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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.


Related in: MedlinePlus

Modes of antiviral action of CW-33 against JEV. In a virucidal assay (A), JEV (106 pfu) was mixed with CW-33, then incubated at 4 °C for 1 h; 1000-fold dilution of the JEV/CW mixture was used to determine the residual infectivity using plaque assay. In the attachment assay (B), JEV (50 pfu) was mixed with CW-33, then immediately added onto the BHK-21 cell monolayer for 1 h at 4 °C. After washing twice with PBS, monolayer was overlaid with 2 mL of a methylcellulose medium for three days at 37 °C in a CO2 incubator. Attachment inhibition was calculated as residual plaques. In time-of-addition assay (C), infected cells were treated with CW-33 1 h prior (pre), simultaneous, or 1 h post-infection and washed with PBS 1 h after treatment; infected cells were also treated with CW-33 in a continuous mode without the washing step, and followed by plaque assay. Inhibitory ratio was calculated from ratio of experimental data to mock-treated control. ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
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ijms-17-01386-f005: Modes of antiviral action of CW-33 against JEV. In a virucidal assay (A), JEV (106 pfu) was mixed with CW-33, then incubated at 4 °C for 1 h; 1000-fold dilution of the JEV/CW mixture was used to determine the residual infectivity using plaque assay. In the attachment assay (B), JEV (50 pfu) was mixed with CW-33, then immediately added onto the BHK-21 cell monolayer for 1 h at 4 °C. After washing twice with PBS, monolayer was overlaid with 2 mL of a methylcellulose medium for three days at 37 °C in a CO2 incubator. Attachment inhibition was calculated as residual plaques. In time-of-addition assay (C), infected cells were treated with CW-33 1 h prior (pre), simultaneous, or 1 h post-infection and washed with PBS 1 h after treatment; infected cells were also treated with CW-33 in a continuous mode without the washing step, and followed by plaque assay. Inhibitory ratio was calculated from ratio of experimental data to mock-treated control. ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.

Mentions: To ascertain whether CW-33 directly acts on JEV particles, virucidal activity assays were examined (Figure 5A). In virucidal activity assays, JEV was pre-incubated with CW-33 at 4 °C for 1 h and the 1000-fold dilution of each mixture was added on to the cell monolayer for quantifying their residual infectivity by plaque assay (Figure 5A). CW-33 exhibited no significant inhibitory effect on residual infectivity compared to controls. Results revealed CW-33 did not directly damage JEV particles. Examining the effect of CW-33 on virus attachment was further performed using viral attachment assay (Figure 5B). JEV was mixed with various concentrations of CW-33, and then immediately added onto the BHK-21 cell monolayer. After incubating at 4 °C for allowing attachment alone, each mixture was removed and the cell monolayer washed with PBS; residual infectivity was determined using plaque assay. The result of viral attachment assay indicated that CW-33 exhibited no significant effect on virus attachment (Figure 5B). A JEV replicon-expressing EGFP system further confirmed that CW-33 did not directly interfere with the infectivity of virus particles and early entry into cells. CW-33 significantly inhibited the transcription and translation during the JEV replication cycle, which reduced the viral RNA genome and proteins in a concentration-dependent manner (Supplementary Figures S1 and S2).


Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload
Modes of antiviral action of CW-33 against JEV. In a virucidal assay (A), JEV (106 pfu) was mixed with CW-33, then incubated at 4 °C for 1 h; 1000-fold dilution of the JEV/CW mixture was used to determine the residual infectivity using plaque assay. In the attachment assay (B), JEV (50 pfu) was mixed with CW-33, then immediately added onto the BHK-21 cell monolayer for 1 h at 4 °C. After washing twice with PBS, monolayer was overlaid with 2 mL of a methylcellulose medium for three days at 37 °C in a CO2 incubator. Attachment inhibition was calculated as residual plaques. In time-of-addition assay (C), infected cells were treated with CW-33 1 h prior (pre), simultaneous, or 1 h post-infection and washed with PBS 1 h after treatment; infected cells were also treated with CW-33 in a continuous mode without the washing step, and followed by plaque assay. Inhibitory ratio was calculated from ratio of experimental data to mock-treated control. ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-17-01386-f005: Modes of antiviral action of CW-33 against JEV. In a virucidal assay (A), JEV (106 pfu) was mixed with CW-33, then incubated at 4 °C for 1 h; 1000-fold dilution of the JEV/CW mixture was used to determine the residual infectivity using plaque assay. In the attachment assay (B), JEV (50 pfu) was mixed with CW-33, then immediately added onto the BHK-21 cell monolayer for 1 h at 4 °C. After washing twice with PBS, monolayer was overlaid with 2 mL of a methylcellulose medium for three days at 37 °C in a CO2 incubator. Attachment inhibition was calculated as residual plaques. In time-of-addition assay (C), infected cells were treated with CW-33 1 h prior (pre), simultaneous, or 1 h post-infection and washed with PBS 1 h after treatment; infected cells were also treated with CW-33 in a continuous mode without the washing step, and followed by plaque assay. Inhibitory ratio was calculated from ratio of experimental data to mock-treated control. ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
Mentions: To ascertain whether CW-33 directly acts on JEV particles, virucidal activity assays were examined (Figure 5A). In virucidal activity assays, JEV was pre-incubated with CW-33 at 4 °C for 1 h and the 1000-fold dilution of each mixture was added on to the cell monolayer for quantifying their residual infectivity by plaque assay (Figure 5A). CW-33 exhibited no significant inhibitory effect on residual infectivity compared to controls. Results revealed CW-33 did not directly damage JEV particles. Examining the effect of CW-33 on virus attachment was further performed using viral attachment assay (Figure 5B). JEV was mixed with various concentrations of CW-33, and then immediately added onto the BHK-21 cell monolayer. After incubating at 4 °C for allowing attachment alone, each mixture was removed and the cell monolayer washed with PBS; residual infectivity was determined using plaque assay. The result of viral attachment assay indicated that CW-33 exhibited no significant effect on virus attachment (Figure 5B). A JEV replicon-expressing EGFP system further confirmed that CW-33 did not directly interfere with the infectivity of virus particles and early entry into cells. CW-33 significantly inhibited the transcription and translation during the JEV replication cycle, which reduced the viral RNA genome and proteins in a concentration-dependent manner (Supplementary Figures S1 and S2).

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 &mu;M. 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 &mu;M, 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.


Related in: MedlinePlus