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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.


Phosphoproteome profiling of JEV-infected cells in response to CW-33. MS/MS spectrum of the phosphopeptide IQEQESpSpGEEDSDLSPEER matched in protein phosphatase 1 inhibitor 2 (I-2) is shown (A); regulated proteins identified in infected cells in response to CW-33 were categorized using PANTHER classification system (B); western blot analysis of lysates from treated and mock cells was performed; the blots were probed with anti-phospho-Nedd4 antibodies (C); infected cells were treated with I-2 for 36 h, then harvested for analyzing sub-G1 phase using PI staining (D). ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
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ijms-17-01386-f008: Phosphoproteome profiling of JEV-infected cells in response to CW-33. MS/MS spectrum of the phosphopeptide IQEQESpSpGEEDSDLSPEER matched in protein phosphatase 1 inhibitor 2 (I-2) is shown (A); regulated proteins identified in infected cells in response to CW-33 were categorized using PANTHER classification system (B); western blot analysis of lysates from treated and mock cells was performed; the blots were probed with anti-phospho-Nedd4 antibodies (C); infected cells were treated with I-2 for 36 h, then harvested for analyzing sub-G1 phase using PI staining (D). ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.

Mentions: To analyze the possible inhibitory mechanism(s) of CW-33 on JEV-induced intracellular Ca2+ that linked with the activation of Akt/mTOR, and Jak/STAT signal, phosphoproteomic profiling of infected cells treated with or without CW-33 was determined using enriched phosphopeptides by the TiO2-PDMS plate and quantitative LC-MS/MS (Supplementary Table S1). Figure 8A showed MS/MS spectra for mapping phosphopeptides of protein phosphatase inhibitor 2 (I-2). Protein class analysis of proteins identified using PANTHER classification system indicated that 21.6% (11/51) belonged to the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, Ras-related GTP-binding protein C, putative 3-phosphoinositide-dependent protein kinase 2, Serine/threonine-protein kinase SIK3, and Serine/threonine-protein kinase N2 (Figure 8B). Western blotting analysis of phosphorylation status of identified proteins like E3 ubiquitin-protein ligase NEDD4-like showed the consistent pattern of protein phosphorylation in treated infected cells with the phosphoproteomic profiling identified by LC-MS/MS (Figure 8C). In addition, protein phosphatase-1 inhibitor-2 (I-2) identified was decreased by nearly 60% in JEV-infected cells, but regained in infected cells post-treatment with CW-33. Moreover, I-2 was used to treat JEV-infected cells for ascertaining the relationship of JEV infection with I-2 (Figure 8D). Interestingly, I-2 at 5 nM significantly reduced the sub-G1 (apoptotic) fraction in JEV-infected cells. The result indicated that CW-33 regulated enzyme modulators like I-2, Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and Ras-related GTP-binding protein, which are involved in the activation of Akt/mTOR and/or Jak/STAT in infected cells.


Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload
Phosphoproteome profiling of JEV-infected cells in response to CW-33. MS/MS spectrum of the phosphopeptide IQEQESpSpGEEDSDLSPEER matched in protein phosphatase 1 inhibitor 2 (I-2) is shown (A); regulated proteins identified in infected cells in response to CW-33 were categorized using PANTHER classification system (B); western blot analysis of lysates from treated and mock cells was performed; the blots were probed with anti-phospho-Nedd4 antibodies (C); infected cells were treated with I-2 for 36 h, then harvested for analyzing sub-G1 phase using PI staining (D). ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
© Copyright Policy
Related In: Results  -  Collection

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ijms-17-01386-f008: Phosphoproteome profiling of JEV-infected cells in response to CW-33. MS/MS spectrum of the phosphopeptide IQEQESpSpGEEDSDLSPEER matched in protein phosphatase 1 inhibitor 2 (I-2) is shown (A); regulated proteins identified in infected cells in response to CW-33 were categorized using PANTHER classification system (B); western blot analysis of lysates from treated and mock cells was performed; the blots were probed with anti-phospho-Nedd4 antibodies (C); infected cells were treated with I-2 for 36 h, then harvested for analyzing sub-G1 phase using PI staining (D). ** p value < 0.01; *** p value < 0.001 compared with mock-treated infected cells.
Mentions: To analyze the possible inhibitory mechanism(s) of CW-33 on JEV-induced intracellular Ca2+ that linked with the activation of Akt/mTOR, and Jak/STAT signal, phosphoproteomic profiling of infected cells treated with or without CW-33 was determined using enriched phosphopeptides by the TiO2-PDMS plate and quantitative LC-MS/MS (Supplementary Table S1). Figure 8A showed MS/MS spectra for mapping phosphopeptides of protein phosphatase inhibitor 2 (I-2). Protein class analysis of proteins identified using PANTHER classification system indicated that 21.6% (11/51) belonged to the enzyme modulator category, such as protein phosphatase inhibitor 2 (I-2), Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, Ras-related GTP-binding protein C, putative 3-phosphoinositide-dependent protein kinase 2, Serine/threonine-protein kinase SIK3, and Serine/threonine-protein kinase N2 (Figure 8B). Western blotting analysis of phosphorylation status of identified proteins like E3 ubiquitin-protein ligase NEDD4-like showed the consistent pattern of protein phosphorylation in treated infected cells with the phosphoproteomic profiling identified by LC-MS/MS (Figure 8C). In addition, protein phosphatase-1 inhibitor-2 (I-2) identified was decreased by nearly 60% in JEV-infected cells, but regained in infected cells post-treatment with CW-33. Moreover, I-2 was used to treat JEV-infected cells for ascertaining the relationship of JEV infection with I-2 (Figure 8D). Interestingly, I-2 at 5 nM significantly reduced the sub-G1 (apoptotic) fraction in JEV-infected cells. The result indicated that CW-33 regulated enzyme modulators like I-2, Rho GTPase-activating protein 35, ARF GTPase-activating protein GIT2, and Ras-related GTP-binding protein, which are involved in the activation of Akt/mTOR and/or Jak/STAT in infected cells.

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.