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Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins.

Yu CY, Liang JJ, Li JK, Lee YL, Chang BL, Su CI, Huang WJ, Lai MM, Lin YL - PLoS Pathog. (2015)

Bottom Line: By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection.MFN1 was required for efficient antiviral retinoic acid-inducible gene I-like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death.Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I-like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.

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DENV infection impairs mitochondrial dynamics.(A and B) Diagram (A) and the results (B) of mitochondria intermixing assay. Mitochondria of A549 cells were labeled by transfection with mitoYFP or mitoCherry to distinguish the origin of each mitochondrion. Cells were mock or infected with DENV serotype 2 (multiplicity of infection [moi] 10) for 24 h before HVJ-E-mediated cell fusion. The fused cell hybrids with or without DENV infection were magnified and analyzed for green and red fluorescence intensity by use of ZEN lite 2011 (Carl Zeiss MicroImaging GmbH). Green: mitoYFP; red: mitoCherry; magenta: DENV NS3. (C to I) Time course study of DENV infection in human A549 cells with moi 10 for the indicated hours. Culture supernatants were harvested for virus titration (C) and release of lactic dehydrogenase (LDH) (D). Cell lysates were harvested for western blot analysis with the indicated antibodies (E) and RT-qPCR (F to I) for the indicated genes. Data are mean ± SD (n = 3 per group). The data for LDH release was compared by one way ANOVA and Bonferroni multiple-comparison test with use of Prism 5 (GraphPad; La Jolla, CA, USA). ns, no significance; *, p<0.05. Nocodazole treatment (NOC; 100 ng/ml for 16 h) served as positive control to induce Drp1 phosphorylation at S616 residue. RQ, relative quantification. (J) STAT1-/- mice were inoculated with DENV (serotype 2, strain NGC-N) by an intraperitoneal plus intracerebral route. The peripheral blood mononuclear cells (PBMC) were isolated on day 0, 1, and 2 as indicated (n = 2 for each time point) and then sampled for western blot analysis with the indicated antibodies.
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ppat.1005350.g001: DENV infection impairs mitochondrial dynamics.(A and B) Diagram (A) and the results (B) of mitochondria intermixing assay. Mitochondria of A549 cells were labeled by transfection with mitoYFP or mitoCherry to distinguish the origin of each mitochondrion. Cells were mock or infected with DENV serotype 2 (multiplicity of infection [moi] 10) for 24 h before HVJ-E-mediated cell fusion. The fused cell hybrids with or without DENV infection were magnified and analyzed for green and red fluorescence intensity by use of ZEN lite 2011 (Carl Zeiss MicroImaging GmbH). Green: mitoYFP; red: mitoCherry; magenta: DENV NS3. (C to I) Time course study of DENV infection in human A549 cells with moi 10 for the indicated hours. Culture supernatants were harvested for virus titration (C) and release of lactic dehydrogenase (LDH) (D). Cell lysates were harvested for western blot analysis with the indicated antibodies (E) and RT-qPCR (F to I) for the indicated genes. Data are mean ± SD (n = 3 per group). The data for LDH release was compared by one way ANOVA and Bonferroni multiple-comparison test with use of Prism 5 (GraphPad; La Jolla, CA, USA). ns, no significance; *, p<0.05. Nocodazole treatment (NOC; 100 ng/ml for 16 h) served as positive control to induce Drp1 phosphorylation at S616 residue. RQ, relative quantification. (J) STAT1-/- mice were inoculated with DENV (serotype 2, strain NGC-N) by an intraperitoneal plus intracerebral route. The peripheral blood mononuclear cells (PBMC) were isolated on day 0, 1, and 2 as indicated (n = 2 for each time point) and then sampled for western blot analysis with the indicated antibodies.

