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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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Related in: MedlinePlus

DENV cleaves human MFN1 and MFN2.(A) Alignment of amino acid sequences surrounding the MFN1 and MFN2 cleavage sites of DENV protease. The wild-type (WT) and mutated MFNs used in this study and the relative position of each domain are illustrated. (B) Western blot analysis of the cotransfection of C-terminal V5-tagged human MFN1 or MFN2 with Flag-tagged WT or mutated viral protease of DENV or JEV in A549 cells for 24 h. WT: wild-type; S135A: protease-dead mutant. (C) Coimmunoprecipitation analysis of A549 cells cotransfected with V5-tagged MFN1 or MFN2 with Flag-tagged NS2B3(S135A) of DENV or JEV for 24 h. (D) Coimmunoprecipitation analysis of A549 cells expressing S135A mutated DENV NS2B3 transfected with V5-tagged WT or mutated MFN1 or MFN2 for 24 h. (E) Western blot analysis of Flag-tagged DENV protease NS2B3 cotransfected with the indicated (WT or mutant) C-terminal V5-tagged MFNs constructs in A549 cells in the absence (lane 1–5) or presence (lane 6–10) of pan-caspase inhibitor (zVAD; 100 μM) for 24 h. Filled arrow: full-length; open arrow: cleaved product; star: non-specific band.
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ppat.1005350.g004: DENV cleaves human MFN1 and MFN2.(A) Alignment of amino acid sequences surrounding the MFN1 and MFN2 cleavage sites of DENV protease. The wild-type (WT) and mutated MFNs used in this study and the relative position of each domain are illustrated. (B) Western blot analysis of the cotransfection of C-terminal V5-tagged human MFN1 or MFN2 with Flag-tagged WT or mutated viral protease of DENV or JEV in A549 cells for 24 h. WT: wild-type; S135A: protease-dead mutant. (C) Coimmunoprecipitation analysis of A549 cells cotransfected with V5-tagged MFN1 or MFN2 with Flag-tagged NS2B3(S135A) of DENV or JEV for 24 h. (D) Coimmunoprecipitation analysis of A549 cells expressing S135A mutated DENV NS2B3 transfected with V5-tagged WT or mutated MFN1 or MFN2 for 24 h. (E) Western blot analysis of Flag-tagged DENV protease NS2B3 cotransfected with the indicated (WT or mutant) C-terminal V5-tagged MFNs constructs in A549 cells in the absence (lane 1–5) or presence (lane 6–10) of pan-caspase inhibitor (zVAD; 100 μM) for 24 h. Filled arrow: full-length; open arrow: cleaved product; star: non-specific band.

Mentions: We noted protein reduction of endogenous and overexpressed MFNs with DENV infection (Figs 1E, 3A and 3E), so we explored whether MFN proteins were downregulated by DENV. We found several potential DENV protease-cleavage recognition sequences, two basic residues followed by a small amino acid [44–47], in human MFN1 and MFN2 proteins (Fig 4A). To test whether DENV protease can cleave MFNs, we cotransfected cells with Flag-tagged DENV NS2B3 plus C-terminal V5-tagged MFN1 or MFN2 for western blot analysis. Besides the full-length MFNs (~100 kDa), smaller protein bands of ~28 kDa were detected by anti-V5 antibody in cells with wild-type (WT) but not protease-dead (S135A) DENV NS2B3 (Fig 4B). The protease from another flavivirus, Japanese encephalitis virus (JEV), failed to cleave MFNs (Fig 4B, lane 3 and 7), even though they share the same cleavage recognition sequences [48] and both of them can physically interact with MFNs (Fig 4C), for unknown reasons.


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 cleaves human MFN1 and MFN2.(A) Alignment of amino acid sequences surrounding the MFN1 and MFN2 cleavage sites of DENV protease. The wild-type (WT) and mutated MFNs used in this study and the relative position of each domain are illustrated. (B) Western blot analysis of the cotransfection of C-terminal V5-tagged human MFN1 or MFN2 with Flag-tagged WT or mutated viral protease of DENV or JEV in A549 cells for 24 h. WT: wild-type; S135A: protease-dead mutant. (C) Coimmunoprecipitation analysis of A549 cells cotransfected with V5-tagged MFN1 or MFN2 with Flag-tagged NS2B3(S135A) of DENV or JEV for 24 h. (D) Coimmunoprecipitation analysis of A549 cells expressing S135A mutated DENV NS2B3 transfected with V5-tagged WT or mutated MFN1 or MFN2 for 24 h. (E) Western blot analysis of Flag-tagged DENV protease NS2B3 cotransfected with the indicated (WT or mutant) C-terminal V5-tagged MFNs constructs in A549 cells in the absence (lane 1–5) or presence (lane 6–10) of pan-caspase inhibitor (zVAD; 100 μM) for 24 h. Filled arrow: full-length; open arrow: cleaved product; star: non-specific band.
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Related In: Results  -  Collection

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ppat.1005350.g004: DENV cleaves human MFN1 and MFN2.(A) Alignment of amino acid sequences surrounding the MFN1 and MFN2 cleavage sites of DENV protease. The wild-type (WT) and mutated MFNs used in this study and the relative position of each domain are illustrated. (B) Western blot analysis of the cotransfection of C-terminal V5-tagged human MFN1 or MFN2 with Flag-tagged WT or mutated viral protease of DENV or JEV in A549 cells for 24 h. WT: wild-type; S135A: protease-dead mutant. (C) Coimmunoprecipitation analysis of A549 cells cotransfected with V5-tagged MFN1 or MFN2 with Flag-tagged NS2B3(S135A) of DENV or JEV for 24 h. (D) Coimmunoprecipitation analysis of A549 cells expressing S135A mutated DENV NS2B3 transfected with V5-tagged WT or mutated MFN1 or MFN2 for 24 h. (E) Western blot analysis of Flag-tagged DENV protease NS2B3 cotransfected with the indicated (WT or mutant) C-terminal V5-tagged MFNs constructs in A549 cells in the absence (lane 1–5) or presence (lane 6–10) of pan-caspase inhibitor (zVAD; 100 μM) for 24 h. Filled arrow: full-length; open arrow: cleaved product; star: non-specific band.
Mentions: We noted protein reduction of endogenous and overexpressed MFNs with DENV infection (Figs 1E, 3A and 3E), so we explored whether MFN proteins were downregulated by DENV. We found several potential DENV protease-cleavage recognition sequences, two basic residues followed by a small amino acid [44–47], in human MFN1 and MFN2 proteins (Fig 4A). To test whether DENV protease can cleave MFNs, we cotransfected cells with Flag-tagged DENV NS2B3 plus C-terminal V5-tagged MFN1 or MFN2 for western blot analysis. Besides the full-length MFNs (~100 kDa), smaller protein bands of ~28 kDa were detected by anti-V5 antibody in cells with wild-type (WT) but not protease-dead (S135A) DENV NS2B3 (Fig 4B). The protease from another flavivirus, Japanese encephalitis virus (JEV), failed to cleave MFNs (Fig 4B, lane 3 and 7), even though they share the same cleavage recognition sequences [48] and both of them can physically interact with MFNs (Fig 4C), for unknown reasons.

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