Hepatitis C virus variants can by-pass PI3K inhibition by enviroxime

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PI4KIII inhibitor enviroxime impedes the replication of the hepatitis C virus by inhibiting PI3 kinases

Objectives: Many positive-stranded RNA viruses, including HCV, drastically remodel intracellular membranes to generate specialized environments for RNA replication. Phosphatidylinositol 4-kinase III (PI4KIII)α plays an essential role in the formation of HCV replication complexes and has therefore been explored as a potential drug target. Here, we characterized the anti-HCV activity of the PI4KIII inhibitors enviroxime and BF738735 and elucidated their mechanism of action. Methods: Antiviral assays were performed using HCV subgenomic replicons and infectious HCV. Enviroxime- and BF738735-resistant HCV replicons were generated by long-term culture with increasing compound concentrations. Intracellular localization of phosphatidylinositol 4-phosphate (PI4P) lipids was analysed by confocal microscopy. Results: HCV subgenomic replicons resistant to either enviroxime or BF738735 proved cross-resistant and carried mutations in the NS3, NS4B and NS5A genes. Knockdown of PI4KIIIβ by small interfering RNA (siRNA) did not affect the replication of the HCV subgenomic replicon in this study. Furthermore, the compounds did not affect PI4P lipid levels at the replication complexes nor the phosphorylation status of NS5A, activities attributed to PI4KIIIα. Interestingly, the broad-spectrum phosphoinositide 3-kinase (PI3K) inhibitor LY294002 proved to be 10-fold less effective against the resistant replicons. In addition, enviroxime and BF738735 inhibited several PI3Ks in enzymatic assays. Conclusions: Contrary to assumptions, our data indicate that PI4KIIIα and PI4KIIIβ are not the main targets for the anti-HCV activity of enviroxime and BF738735. Instead, we demonstrated that both molecules impede HCV replication at least partially by an inhibitory effect on PI3Ks. Moreover, HCV is able to bypass PI3K inhibition by acquiring mutations in its genome.status: publishe

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

Tyrphostin AG1478 inhibits encephalomyocarditis virus and hepatitis C virus by targeting phosphatidylinositol 4-kinase IIIα

Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known EGFR inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV, but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously only regarded as cancer chemotherapy agent

Tyrphostin AG1478 inhibits encephalomyocarditis virus and hepatitis C virus by targeting phosphatidylinositol 4-kinase IIIα

Encephalomyocarditis virus (EMCV), like hepatitis C virus (HCV), requires phosphatidylinositol 4-kinase IIIα (PI4KA) for genome replication. Here, we demonstrate that tyrphostin AG1478, a known EGFR inhibitor, also inhibits PI4KA activity, both in vitro and in cells. AG1478 impaired replication of EMCV and HCV, but not that of an EMCV mutant previously shown to escape PI4KA inhibition. This work uncovers novel cellular and antiviral properties of AG1478, a compound previously only regarded as cancer chemotherapy agent

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

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

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

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