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The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.

Reiss S, Harak C, Romero-Brey I, Radujkovic D, Klein R, Ruggieri A, Rebhan I, Bartenschlager R, Lohmann V - PLoS Pathog. (2013)

Bottom Line: Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα.In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status.Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

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NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction.A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.
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ppat-1003359-g008: NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction.A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.

Mentions: Since alteration of NS5A phosphorylation and PI4P induction seemed to be intimately linked in case of the PFIS mutants, we next tried to dissect the requirements for both parameters by using transcomplementation assays. Previous studies have shown that replication-deficient mutants of NS5A, in particular those with defects in phosphorylation, can be rescued by expression of a wt protein in trans [50], [51], [57]. Minimal requirement for transcomplementation was the expression of NS5A in the context of a NS3-5A polyprotein, which was necessary and sufficient for NS5A hyperphosphorylation. In contrast, the sole expression of NS5A was not capable of rescuing deficient mutants [50], most likely due to aberrant phosphorylation, indicated by the lack of p58 [38]. Therefore, we generated six Huh7-Lunet derivatives, either containing a subgenomic replicon or constitutively expressing NS3-5A or NS5A, each based on JFH-1 or Con1 ET, respectively, to analyze which of these settings was capable of rescuing replication of a representative PFIS mutant (HIT) in the context of JFH-1 and Con1 ET reporter replicons (Fig. 8A). Wt replicons of both genotypes as well as a NS5B mutant (ΔGDD), which cannot be complemented in trans [50], were included as positive and negative controls, respectively. We first analyzed each rescue setting for induction of PI4P synthesis and NS5A phosphorylation in the absence of the transfected mutant replicons (Fig. 8B–D). Both replicon cell lines contained functional NS5A by definition, although in case of Con1 ET only p56 was clearly detectable (Fig. 8D). Both cell lines expressing NS3-5A contained two NS5A species of the expected sizes, in contrast to the cells expressing NS5A only (Fig. 8D). The replicon cell lines also contained elevated levels of PI4P (Fig. 8B, C). Surprisingly, neither expression of NS3-5A nor of NS5A increased intracellular PI4P abundance (Fig. 8B, C), despite similar NS5A levels compared to replicon cells (Fig. 8D). The lack of PI4P induction in cells expressing NS3-5A suggested that activation of PI4KIIIα requires not only NS5A, but also NS5B, which has been shown to interact with this lipid kinase as well [11].


The lipid kinase phosphatidylinositol-4 kinase III alpha regulates the phosphorylation status of hepatitis C virus NS5A.

Reiss S, Harak C, Romero-Brey I, Radujkovic D, Klein R, Ruggieri A, Rebhan I, Bartenschlager R, Lohmann V - PLoS Pathog. (2013)

NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction.A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.
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getmorefigures.php?uid=PMC3649985&req=5

