Phosphatidylinositol 4,5-Bisphosphate Is an HCV NS5A Ligand and Mediates Replication of the Viral Genome

BACKGROUND & AIMS: Phosphoinositides (PIs) bind and regulate localization of proteins via a variety of structural motifs. PI 4,5-bisphosphate (PI[4,5]P-2) interacts with and modulates the function of several proteins involved in intracellular vesicular membrane trafficking. We investigated interactions between PI(4,5)P-2 and hepatitis C virus (HCV) nonstructural protein 5A (NS5A) and effects on the viral life cycle. METHODS: We used a combination of quartz crystal microbalance, circular dichroism, molecular genetics, and immunofluorescence to study specific binding of PI(4,5)P-2 by the HCV NS5A protein. We evaluated the effects of PI(4,5)P-2 on the function of NS5A by expressing wild-type or mutant forms of Bart79I or FL-J6/ JFH-5'C19Rluc2AUbi21 RNA in Huh7 cells. We also studied the effects of strategies designed to inhibit PI(4,5)P-2 on HCV replication in these cells. RESULTS: The N-terminal amphipathic helix of NS5A bound specifically to PI(4,5)P-2, inducing a conformational change that stabilized the interaction between NS5A and TBC1D20, which is required for HCV replication. A pair of positively charged residues within the amphipathic helix (the basic amino acid PI(4,5)P-2 pincer domain) was required for PI(4,5)P-2 binding and replication of the HCV-RNA genome. A similar motif was found to be conserved across all HCV isolates, as well as amphipathic helices of many pathogens and apolipoproteins. CONCLUSIONS: PI(4,5)P-2 binds to HCV NS5A to promote replication of the viral RNA genome in hepatocytes. Strategies to disrupt this interaction might be developed to inhibit replication of HCV and other viruses.