Hepatitis C Virus Stimulates the Phosphatidylinositol 4-Kinase III Alpha-Dependent Phosphatidylinositol 4-Phosphate Production That Is Essential for Its Replication

Abstract

Phosphatidylinositol 4-kinase III alpha (PI4KA) is an essential cofactor of hepatitis C virus (HCV) replication. We initiated this study to determine whether HCV directly engages PI4KA to establish its replication. PI4KA kinase activity was found to be absolutely required for HCV replication using a small interfering RNA transcomplementation assay. Moreover, HCV infection or subgenomic HCV replicons produced a dramatic increase in phosphatidylinositol 4-phosphate (PI4P) accumulation throughout the cytoplasm, which partially colocalized with the endoplasmic reticulum. In contrast, the majority of PI4P accumulated at the Golgi bodies in uninfected cells. The increase in PI4P was not observed after infection with UV-inactivated HCV and did not reflect changes in PI4KA protein or RNA abundance. In an analysis of U2OS cell lines with inducible expression of the HCV polyprotein or individual viral proteins, viral polyprotein expression resulted in enhanced cytoplasmic PI4P production. Increased PI4P accumulation following HCV protein expression was precluded by silencing the expression of PI4KA, but not the related PI4KB. Silencing PI4KA also resulted in aberrant agglomeration of viral replicase proteins, including NS5A, NS5B, and NS3. NS5A alone, but not other viral proteins, stimulated PI4P production in vivo and enhanced PI4KA kinase activity in vitro. Lastly, PI4KA coimmunoprecipitated with NS5A from infected Huh-7.5 cells and from dually transfected 293T cells. In sum, these results suggest that HCV NS5A modulation of PI4KA-dependent PI4P production influences replication complex formation.Hepatitis C virus (HCV) is a major public health problem with limited therapeutic options. Approximately 3% of the world's population is chronically infected with HCV (46) and is at risk for progression to end-stage liver disease and hepatocellular carcinoma. The current standard of care is treatment with pegylated interferon-alpha plus ribavirin, which is successful in only half of treated patients (12). An intensive and prolonged effort to develop therapeutics that target HCV enzymes, including the nonstructural (NS) 3 protease and NS5B RNA-dependent RNA polymerase, is progressing to the clinic. More recently, a drug that appears to target another viral protein, NS5A, has been shown to have rather spectacular properties in terms of viral replication inhibition at picomolar concentrations (13). The first HCV-specific therapeutics targeting NS3 protease activity are likely to become widely available in the near future, with projected sustained virologic responses near 68% for telaprevir and 75% for boceprevir when either is combined with the current standard of care (18,21,27,28). Despite this critical advance, concerns remain over a significant rate of nonresponse and emergence of antiviral resistance (38). For this reason, there is interest in identifying novel drug targets.A number of groups have used small interfering RNA (siRNA) screens to identify cellular cofactors of HCV infection. This approach has a two-pronged benefi...

A Proline-Tryptophan Turn in the Intrinsically Disordered Domain 2 of NS5A Protein Is Essential for Hepatitis C Virus RNA Replication

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Coxsackievirus mutants that can bypass host factor PI4KIIIβ and the need for high levels of PI4P lipids for replication

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The phospholipid code: a key component of dying cell recognition, tumor progression and host–microbe interactions

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