Hepatitis C virus (HCV) non-structural protein 5A (NS5A) plays essential roles in HCV RNA replication and virus assembly. In recent studies several NS5A phosphorylation sites have been mapped in the context of active viral replication and a picture of sequential phosphorylation events contributing to conformational and functional changes in NS5A is emerging. Here we report the unambiguous identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that a phosphomimetic mutant of this residue (S235D) causes redistribution of NS5A to large juxta-nuclear foci that display increased colocalization with NS3 but decreased colocalization with VAP-A and dsRNA. Using electron microscopy (EM) we found that S235D induces less expansive virus-induced cytoplasmic membrane rearrangements while EM analysis using tagged viruses encoding an ‘APEX2’ peroxidase insertion in NS5A demonstrated that the S235D mutation induces the enrichment of NS5A in irregular membrane-wrapped foci. Furthermore, using a novel bioluminescence resonance energy transfer (BRET) biosensor we found that S235D alters NS5A conformation. Finally, using a customised siRNA screen of host kinases with proven or predicted roles in HCV RNA replication and subsequent analysis using an antibody against phosphorylated Ser-235, we demonstrate that phosphatidylinositol-4 kinase III alpha (PI4KIIIalpha) is required for NS5A Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by the host kinase PI4KIIIalpha.