Limits...
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.

Show MeSH

Related in: MedlinePlus

Model of the interplay between NS5A and PI4KIIIα.A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database (http://hcv.lanl.gov). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral RNA replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3649985&req=5

ppat-1003359-g010: Model of the interplay between NS5A and PI4KIIIα.A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database (http://hcv.lanl.gov). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral RNA replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.

Mentions: Our initial deletion analysis identified aa 187–213 of NS5A to be involved in PI4KIIIα binding (PBR), which could be narrowed down to a sequence of 7–9 aa (PFIS) crucial for PI4KIIIα interaction (Fig. 10A). The PFIS encompasses the very C-terminus of NS5A D1 (aa 202–210) and is highly conserved among all HCV genotypes, in line with the essential function of PI4KIIIα in HCV replication and with previous mapping studies [11], [61]. No function has been assigned yet to this region [4] and, unfortunately, this motif is not included in published crystal structures of NS5A D1 [33], [62]. However, a very recent study suggests that the PFIS partially overlaps with a region adopting an α-helical structure, which might be induced upon interaction with other proteins [63]. It also seems likely that the interaction of NS5A with PI4KIIIα is not restricted to the PFIS since almost all mutations in the PBR affected binding to PI4KIIIα to various extents and NS5A lacking the entire PBR or even D1 retained some PI4KIIIα binding (Fig. 1, ΔS3 and ΔD1). Our results furthermore strongly indicate that the interaction with NS5B is also essential for functional PI4KIIIα interaction, since expression of an NS3 to NS5A polyprotein was not capable of activating PI4KIIIα. Still, according to our data, the PFIS is important for PI4KIIIα binding and indispensable for the activation of the lipid kinase activity. All triple alanine mutations in this region had almost identical phenotypes, very similar to PI4KIIIα knockdown (Table 1), correlated with a strong impairment in PI4KIIIα binding and blocked HCV replication. This correlation argued for a replication defect mediated by interference with functional NS5A-PI4KIIIα interaction, which was furthermore supported by the partial rescue of RNA replication of PFIS mutant PPH upon PI4KIIIα overexpression, probably compensating the reduced binding efficiency. Replication defects of other PFIS mutants (MLT, TDP and HIT) were not compensated by PI4KIIIα overexpression, suggesting that the functional interaction of these NS5A mutants with PI4KIIIα was more severely impaired as for mutant PPH. Indeed, the PPH mutant was slightly less impaired in PI4P induction (Fig. 4C), induced MW clusters in a lower number of cells (Fig. 4B) and had a slightly reduced p58/p56 ratio (Fig. 6) compared to the other replication dead PFIS mutants. Alternatively, mutants MLT, TDP and HIT could require higher PI4KIIIα expression levels to compensate the functional interaction defects, which cannot be achieved by our lentiviral transduction system. We furthermore cannot rule out that mutations in the highly conserved PFIS motif might affect other important functions of NS5A independent from PI4KIIIα, like RNA binding, dimerization etc. and therefore cannot be rescued solely by PI4KIIIα overexpression.


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)

Model of the interplay between NS5A and PI4KIIIα.A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database (http://hcv.lanl.gov). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral RNA replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3649985&req=5

ppat-1003359-g010: Model of the interplay between NS5A and PI4KIIIα.A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database (http://hcv.lanl.gov). Green numbers in the top line refer to the degree of conservation (rounded). Numbers on the left and right refer to the positions of the flanking amino acids within NS5A. Variations from the consensus are listed according to their frequency. A proline found in the JFH-1 PFIS is marked in blue. Variants found only once are not shown. B: NS5A (light brown) interacts with PI4KIIIα (red). This interaction regulates NS5A phosphorylation status directly or indirectly. Active kinase promotes NS5A p56 formation, a fraction of which is hyperphosphorylated resulting in p58. P56 might positively influence viral RNA replication either directly or by affecting the morphology of the replication sites, for which additional host factors are probably required. PI4KIIIα interaction with NS5A and NS5B is required to trigger lipid kinase activity. This leads to formation of new PI4P pools, presumably involved in membranous web morphology.
Mentions: Our initial deletion analysis identified aa 187–213 of NS5A to be involved in PI4KIIIα binding (PBR), which could be narrowed down to a sequence of 7–9 aa (PFIS) crucial for PI4KIIIα interaction (Fig. 10A). The PFIS encompasses the very C-terminus of NS5A D1 (aa 202–210) and is highly conserved among all HCV genotypes, in line with the essential function of PI4KIIIα in HCV replication and with previous mapping studies [11], [61]. No function has been assigned yet to this region [4] and, unfortunately, this motif is not included in published crystal structures of NS5A D1 [33], [62]. However, a very recent study suggests that the PFIS partially overlaps with a region adopting an α-helical structure, which might be induced upon interaction with other proteins [63]. It also seems likely that the interaction of NS5A with PI4KIIIα is not restricted to the PFIS since almost all mutations in the PBR affected binding to PI4KIIIα to various extents and NS5A lacking the entire PBR or even D1 retained some PI4KIIIα binding (Fig. 1, ΔS3 and ΔD1). Our results furthermore strongly indicate that the interaction with NS5B is also essential for functional PI4KIIIα interaction, since expression of an NS3 to NS5A polyprotein was not capable of activating PI4KIIIα. Still, according to our data, the PFIS is important for PI4KIIIα binding and indispensable for the activation of the lipid kinase activity. All triple alanine mutations in this region had almost identical phenotypes, very similar to PI4KIIIα knockdown (Table 1), correlated with a strong impairment in PI4KIIIα binding and blocked HCV replication. This correlation argued for a replication defect mediated by interference with functional NS5A-PI4KIIIα interaction, which was furthermore supported by the partial rescue of RNA replication of PFIS mutant PPH upon PI4KIIIα overexpression, probably compensating the reduced binding efficiency. Replication defects of other PFIS mutants (MLT, TDP and HIT) were not compensated by PI4KIIIα overexpression, suggesting that the functional interaction of these NS5A mutants with PI4KIIIα was more severely impaired as for mutant PPH. Indeed, the PPH mutant was slightly less impaired in PI4P induction (Fig. 4C), induced MW clusters in a lower number of cells (Fig. 4B) and had a slightly reduced p58/p56 ratio (Fig. 6) compared to the other replication dead PFIS mutants. Alternatively, mutants MLT, TDP and HIT could require higher PI4KIIIα expression levels to compensate the functional interaction defects, which cannot be achieved by our lentiviral transduction system. We furthermore cannot rule out that mutations in the highly conserved PFIS motif might affect other important functions of NS5A independent from PI4KIIIα, like RNA binding, dimerization etc. and therefore cannot be rescued solely by PI4KIIIα overexpression.

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