ppat-1003359-g008: NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction.A: Experimental setup of transcomplementation experiments: Huh7-Lunet cells bearing either a persistent HCV replicon (I) or constitutively expressing HCV NS3 to NS5A (II) or NS5A (III), respectively, are transfected with luciferase reporter replicons harboring the HIT triple alanine mutation to analyze for conditions rescuing RNA replication. Wt replicons and a replication deficient NS5B mutant (ΔGDD) are used for positive and negative control, respectively. B. Huh7-Lunet cells with persistent replicons (neoJFH) or constitutive expression protein of genotype 2a NS3-5A or NS5A, respectively, were subjected to immunofluorescence analysis. NS5A (red) or PI4P (green), respectively, were detected with specific antibodies and DAPI was used to stain nuclei (blue). C. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel B and on equivalent cell lines harboring genotype 1b (Con1) replicons and proteins. PI4P levels were normalized to the mean value of naïve Huh7-Lunet cells. Data represent mean +/− SD of 35 NS5A positive cells analyzed per condition. D. Detection of NS5A and β-actin in Huh7-Lunet cells with persistent replicons or constitutive expression of NS3-5A or NS5A of genotype (Gt) 1b and 2a, respectively, by western-blot. Equal amounts of NS5A were loaded to judge variations in NS5A phosphorylation. Differences in β-actin, therefore, reflect varying NS5A expression levels. E, F: Wt (dark color), repHIT (medium color) and ΔGDD reporter replicons (light color) of either genotype 2a (panel E, JFH-1, red) or genotype 1b (panel F, Con1ET, blue) were transfected into Huh7-Lunet cell lines harboring a persistent replicon (Repl.) or constitutively expressing NS3-5A (3-5A) or NS5A (5A) of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of at least two experiments performed in duplicates.
Mentions: Since alteration of NS5A phosphorylation and PI4P induction seemed to be intimately linked in case of the PFIS mutants, we next tried to dissect the requirements for both parameters by using transcomplementation assays. Previous studies have shown that replication-deficient mutants of NS5A, in particular those with defects in phosphorylation, can be rescued by expression of a wt protein in trans [50], [51], [57]. Minimal requirement for transcomplementation was the expression of NS5A in the context of a NS3-5A polyprotein, which was necessary and sufficient for NS5A hyperphosphorylation. In contrast, the sole expression of NS5A was not capable of rescuing deficient mutants [50], most likely due to aberrant phosphorylation, indicated by the lack of p58 [38]. Therefore, we generated six Huh7-Lunet derivatives, either containing a subgenomic replicon or constitutively expressing NS3-5A or NS5A, each based on JFH-1 or Con1 ET, respectively, to analyze which of these settings was capable of rescuing replication of a representative PFIS mutant (HIT) in the context of JFH-1 and Con1 ET reporter replicons (Fig. 8A). Wt replicons of both genotypes as well as a NS5B mutant (ΔGDD), which cannot be complemented in trans [50], were included as positive and negative controls, respectively. We first analyzed each rescue setting for induction of PI4P synthesis and NS5A phosphorylation in the absence of the transfected mutant replicons (Fig. 8B–D). Both replicon cell lines contained functional NS5A by definition, although in case of Con1 ET only p56 was clearly detectable (Fig. 8D). Both cell lines expressing NS3-5A contained two NS5A species of the expected sizes, in contrast to the cells expressing NS5A only (Fig. 8D). The replicon cell lines also contained elevated levels of PI4P (Fig. 8B, C). Surprisingly, neither expression of NS3-5A nor of NS5A increased intracellular PI4P abundance (Fig. 8B, C), despite similar NS5A levels compared to replicon cells (Fig. 8D). The lack of PI4P induction in cells expressing NS3-5A suggested that activation of PI4KIIIα requires not only NS5A, but also NS5B, which has been shown to interact with this lipid kinase as well [11].

Bottom Line: Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα.In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status.Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Infectious Diseases, Molecular Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an essential host factor of hepatitis C virus (HCV) replication. PI4KIIIα catalyzes the synthesis of phosphatidylinositol 4-phosphate (PI4P) accumulating in HCV replicating cells due to enzyme activation resulting from its interaction with nonstructural protein 5A (NS5A). This study describes the interaction between PI4KIIIα and NS5A and its mechanistic role in viral RNA replication. We mapped the NS5A sequence involved in PI4KIIIα interaction to the carboxyterminal end of domain 1 and identified a highly conserved PI4KIIIα functional interaction site (PFIS) encompassing seven amino acids, which are essential for viral RNA replication. Mutations within this region were also impaired in NS5A-PI4KIIIα binding, reduced PI4P levels and altered the morphology of viral replication sites, reminiscent to the phenotype observed by silencing of PI4KIIIα. Interestingly, abrogation of RNA replication caused by mutations in the PFIS correlated with increased levels of hyperphosphorylated NS5A (p58), indicating that PI4KIIIα affects the phosphorylation status of NS5A. RNAi-mediated knockdown of PI4KIIIα or pharmacological ablation of kinase activity led to a relative increase of p58. In contrast, overexpression of enzymatically active PI4KIIIα increased relative abundance of basally phosphorylated NS5A (p56). PI4KIIIα therefore regulates the phosphorylation status of NS5A and viral RNA replication by favoring p56 or repressing p58 synthesis. Replication deficiencies of PFIS mutants in NS5A could not be rescued by increasing PI4P levels, but by supplying functional NS5A, supporting an essential role of PI4KIIIα in HCV replication regulating NS5A phosphorylation, thereby modulating the morphology of viral replication sites. In conclusion, we demonstrate that PI4KIIIα activity affects the NS5A phosphorylation status. Our results highlight the importance of PI4KIIIα in the morphogenesis of viral replication sites and its regulation by facilitating p56 synthesis.

Show MeSH
Related in: MedlinePlus