Interferon-inducible cholesterol-25-hydroxylase restricts hepatitis C virus replication via blockage of membranous web formation.

Hepatitis C virus (HCV) is a positive strand RNA virus that primarily infects human hepatocytes. HCV infections constitute a global health problem with 80 million people currently chronically infected. Recent studies reported that cholesterol 25-hydroxylase (CH25H) is expressed as an interferon-stimulated gene (ISG) and mediates antiviral activities against different enveloped viruses through the production of 25-hydroxycholesterol (25HC). However, the intrinsic regulation of human CH25H (hCH25H) expression within the liver as well as its mechanistic effects on HCV infectivity remains elusive. In this study, we characterized the expression of hCH25H using liver biopsies and primary human hepatocytes (PHH). In addition, the antiviral properties of this protein and its enzymatic product, 25HC, were further characterized against HCV in tissue culture. hCH25H mRNA levels were significantly upregulated both in HCV-positive liver biopsies and HCV-infected PHH. The expression of hCH25H in PHH was primarily and transiently induced by type-I interferon (IFN). Transient expression of hCH25H in human hepatoma cells restricted HCV infection in a genotype-independent manner. This inhibition required the enzymatic activity of CH25H. We observed an inhibition of viral membrane fusion during the entry process by 25HC, which was not due to a virucidal effect. Yet the primary effect by 25HC on HCV was at the level of RNA replication which was observed by using subgenomic replicons of two different genotypes. Further analysis using electron microscopy revealed that 25HC inhibited formation of the membranous web (MW), the HCV replication factory, independent of RNA replication. In conclusion, HCV infection causes the upregulation of interferon-inducible CH25H in vivo. Its product 25HC restricts HCV primarily at the level of RNA replication by preventing the formation of viral replication factory. This article is protected by copyright. All rights reserved.