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Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

View Article: PubMed Central - PubMed

ABSTRACT

--: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Timeofaddition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

No MeSH data available.


Effect of CW-33 on intracellular Ca2+ and mitochondrial membrane potential (ΔΨM) in JEV-infected cells. Infected cells were treated with CW-33 for 36 h, harvested and stained using Fluo-3/AM, and then analyzed by flow cytometry with excitation and emission spectra of 506 and 526 nm, respectively (A). Cells were also stained using a JC-1 dye, and then the green fluorescence of JC-1 monomer in the cells was photographed using a fluorescent microscope (B), scale bars = 50 µm.
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ijms-17-01386-f006: Effect of CW-33 on intracellular Ca2+ and mitochondrial membrane potential (ΔΨM) in JEV-infected cells. Infected cells were treated with CW-33 for 36 h, harvested and stained using Fluo-3/AM, and then analyzed by flow cytometry with excitation and emission spectra of 506 and 526 nm, respectively (A). Cells were also stained using a JC-1 dye, and then the green fluorescence of JC-1 monomer in the cells was photographed using a fluorescent microscope (B), scale bars = 50 µm.

Mentions: Since calcium overload could be involved in the regulation of apoptosis via mitochondrial damage to activate caspase cascade [17,18,19], analysis of intracellular Ca2+ accumulation and mitochondria membrane potential (MMP) was performed using flow cytometry (Figure 6A) and JC-1 staining (Figure 6B). Flow cytometric analysis of intracellular Ca2+ with the dye FLUO3/AM revealed JEV causing the release of ER Ca2+ into cytosol, as well as CW-33 decreasing the cytosolic Ca2+ concentration in both cell types infected with JEV (Figure 6A). Similarly, JC-1 staining indicated that JEV infection triggered the green fluorescence of JC-1 monomer increase in cells as low MMP, while CW-33 significantly reduced the green fluorescence of JC-1 monomer in infected cells, indicating the increase of MMP (Figure 6B). Results revealed CW-33 reducing JEV-induced calcium overload, and then recovering the MMP of infected cells, which corrected the inhibitory effect of CW-33 on JEV-induced apoptosis.


Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload
Effect of CW-33 on intracellular Ca2+ and mitochondrial membrane potential (ΔΨM) in JEV-infected cells. Infected cells were treated with CW-33 for 36 h, harvested and stained using Fluo-3/AM, and then analyzed by flow cytometry with excitation and emission spectra of 506 and 526 nm, respectively (A). Cells were also stained using a JC-1 dye, and then the green fluorescence of JC-1 monomer in the cells was photographed using a fluorescent microscope (B), scale bars = 50 µm.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5037666&req=5

ijms-17-01386-f006: Effect of CW-33 on intracellular Ca2+ and mitochondrial membrane potential (ΔΨM) in JEV-infected cells. Infected cells were treated with CW-33 for 36 h, harvested and stained using Fluo-3/AM, and then analyzed by flow cytometry with excitation and emission spectra of 506 and 526 nm, respectively (A). Cells were also stained using a JC-1 dye, and then the green fluorescence of JC-1 monomer in the cells was photographed using a fluorescent microscope (B), scale bars = 50 µm.
Mentions: Since calcium overload could be involved in the regulation of apoptosis via mitochondrial damage to activate caspase cascade [17,18,19], analysis of intracellular Ca2+ accumulation and mitochondria membrane potential (MMP) was performed using flow cytometry (Figure 6A) and JC-1 staining (Figure 6B). Flow cytometric analysis of intracellular Ca2+ with the dye FLUO3/AM revealed JEV causing the release of ER Ca2+ into cytosol, as well as CW-33 decreasing the cytosolic Ca2+ concentration in both cell types infected with JEV (Figure 6A). Similarly, JC-1 staining indicated that JEV infection triggered the green fluorescence of JC-1 monomer increase in cells as low MMP, while CW-33 significantly reduced the green fluorescence of JC-1 monomer in infected cells, indicating the increase of MMP (Figure 6B). Results revealed CW-33 reducing JEV-induced calcium overload, and then recovering the MMP of infected cells, which corrected the inhibitory effect of CW-33 on JEV-induced apoptosis.

View Article: PubMed Central - PubMed

ABSTRACT

--: Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, has five genotypes (I, II, III, IV, and V). JEV genotype I circulates widely in some Asian countries. However, current JEV vaccines based on genotype III strains show low neutralizing capacities against genotype I variants. In addition, JE has no specific treatment, except a few supportive treatments. Compound CW-33, an intermediate synthesized derivative of furoquinolines, was investigated for its antiviral activities against JEV in this study. CW-33 exhibited the less cytotoxicity to Syrian baby hamster kidney (BHK-21) and human medulloblastoma (TE761) cells. CW-33 dose-dependently reduced the cytopathic effect and apoptosis of JEV-infected cells. Supernatant virus yield assay pinpointed CW-33 as having potential anti-JEV activity with IC50 values ranging from 12.7 to 38.5 μM. Timeofaddition assay with CW-33 indicated that simultaneous and post-treatment had no plaque reduction activity, but continuous and simultaneous treatments proved to have highly effective antiviral activity, with IC50 values of 32.7 and 48.5 μM, respectively. CW-33 significantly moderated JEV-triggered Ca2+ overload, which correlated with the recovery of mitochondria membrane potential as well as the activation of Akt/mTOR and Jak/STAT1 signals in treated infected cells. Phosphopeptide profiling by LC-MS/MS revealed that CW-33 upregulated proteins from the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and putative 3-phosphoinositide-dependent protein kinase 2. These enzyme modulators identified were associated with the activation of Akt/mTOR and Jak/STAT1 signals. Meanwhile, I-2 treatment substantially inhibited the apoptosis of JEV-infected cells. The results demonstrated that CW-33 exhibited a significant potential in the development of anti-JEV agents.

No MeSH data available.