Mentions: To explore whether DENV can affect mitochondrial dynamics, we performed a mitochondrial intermixing experiment, which has been used to investigate mitochondrial fusion/fission events by fusing two individual cells [37,38]. We established two stable A549 cell lines with either mitochondria-targeted YFP (mitoYFP) or RFP (mitoCherry) and fused them by using an HVJ Envelope Cell fusion kit (Fig 1A). In mock-infected cells, continuous mitochondrial fusion/fission led to an even redistribution and colocalization of mitoYFP and mitoCherry in the fused cells, whereas much less colocalization of mitoYFP and mitoCherry was noted in the fused DENV-infected cells (Fig 1B) at 24 h post infection (p.i.) when DENV replicated to high titer (Fig 1C) but without significant cytotoxicity (Fig 1D). The delayed redistribution of mitochondria might be resulted from reduced mobility, enhanced fission or suppressed fusion. We found no obvious difference in the mitochondrial movement between mock- and DENV-infected cells by the live-confocal microscopy analysis (S1 Movie and S1 Fig). We also checked the phosphorylation level of the fission-related dynamin-related protein 1 (Drp1) at Ser616 (pDrp1), which was induced by hepatitis B virus (HBV) and HCV to promote mitochondrial fission [39,40], as well as the endogenous protein levels of several mitochondria fusion/fission regulators. Levels of fission-related proteins, Drp1, pDrp1, optic atrophy 1 (OPA1) and mitochondrial fission 1 (Fis1) were similar during the course of DENV infection (Fig 1E and S2 Fig), while reduced fusion-related MFN1 and MFN2 proteins were detected in DENV-infected cells (Fig 1E). The mRNA levels of MFN1 and MFN2 remained constant in DENV-infected cells when viral RNA and IFNβ were induced exponentially (Fig 1F–1I), so the downregulation of MFNs proteins likely occurred at post-transcriptional level. Moreover, MFN2 protein levels were reduced in the peripheral blood mononuclear cells (PBMC) isolated from STAT1-deficient mice during the course of DENV infection (Fig 1J). Overall, despite the role of mitochondrial fission-related molecules cannot be completely excluded, our data indicated that mitochondrial fusion might be affected in DENV-infected cells.


Dengue Virus Impairs Mitochondrial Fusion by Cleaving Mitofusins.

Yu CY, Liang JJ, Li JK, Lee YL, Chang BL, Su CI, Huang WJ, Lai MM, Lin YL - PLoS Pathog. (2015)

