Enzymatic activity of Phosphatidyl-inositol-4 kinase III alpha (PI4KIIIα) is critically involved in HCV replication and regulated by HCV NS5A

PI4KIIIα has been identified as important host factor for HCV replication (1). To gain insight into the mechanism of action, we generated cell lines with a stable knock-down of PI4KIIIα, resulting in a significant reduction of HCV replication upon infection with replication competent genomes. Interestingly, upon expression of non-structural proteins NS3–5B the structure of HCV induced membrane alterations was different in stable knock-down cells changing from a dot-like distribution to large membrane clusters. HCV replication as well as the induction of typical membrane alterations could be rescued by expression of wildtype PI4KIIIα but not by an enzymatic inactive point mutant. Since the enzymatic activity of PI4KIIIα is catalyzing the production of phosphatidyl-inositol-4-phosphate (PI4P) from PI, we analyzed the distribution of this lipid. In naive Huh-7 cells PI4P mainly localized on Golgi-derived membranes. In contrast, HCV infection resulted in an increase in PI4P levels and redistribution into dot like compartments partially co-localizing to the HCV non-structural proteins. Quantitation by immunofluorescence and lipid extraction revealed higher PI4P levels in cells harboring replicating virus or subgenomic replicons. Increasing PI4P staining could also be detected in liver cryosections of chronically infected HCV patients. PI4P pattern was detected in the same areas of consecutive sections as NS5A. We were able to identify domain 1 of NS5A to be responsible for the generation of new intracellular PI4P pools as well as for the direct interaction with the kinase. Strikingly purified NS5A was able to stimulate PI4KIIIα activity resulting in higher PI4P synthesis in vitro. Taken together our data indicate that HCV replication requires PI4KIIIα enzymatic activity, resulting in an increase and redistribution of intracellular PI4P pools. NS5A is involved in recruitment and activation of PI4KIIIα, which might play a role in the establishment of the membranous web.

Literature:

(1) Potential roles for cellular cofactors in hepatitis C virus replication complex formation. Berger KL, Randall G.Commun Integr Biol. 2009 Nov;2(6):471-3.