Hepatitis C Virus NS5A Competes With PI4KB For Binding To ACBD3 In A Genotype-dependent Manner

Upregulating of intracellular phosphatidylinositol 4-phosphate (PI4P) level has been demonstrated to be an essential step for hepatitis C virus (HCV) replication. Although PI4KB contributes to the induction of PI4P and promotes viral replication for picornaviruses such as poliovirus and Aichi virus, the involvement of PI4KB in HCV replication is controversial. Some studies reported that PI4KB is required for HCV replication and others suggested that it is not. This inconsistency might be due to the study of different HCV genotypes. Interestingly, viral replication of genotype 1b (GT1b) HCV were more greatly reduced than that of genotype 2a (GT2a) HCV upon silencing PI4KB. The mechanism underlying this genotype difference remains elusive.To elucidate the role of ACBD3 in HCV replication, we treated OR6 cells harboring a full-length GT1b HCV RNA and Renilla luciferase reporter with small interfering RNA (siRNA) targeting ACBD3 or control siRNA and monitored the expression level of viral proteins or Renilla luciferase reporter activities. Our data demonstrate that ACBD3 plays a negative role in HCV replication. NS5A preferentially recognizes the GST-ACBD3 (116-327) fragment. We compared the binding affinity of ACBD3(116-327) to NS5A between GT1b and GT2a, and found that NS5A from GT1b had higher binding affinity with ACBD3(116-327) than that from GT2a. Earlier study suggests that GST-ACBD3 (116-327) fragment is the region associated with PI4KB.Thus PI4KB and NS5A share a common binding region on ACBD3.As a consequence, more PI4KB was released from ACBD3 in GT1b HCV than GT2a HCV, which will explain why PI4KB plays more dependency role in GT1b virus than in GT2a virus. These results suggested that NS5A could rob the preexisting ACBD3/PI4KB complex to form NS5A/ACBD3 complex and PI4KB could relocate to the viral RNA replication sites to facilitate HCV replication. Our findings not only revealed the anti-HCV function of ACBD3, but also shed mechanistic light on how ACBD3 was manipulated by NS5A from different GT of HCV.