The Lipid Kinase Phosphatidylinositol-4 Kinase III Alpha Regulates the Phosphorylation Status of Hepatitis C Virus NS5A

<div><p>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.</p></div

NS5A phosphorylation is influenced by PI4KIIIα enzymatic activity.

<p>A: Huh7-Lunet T7 cells expressing shRNA directed against PI4KIIIα (shPI4K) or a non-targeting control (shNT) were transfected with plasmids encoding the NS3 to NS5B polyprotein of HCV genotype 2a (JFH-1) or variants of genotype 1b (Con1 wt, ET, mutHIT). PI4KIIIα expression in shPI4K cells was reconstituted by expression of a knockdown-resistant escape variant of PI4KIIIα (sh+Esc) to exclude off-target effects. B: Quantitative analysis of the ratio of NS5A p58 and p56 obtained by phosphoimaging of experiments as shown in panel A. C: Naïve Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of JFH-1 wt, Con1 wt or Con1 ET. Starting at 7 h post transfection, cells were incubated with indicated concentrations of AL-9. D: Quantitative analysis of the ratio of NS5A p58 and p56 obtained by phosphoimaging of experiments as shown in panel C. E: Huh7-Lunet T7 cells were cotransfected with plasmids encoding the NS3 to NS5B polyprotein of HCV genotype 2a (JFH-1) or variants of genotype 1b (Con1 wt, ET, mutHIT) and empty constructs (−) or plasmids encoding HA-tagged wt (wt) or inactive mutant (D1957A) PI4KIIIα. F: Quantitative analysis of the ratio of NS5A p58 and p56 obtained by phosphoimaging of experiments as shown in panel E. A, C, E: Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitations using NS5A specific antibodies. Immunocomplexes were analyzed by 10% SDS-PAGE and autoradiography. B, D, F: Data represent mean values +/− SD from 2 independent experiments analyzed in duplicates. Significances were calculated by paired t-tests. *, p<0.05; **, p<0.01; ***, p<0.001.</p

New PI4P pools emerge as a consequence of HCV RNA replication.

<p>A: Scheme of the transcomplementation experiments shown in panel B–D: Huh7-Lunet cells constitutively expressing HCV NS3 to NS5A (I) or NS5A (II), respectively, were transfected with reporter replicons containing luciferase and eGFP genes as indicated to analyze for conditions rescuing RNA replication. B: Wiltype (wt-eGFP), repHIT (repHIT-eGFP) and ΔGDD reporter replicons of genotype 2a (JFH-1) were transfected into Huh7-Lunet cell lines constitutively expressing NS3-5A or NS5A of genotype 1b or 2a, as indicated. RNA replication of replicons was determined by measuring luciferase activity in cell lysates at 24 h, 48 h and 72 h post transfection relative to 4 h to normalize for transfection efficiency. Diagrams show mean values +/−SD of a representative of two experiments performed in duplicates. C. Immunofluorescence analysis of the experiment shown in panel B at 48 h post electroporation. GFP (green) or PI4P (red), respectively, was detected with specific antibodies and DAPI was used to stain nuclei (blue). D. Quantitation of intracellular PI4P levels by measuring PI4P fluorescence intensity using ImageJ analysis (IntDen read-out) on cells as shown in panel C. Data represent mean arbitrary units (AU) +/− SD of 35 GFP positive cells analyzed per condition. In case of ΔGDD, cells were randomly chosen due to the lack of GFP signals. Significance was calculated by a paired students t-test. ***, p<0.001.</p

Reduced PI4KIIIα interaction and RNA replication deficiency correlate with relative increase of NS5A hyperphosphorylation.

<p>A: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing a wt sequence or triple alanine mutants as indicated or with empty plasmid (mock). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A specific antibodies. Immunocomplexes were analyzed by SDS-PAGE and autoradiography. B: Quantitative analysis of the NS5A p58/p56 ratio. Bands corresponding to NS5A p58 and p56, respectively, as shown in panel A were individually quantified by phosphoimaging to obtain a p58/p56 ratio. Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was compared to the wt polyprotein and calculated by a paired t-test. *, p<0.05; **, p<0.01; ***, p<0.001. A blow up of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g006" target="_blank">figure 6A</a> focusing on the NS5A phosphorylation of PFIS mutants in comparison to the wildtype polyprotein is shown below.</p

NS5A but not PI4P can transcomplement replication-deficient replicons with NS5A mutations affecting PI4KIIIα interaction.

<p>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.</p

Model of the interplay between NS5A and PI4KIIIα.

