Phosphoinositides function as fundamental signaling molecules and play roles in diverse cellular processes. Certain types of viruses may employ host cell phosphoinositide signaling systems to facilitate their replication cycles. Here we demonstrate that the beta isoform of class II PI3K (PI3K-C2 beta) plays an indispensable role in hepatitis C virus (HCV) propagation in human hepatocellular carcinoma cells. Knockdown of PI3K-C2 beta abrogated HCV propagation in the cell. Using an HCV replicon system, we found that knockdown of PI3K-C2 beta substantially repressed the full-genome replication, while showing relatively small reductions in sub-genome replication, in which structural proteins including core protein were deleted. We also found that HCV core protein showed the binding activity towards D4-phosphorylated phosphoinositides and overlapped localization with phosphatidylinositol 3,4-bisphosphate in the cell. These results suggest that the phosphoinositide generated by PI3K-C2 beta plays an indispensable role in the HCV replication cycle through the binding to HCV core protein. (C) 2013 Elsevier Inc. All rights reserved.