DENV infection impairs mitochondrial dynamics.(A and B) Diagram (A) and the results (B) of mitochondria intermixing assay. Mitochondria of A549 cells were labeled by transfection with mitoYFP or mitoCherry to distinguish the origin of each mitochondrion. Cells were mock or infected with DENV serotype 2 (multiplicity of infection [moi] 10) for 24 h before HVJ-E-mediated cell fusion. The fused cell hybrids with or without DENV infection were magnified and analyzed for green and red fluorescence intensity by use of ZEN lite 2011 (Carl Zeiss MicroImaging GmbH). Green: mitoYFP; red: mitoCherry; magenta: DENV NS3. (C to I) Time course study of DENV infection in human A549 cells with moi 10 for the indicated hours. Culture supernatants were harvested for virus titration (C) and release of lactic dehydrogenase (LDH) (D). Cell lysates were harvested for western blot analysis with the indicated antibodies (E) and RT-qPCR (F to I) for the indicated genes. Data are mean ± SD (n = 3 per group). The data for LDH release was compared by one way ANOVA and Bonferroni multiple-comparison test with use of Prism 5 (GraphPad; La Jolla, CA, USA). ns, no significance; *, p<0.05. Nocodazole treatment (NOC; 100 ng/ml for 16 h) served as positive control to induce Drp1 phosphorylation at S616 residue. RQ, relative quantification. (J) STAT1-/- mice were inoculated with DENV (serotype 2, strain NGC-N) by an intraperitoneal plus intracerebral route. The peripheral blood mononuclear cells (PBMC) were isolated on day 0, 1, and 2 as indicated (n = 2 for each time point) and then sampled for western blot analysis with the indicated antibodies.
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ppat.1005350.g001: DENV infection impairs mitochondrial dynamics.(A and B) Diagram (A) and the results (B) of mitochondria intermixing assay. Mitochondria of A549 cells were labeled by transfection with mitoYFP or mitoCherry to distinguish the origin of each mitochondrion. Cells were mock or infected with DENV serotype 2 (multiplicity of infection [moi] 10) for 24 h before HVJ-E-mediated cell fusion. The fused cell hybrids with or without DENV infection were magnified and analyzed for green and red fluorescence intensity by use of ZEN lite 2011 (Carl Zeiss MicroImaging GmbH). Green: mitoYFP; red: mitoCherry; magenta: DENV NS3. (C to I) Time course study of DENV infection in human A549 cells with moi 10 for the indicated hours. Culture supernatants were harvested for virus titration (C) and release of lactic dehydrogenase (LDH) (D). Cell lysates were harvested for western blot analysis with the indicated antibodies (E) and RT-qPCR (F to I) for the indicated genes. Data are mean ± SD (n = 3 per group). The data for LDH release was compared by one way ANOVA and Bonferroni multiple-comparison test with use of Prism 5 (GraphPad; La Jolla, CA, USA). ns, no significance; *, p<0.05. Nocodazole treatment (NOC; 100 ng/ml for 16 h) served as positive control to induce Drp1 phosphorylation at S616 residue. RQ, relative quantification. (J) STAT1-/- mice were inoculated with DENV (serotype 2, strain NGC-N) by an intraperitoneal plus intracerebral route. The peripheral blood mononuclear cells (PBMC) were isolated on day 0, 1, and 2 as indicated (n = 2 for each time point) and then sampled for western blot analysis with the indicated antibodies.
Mentions: To explore whether DENV can affect mitochondrial dynamics, we performed a mitochondrial intermixing experiment, which has been used to investigate mitochondrial fusion/fission events by fusing two individual cells [37,38]. We established two stable A549 cell lines with either mitochondria-targeted YFP (mitoYFP) or RFP (mitoCherry) and fused them by using an HVJ Envelope Cell fusion kit (Fig 1A). In mock-infected cells, continuous mitochondrial fusion/fission led to an even redistribution and colocalization of mitoYFP and mitoCherry in the fused cells, whereas much less colocalization of mitoYFP and mitoCherry was noted in the fused DENV-infected cells (Fig 1B) at 24 h post infection (p.i.) when DENV replicated to high titer (Fig 1C) but without significant cytotoxicity (Fig 1D). The delayed redistribution of mitochondria might be resulted from reduced mobility, enhanced fission or suppressed fusion. We found no obvious difference in the mitochondrial movement between mock- and DENV-infected cells by the live-confocal microscopy analysis (S1 Movie and S1 Fig). We also checked the phosphorylation level of the fission-related dynamin-related protein 1 (Drp1) at Ser616 (pDrp1), which was induced by hepatitis B virus (HBV) and HCV to promote mitochondrial fission [39,40], as well as the endogenous protein levels of several mitochondria fusion/fission regulators. Levels of fission-related proteins, Drp1, pDrp1, optic atrophy 1 (OPA1) and mitochondrial fission 1 (Fis1) were similar during the course of DENV infection (Fig 1E and S2 Fig), while reduced fusion-related MFN1 and MFN2 proteins were detected in DENV-infected cells (Fig 1E). The mRNA levels of MFN1 and MFN2 remained constant in DENV-infected cells when viral RNA and IFNβ were induced exponentially (Fig 1F–1I), so the downregulation of MFNs proteins likely occurred at post-transcriptional level. Moreover, MFN2 protein levels were reduced in the peripheral blood mononuclear cells (PBMC) isolated from STAT1-deficient mice during the course of DENV infection (Fig 1J). Overall, despite the role of mitochondrial fission-related molecules cannot be completely excluded, our data indicated that mitochondrial fusion might be affected in DENV-infected cells.

Bottom Line: By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection.MFN1 was required for efficient antiviral retinoic acid-inducible gene I-like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death.Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, National Cheng Kung University, Tainan, Taiwan.

ABSTRACT
Mitochondria are highly dynamic subcellular organelles participating in many signaling pathways such as antiviral innate immunity and cell death cascades. Here we found that mitochondrial fusion was impaired in dengue virus (DENV) infected cells. Two mitofusins (MFN1 and MFN2), which mediate mitochondrial fusion and participate in the proper function of mitochondria, were cleaved by DENV protease NS2B3. By knockdown and overexpression approaches, these two MFNs showed diverse functions in DENV infection. MFN1 was required for efficient antiviral retinoic acid-inducible gene I-like receptor signaling to suppress DENV replication, while MFN2 participated in maintaining mitochondrial membrane potential (MMP) to attenuate DENV-induced cell death. Cleaving MFN1 and MFN2 by DENV protease suppressed mitochondrial fusion and deteriorated DENV-induced cytopathic effects through subverting interferon production and facilitating MMP disruption. Thus, MFNs participate in host defense against DENV infection by promoting the antiviral response and cell survival, and DENV regulates mitochondrial morphology by cleaving MFNs to manipulate the outcome of infection.

Show MeSH
Related in: MedlinePlus