reduced LGTV loads in density gradient EVs fractions, which correlated with decreased EVs concentration in those fractions. These data not only indicate that GW4869 affects LGTV replication, but that it also interferes with EV secretion and release from tick cells. Lastly, we found that GW4869 inhibits LGTV replication in tick cells but does not directly affect the infectivity of LGTV viral particles. Overall, our study suggests that GW4869 is a potential therapeutic inhibitor in controlling tick-borne diseases.

Keywords: EVs secretion; GW4869; Langat virus; cytotoxicity; tick cells; viral dilution; viral particle infectivity; viral replication.

Figure 1

LDH-Glo cytotoxicity and MTT assays performed in tick cells treated with GW4869. (A) Uninfected tick cells treated with GW4869 (at 0–150 µM doses) or mock (1.5% DMSO solution), showing LDH release measured by absorbance. (B) Cytotoxicity measurement at 150 µM of GW4869 treatment is shown independently, as a bar graph, in comparison to the 1.5% DMSO mock control. In both panels, (A,B), ns indicates not significant. (C) Microscopic images showing morphology of uninfected tick cells treated with GW4869 (at 50 or 150 µM doses), or mock control (1.5% DMSO) from untreated (UT), or 3 days post-GW4869-treatment groups. Tick cell images were obtained using the Cytation7 imager. Scale bar indicates 200 µm. (D) MTT assay performed on uninfected tick cells treated with GW4869 (at 50, or 150 µM doses) or mock control (1.5% DMSO) is shown. Each circle (open/close) or rhombus represents one experimental replicate. A sample size of n = 6 was used, with each replicate run in duplicate (in panels (A,B,D)). The p-value less than 0.05 is considered statistically significant.

Figure 2

GW4869 effects on free LGTV present in laboratory viral stock suspensions. (A) Schematics showing experimental plan for the incubation of LGTV laboratory viral stocks (undiluted suspension with 1 × 10⁸ pfu/mL or diluted as MOI 1) with GW4869 (1 µM) or with mock (0.01% DMSO) control (for 4 h at 37 °C incubation), followed by isolation of viral RNA. (B) RT-qPCR analysis showing LGTV viral loads in mock/GW4869-treated undiluted or diluted virus stocks. LGTV prM transcript levels were normalized to total viral RNA levels. Solid circles/triangles denote the mock-treated groups, whereas the squares/inverted triangles represent the GW4869-treated groups. Each circle/triangle/square/inverted triangle represents one experimental replicate (100 µL) of the respective virus suspension. The p-value less than 0.05 is considered statistically significant.

Figure 3

Determination of LGTV infectious dose in GW4869-treated (at 150 µM) tick cells. Bright-field (BF) and fluorescence images collected from viral dilution assay of LGTV-infected tick cells treated with either mock (1.5% DMSO) control (A) or with GW4869 (150 µM) treatment (B) from dilutions of 10⁻², 10⁻³ and 10⁻⁴ are shown. Blue is DAPI staining showing cell nuclei, and red staining detects LGTV envelope (E) protein. Differences in viral titers determined from mock or GW4869-treated tick cells are shown below images as Mock/GW4869 group TCID₅₀ values in PFU (plaque-forming units) per ml. Tick cell images were obtained using Cytation7 imager and a scale bar of 200 µm is shown for each image.

Figure 4

LGTV loads upon GW4869 treatment (low dose, 1 µM) in tick cells/EVs and EV release from tick cells. (A) RT-qPCR analysis showing LGTV loads in mock (0.01% DMSO) or GW4869-treated (1 µM, for 4 h) tick cells and EVs derived from these infected tick cells (MOI 1, 3 days post-infection, p.i.). (B) IsSMase transcript levels in mock/GW4869-treated tick cells infected with LGTV (MOI 1, 3 days p.i.) are shown. (C) Concentration of EVs determined by nCS1 analysis from mock/GW4869-treated tick cells infected with LGTV is shown. Graphical representations showing EV quantification by nCS1 analyzer in mock- (D) or GW4869-treated (E) tick cells infected with LGTV. The diameter of each particle and concentration of EVs was analyzed. Five-six independent replicates in duplicate were considered for RT-qPCR analysis and four independent replicates in duplicate were used for measuring EV concentration. LGTV and IsSMase transcripts were normalized with either total RNA or tick beta-actin transcript levels, respectively. Each solid circle denotes the mock-treated group, whereas each closed square represents the GW4869-treated groups (in panels (A,B)). Both groups were infected with LGTV. Each circle/square denotes data value from one independent culture well. The p-value less than 0.05 is considered as statistically significant.