<p>A: Consensus sequence (red) of the PI4KIIIα interaction site (PFIS) derived from 672 NS5A sequences of all genotypes in the Los Alamos HCV database (<a href="http://hcv.lanl.gov" target="_blank">http://hcv.lanl.gov</a>). 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.</p

Triple alanine mutants induce ultrastructural changes similar to membranous web structures in PI4KIIIα knockdown cells.

<p>Huh7-Lunet T7 cells (A) or Huh7-Lunet T7 cells with stable PI4KIIIα knockdown (B) were transfected with pTM constructs expressing wt or mutant NS3 to NS5B polyproteins or eGFP. Cells were fixed and prepared for EM analysis 24 h post transfection. Consecutive enlargements of the boxed areas are shown from left to right. Note the heterogeneous membranous web (MW, yellow arrows) in cells expressing the wt polyprotein and the clusters of smaller double-membrane vesicles (DMVs) in shPI4KIIIα cells (B) and in cells expressing mutant polyproteins. Scale bars are given in the lower right of each panel. N, nucleus; LD, lipid droplet; rER, rough endoplasmic reticulum; m, mitochondrium. The number in the upper left of right panels shows the average diameter of 70 double-membranous vesicles (DMV) +/− SD measured for each condition. (C) Average diameter of DMVs detected in cells that had been transfected with constructs and conditions specified on the left and shown in panel A and B. Error bars indicate the mean +/− SD of seventy vesicles. Significance of differences in DMV sizes was measured by a paired t-test and is indicated ***, p<0.001.</p

PI4KIIIα binding to NS5A triple alanine mutants of the PI4KIIIα binding region (PBR).

<p>A: Schematic representation of NS5A and NS5A D1-LCS1. The sequence encompassing deletions ΔS3B and ΔS3C (186–212) is named PI4KIIIα binding region (PBR) and is highlighted in red. PBR was subjected to closer analysis by generation of triple alanine substitutions. Mutants were designated according to the respective wt sequence as exemplified. The PI4KIIIα functional interaction site (PFIS) is indicated by a dark red line. Small letters indicate amino acids specifically found in the JFH-1 isolate. For details refer to the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g001" target="_blank">Fig. 1A</a>. B: Huh7-Lunet T7 cells were cotransfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) containing triple alanine mutations in NS5A domain 1 as indicated and HA-tagged PI4KIIIα (HA-PI4K). Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using NS5A (lower panel) or HA-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography. C: Quantitative analysis of PI4KIIIα pull-down. Experiments as shown in panel B were quantified by phosphoimaging. Coprecipitation efficiency was normalized to the total amounts of HA-PI4K and calculated relative to PI4KIIIα pull-down by NS5A wt. Note that data were not normalized to input NS5A levels due to a 20–100fold molar excess of NS5A compared to PI4KIIIα (data not shown). Error bars indicate mean values +/− SD of two independent experiments analyzed in duplicates. Significance was calculated by a paired t-test. *, p<0.05; **, p<0.01; ***, p<0.001. D: Huh7-Lunet T7 cells were transfected with plasmids encoding the NS3 to NS5B polyprotein of genotype 2a (JFH-1) and/or HA-tagged PI4KIIIα (HA-PI4K) or mock transfected as indicated. Newly synthesized proteins were radiolabeled and cell lysates subjected to immunoprecipitation using HA- (lower panel) or NS5A-specific antibodies (upper panel). Samples were analyzed by SDS-PAGE and autoradiography.</p

Summary of phenotypes observed for NS5A mutants and PI4KIIIα silencing.

<p>Silencing of shPI4KIIIα is given in italics.</p><p>PFIS mutants are given in bold italics.</p>1<p>according to data obtained from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g003" target="_blank">figure 3B</a>; +++: wt type or higher; ++: 10–99% wt; +: 1–9% wt; +/−: residual low level replication; −: no replication.</p>2<p>according to data obtained from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g002" target="_blank">figure 2C</a>; +++:wt or higher; ++: 50–99%wt; +: 26–49% wt; +/−: 20–25% wt; −: below 20% wt.</p>3<p>according to data obtained from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g004" target="_blank">figure 4B</a>; +++:>50%; ++: 26–50%; +: 11–25%; +/−: >5–10%; −: <5% of cells with MW clusters visible in IF.</p>4<p>according to data obtained from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g004" target="_blank">figure 4C</a>; +++:>4fold mock; ++: 3–4 fold mock; +: 2–3 fold mock; +/−: >1.5–2fold mock; −: 1–1.5fold mock.</p>5<p>according to data obtained from <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003359#ppat-1003359-g006" target="_blank">figure 6B</a>; +++: 1 or higher; ++: 0.5–0.9; +: 0.1–0.4.</p>§<p>Data not shown.</p>*<p>According to Reiss et al., CHM 2011.</p