Modulation of the transforming growth factor-β signal transduction pathway by hepatitis C virus nonstructural 5A protein

Transforming growth factor-beta (TGF-beta) is implicated in the pathogenesis of liver disease. TGF-beta is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-beta-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-beta-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-beta signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-beta receptor I (T beta R-I) in hepatoma cell lines. This interaction was mapped to amino acids 148 - 238 of NS5A. We also found that NS5A protein co-localized with T beta R-I in the cytoplasm of Huh7 cells and inhibited TGF-beta-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-beta signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-beta-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-beta signaling through interaction with T beta R-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis.