Hepatitis C virus NS5A-regulated gene expression and signaling revealed via microarray and comparative promoter analyses.
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-beta 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 kappaB (NF-kappaB) binding sites within their promoter region. Using the Gibbs sampling method, we also detected enrichment of NF-kappaB consensus binding sites in the upstream regions of the 43 coexpressed genes. Activation of NF-kappaB by NS5A was subsequently demonstrated in luciferase reporter assays. Adenovirus-mediated expression of IkappaBalpha reverted NS5A mediated up-regulation of gene expression. In conclusion, this study suggests a role of NS5A and NF-kappaB in HCV pathogenesis and related liver disease. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html).
Girard
Sophie
S
Department of Microbiology, University of Michigan, Ann Arbor, MI, USA.
Vossman
Erik
E
Misek
David E
DE
Podevin
Philippe
P
Hanash
Samir
S
Bréchot
Christian
C
Beretta
Laura
L
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D006528:Carcinoma, Hepatocellular / Q000235:genetics; D002352:Carrier Proteins / Q000502:physiology; D020869:Gene Expression Profiling; D015967:Gene Expression Regulation, Viral; D006526:Hepatitis C / Q000209:etiology*; D006801:Humans; D016898:Interferon-alpha / Q000627:therapeutic use; D007375:Interleukin-1 / Q000502:physiology; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D050356:Lipid Metabolism; D008113:Liver Neoplasms / Q000235:genetics; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D015398:Signal Transduction / Q000502:physiology*; D016212:Transforming Growth Factor beta / Q000502:physiology; D053773:Transforming Growth Factor beta1; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D012324:RNA-Dependent RNA Polymerase
D002118:Calcium; D002352:Carrier Proteins; D016898:Interferon-alpha; D007375:Interleukin-1; D047908:Intracellular Signaling Peptides and Proteins; D053779:Latent TGF-beta Binding Proteins; D016328:NF-kappa B; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D017361:Viral Nonstructural Proteins; C508960:TGFB1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase