Metabolism of Phosphatidylinositol 4-Kinase IIIα-Dependent PI4P Is Subverted by HCV and Is Targeted by a 4-Anilino Quinazoline with Antiviral Activity

4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase alpha (PI4KIII alpha) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIa and, to a lesser extent, PI4KIII beta. In Huh7.5 cells, PI4KIII alpha is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIII alpha also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIII beta isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIII alpha by RNA interference. In light of our findings, we propose that the antiviral effect of 4anilino quinazoline compounds is mediated by the inhibition of PI4KIII alpha and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV - by recruiting PI4KIII alpha in the RNA replication complex - hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIII alpha product.