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Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Amako Y, Igloi Z, Mankouri J, Kazlauskas A, Saksela K, Dallas M, Peers C, Harris M - J. Biol. Chem. (2013)

Bottom Line: We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1.An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis.We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

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NS5A binds to MLK3 in a polyproline motif (P2)-dependent manner.A, lysates from Huh7 cells harboring a JFH-1 derived subgenomic replicon (SGR-JFH1) were immunoprecipitated with an anti-MLK3 monoclonal antibody and analyzed by Western blot for the presence of MLK3 and NS5A. The top panel shows the lysates and the bottom panel the immunoprecipitates, IgG LC: light chain of the immunoprecipitating antibody. B, lysates from Huh7 cells harboring an SGR with a One-STrEP-tagged NS5A were purified by affinity column capture with Strep-Tactin beads, prior to Western blotting for the presence of NS5A and MLK3. C, SGR-JFH1 cells were analyzed by indirect immunofluorescence with antibodies to NS5A (red) and MLK3 (green). The merged image shows extensive colocalization of the two proteins. Scale bar, 20 μm. D, ectopic expression of NS5A and MLK3, co-immunoprecipitation/Western blotting to demonstrate protein-protein interaction between NS5A and MLK3. Huh7 cells were co-transfected with a combination of plasmid vectors expressing NS5A (wild type or PA2 mutant) and FLAG-tagged MLK3 (wild type or K144E kinase-dead mutant, MLK3 KD) as indicated. Lysates were immunoprecipitated with an anti-FLAG antibody and Western blotted for the presence of NS5A or MLK3. IgG LC: light chain of the immunoprecipitating antibody. E, Huh7 cells were transfected with plasmid vectors expressing NS5A (top row), FLAG-tagged MLK3 (middle row), or both (bottom row). Indirect immunofluorescence microscopy reveals colocalization of NS5A and MLK3 in the cotransfected cells.
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Figure 2: NS5A binds to MLK3 in a polyproline motif (P2)-dependent manner.A, lysates from Huh7 cells harboring a JFH-1 derived subgenomic replicon (SGR-JFH1) were immunoprecipitated with an anti-MLK3 monoclonal antibody and analyzed by Western blot for the presence of MLK3 and NS5A. The top panel shows the lysates and the bottom panel the immunoprecipitates, IgG LC: light chain of the immunoprecipitating antibody. B, lysates from Huh7 cells harboring an SGR with a One-STrEP-tagged NS5A were purified by affinity column capture with Strep-Tactin beads, prior to Western blotting for the presence of NS5A and MLK3. C, SGR-JFH1 cells were analyzed by indirect immunofluorescence with antibodies to NS5A (red) and MLK3 (green). The merged image shows extensive colocalization of the two proteins. Scale bar, 20 μm. D, ectopic expression of NS5A and MLK3, co-immunoprecipitation/Western blotting to demonstrate protein-protein interaction between NS5A and MLK3. Huh7 cells were co-transfected with a combination of plasmid vectors expressing NS5A (wild type or PA2 mutant) and FLAG-tagged MLK3 (wild type or K144E kinase-dead mutant, MLK3 KD) as indicated. Lysates were immunoprecipitated with an anti-FLAG antibody and Western blotted for the presence of NS5A or MLK3. IgG LC: light chain of the immunoprecipitating antibody. E, Huh7 cells were transfected with plasmid vectors expressing NS5A (top row), FLAG-tagged MLK3 (middle row), or both (bottom row). Indirect immunofluorescence microscopy reveals colocalization of NS5A and MLK3 in the cotransfected cells.

Mentions: We sought to confirm this interaction by co-immunoprecipitation. Endogenous MLK3 was immunoprecipitated from lysates of Huh7 cells stably harboring a JFH1-derived subgenomic replicon, Western blot analysis of the immunoprecipitates demonstrated the presence of both MLK3 and NS5A (Fig. 2A), while immunoprecipitation with a control IgG did not capture either MLK3 or NS5A. We undertook the reciprocal experiment by affinity capture purification from lysates of Huh7 cells harboring a subgenomic replicon containing a one-STrEP-tag within NS5A. Lysates from wild type (untagged), or tagged replicon cells (NS5A wild type or PA2 mutation) were subjected to STrEP-tactin Sepharose column purification. Comparable amounts of NS5A were expressed in all three cell lines as seen in Fig. 2B (left), however as expected after affinity capture purification, NS5A was only precipitated and eluted when tagged. MLK3 was co-purified with either wild type or PA2 NS5A, but densitometry revealed that a lesser amount of MLK3 was associated with PA2 NS5A, (Fig. 2B, right). In support of the coimmunoprecipitation data, indirect immunofluorescence analysis revealed that NS5A and MLK3 were colocalized in perinuclear regions of subgenomic replicon harboring cells (Fig. 2C).


Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Amako Y, Igloi Z, Mankouri J, Kazlauskas A, Saksela K, Dallas M, Peers C, Harris M - J. Biol. Chem. (2013)

NS5A binds to MLK3 in a polyproline motif (P2)-dependent manner.A, lysates from Huh7 cells harboring a JFH-1 derived subgenomic replicon (SGR-JFH1) were immunoprecipitated with an anti-MLK3 monoclonal antibody and analyzed by Western blot for the presence of MLK3 and NS5A. The top panel shows the lysates and the bottom panel the immunoprecipitates, IgG LC: light chain of the immunoprecipitating antibody. B, lysates from Huh7 cells harboring an SGR with a One-STrEP-tagged NS5A were purified by affinity column capture with Strep-Tactin beads, prior to Western blotting for the presence of NS5A and MLK3. C, SGR-JFH1 cells were analyzed by indirect immunofluorescence with antibodies to NS5A (red) and MLK3 (green). The merged image shows extensive colocalization of the two proteins. Scale bar, 20 μm. D, ectopic expression of NS5A and MLK3, co-immunoprecipitation/Western blotting to demonstrate protein-protein interaction between NS5A and MLK3. Huh7 cells were co-transfected with a combination of plasmid vectors expressing NS5A (wild type or PA2 mutant) and FLAG-tagged MLK3 (wild type or K144E kinase-dead mutant, MLK3 KD) as indicated. Lysates were immunoprecipitated with an anti-FLAG antibody and Western blotted for the presence of NS5A or MLK3. IgG LC: light chain of the immunoprecipitating antibody. E, Huh7 cells were transfected with plasmid vectors expressing NS5A (top row), FLAG-tagged MLK3 (middle row), or both (bottom row). Indirect immunofluorescence microscopy reveals colocalization of NS5A and MLK3 in the cotransfected cells.
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Figure 2: NS5A binds to MLK3 in a polyproline motif (P2)-dependent manner.A, lysates from Huh7 cells harboring a JFH-1 derived subgenomic replicon (SGR-JFH1) were immunoprecipitated with an anti-MLK3 monoclonal antibody and analyzed by Western blot for the presence of MLK3 and NS5A. The top panel shows the lysates and the bottom panel the immunoprecipitates, IgG LC: light chain of the immunoprecipitating antibody. B, lysates from Huh7 cells harboring an SGR with a One-STrEP-tagged NS5A were purified by affinity column capture with Strep-Tactin beads, prior to Western blotting for the presence of NS5A and MLK3. C, SGR-JFH1 cells were analyzed by indirect immunofluorescence with antibodies to NS5A (red) and MLK3 (green). The merged image shows extensive colocalization of the two proteins. Scale bar, 20 μm. D, ectopic expression of NS5A and MLK3, co-immunoprecipitation/Western blotting to demonstrate protein-protein interaction between NS5A and MLK3. Huh7 cells were co-transfected with a combination of plasmid vectors expressing NS5A (wild type or PA2 mutant) and FLAG-tagged MLK3 (wild type or K144E kinase-dead mutant, MLK3 KD) as indicated. Lysates were immunoprecipitated with an anti-FLAG antibody and Western blotted for the presence of NS5A or MLK3. IgG LC: light chain of the immunoprecipitating antibody. E, Huh7 cells were transfected with plasmid vectors expressing NS5A (top row), FLAG-tagged MLK3 (middle row), or both (bottom row). Indirect immunofluorescence microscopy reveals colocalization of NS5A and MLK3 in the cotransfected cells.
Mentions: We sought to confirm this interaction by co-immunoprecipitation. Endogenous MLK3 was immunoprecipitated from lysates of Huh7 cells stably harboring a JFH1-derived subgenomic replicon, Western blot analysis of the immunoprecipitates demonstrated the presence of both MLK3 and NS5A (Fig. 2A), while immunoprecipitation with a control IgG did not capture either MLK3 or NS5A. We undertook the reciprocal experiment by affinity capture purification from lysates of Huh7 cells harboring a subgenomic replicon containing a one-STrEP-tag within NS5A. Lysates from wild type (untagged), or tagged replicon cells (NS5A wild type or PA2 mutation) were subjected to STrEP-tactin Sepharose column purification. Comparable amounts of NS5A were expressed in all three cell lines as seen in Fig. 2B (left), however as expected after affinity capture purification, NS5A was only precipitated and eluted when tagged. MLK3 was co-purified with either wild type or PA2 NS5A, but densitometry revealed that a lesser amount of MLK3 was associated with PA2 NS5A, (Fig. 2B, right). In support of the coimmunoprecipitation data, indirect immunofluorescence analysis revealed that NS5A and MLK3 were colocalized in perinuclear regions of subgenomic replicon harboring cells (Fig. 2C).

Bottom Line: We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1.An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis.We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

Show MeSH
Related in: MedlinePlus