Figure 5

LGTV loads upon GW4869 treatment (high dose, 150 µM) in tick cells/EVs and EV release from tick cells. (A) RT-qPCR analysis showing LGTV loads in mock (1.5% DMSO) or GW4869-treated (150 µM, for 4 h) tick cells and (B) in EVs derived from these infected tick cells (MOI 1, 3 days post-infection, p.i.). (C) Concentration of EVs determined by nCS1 analysis from mock/GW4869-treated (150 µM) tick cells infected with LGTV is shown. Graphical representations showing EV quantification by nCS1 analyzer in mock (D) or GW4869- (E) treated (150 µM) tick cells infected with LGTV. The diameter of each particle and concentration of EVs was analyzed. Five-six independent replicates in duplicate were considered for RT-qPCR analysis and five independent replicates in duplicate were used for measuring EV concentration. LGTV transcripts were normalized to total RNA levels, respectively. Each solid circles denotes a mock-treated group, whereas each closed square represents the GW4869-treated groups (in panels (A,B)). Both groups are infected with LGTV. Each circle/square denotes data value from one independent culture well. The p-value less than 0.05 is considered as statistically significant.

Figure 6

LGTV loads upon GW4869 treatment in EV fractions collected from density gradients. RT-qPCR analysis showing LGTV loads in mock-treated (0.01% DMSO) (A) or GW4869-treated (1 µM, for 4 h) (B) EV fractions (I–VI), derived from LGTV-infected-tick cells (with MOI 1, and 3 days post-infection). Both mock- and GW4869-treated groups were infected with LGTV. (C) Comparison of LGTV loads in fractions IV and V collected from mock- or GW4869-treated EVs derived from infected tick cells is shown. LGTV transcript levels were normalized to total RNA levels. Each closed-circle/square/triangle/inverted triangle or open circles denotes the mock- or GW4869-treated group in panels (A–C). The p-value less than 0.05 is considered as statistically significant, and letter b on fraction IV (in the mock-treated group) denotes significance. Immunoblotting analysis showing LGTV NS1 protein levels in mock- or GW4869-treated EVs fractions (I–VI) collected from infected tick cells (D,E). Total protein-profile gel images serve as loading controls (D,E). Densitometric analysis showing the differences in band intensities between different fractions (1–6) from LGTV-infected mock- (F) or GW4869-treated (G) EVs derived from tick cells.

Figure 7

Determination of EV concentrations in fraction IV upon GW4869 treatment. Concentration of EVs in fraction IV (A) determined by nCS1 analysis from mock/GW4869-treated (1 µM, for 4 h) tick cells infected with LGTV is shown. Graphical representation (generated by the instrument) showing EV quantification by nCS1 analyzer in mock- (B) or GW4869-treated (C) tick cells infected with LGTV. The diameter of each particle and concentration of EVs were analyzed at the same time. Three independent replicates (in duplicates) were considered for measuring EV concentration. Both mock- and GW4869-treated groups were infected with LGTV. In panel A, p-value less than 0.05 is considered as statistically significant in (A).

Figure 8

LGTV does not affect the viral particle infectivity in naïve tick cells and the model proposed for GW4869 effects in tick cells. RT-qPCR analysis showing LGTV viral loads at 24 h (A) or at 72 h (B) in naïve/uninfected tick cells that were infected via incubation with LGTV viral particles, treated with mock (1.5% DMSO, for 4 h) or with GW4869-treated (150 µM, for 4 h), virus groups. LGTV transcript levels were normalized to total RNA levels. Each open or closed circle denotes the mock-treated or GW4869-treated groups, respectively, in panels (A,B). The p-value less than 0.05 is considered as statistically significant. (C) LGTV induces the production of EVs and inhibits IsSMase sphingomyelinase. GW4869 inhibits LGTV viral replication and hampers the LGTV transmission/transport into EVs derived from infected tick cells. GW4869 reduces the EV numbers and concentrations by blocking the production and secretion of EVs. The numbers shown in the figure indicate the steps described.