Hepatitis C virus NS5A-regulated gene expression and signaling revealed via microarray and comparative promoter analyses

Abstract

Most individuals exposed to hepatitis C virus (HCV) become chronically infected and are predisposed to liver disease. The mechanisms underlying viral persistence and disease progression are unknown. A role for the HCV NS5A protein in viral replication and interferon resistance has been demonstrated. To identify mechanisms affected by NS5A, we analyzed the gene expression of Huh7 cells expressing NS5A and control cells using oligonucleotide microarrays. A set of 103 genes (43 up-regulated, 60 down-regulated) whose expression was modified by at least twofold was selected. These included genes involved in cell adhesion and motility, calcium homeostasis, lipid transport and metabolism, and genes regulating immune responses. The finding of modulated expression of genes related to the TGF-Β superfamily and liver fibrosis was observed. Interestingly, both the tumor necrosis factor and lymphotoxin beta receptors were down-regulated by NS5A. Similar data were obtained following expression of four NS5A mutants obtained from patients who were not responsive or were sensitive to interferon therapy. Through computational analysis, we determined that 39 of the 43 genes up-regulated by NS5A contained one or more nuclear factor ΚB (NF-ΚB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-ΚB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-ΚB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IΚBΑ reverted NS5A mediated up-regulation of gene expression. In conclusion , this study suggests a role of NS5A and NF-ΚB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ). (H EPATOLOGY 2004;40:708–718.)