TGF-signaling induces epithelial to mesenchymal transition (EMT) and plays an important role in hepatocellular carcinoma (HCC) development. Clinical observations indicate that hepatitis C virus (HCV) chronic infection, which is a major cause of HCC, induces TGF-signaling perturbations. Here, we investigate the mechanisms by which HCV nonstruc-tural proteins interfere with TGF-signaling, in human hepatoma cell lines expressing HCV subgenomic repli-con. A transcriptomic study showed that TGF-stimulation of these cells resulted in a protumoral gene expression profile and in up-regulation of EMT-related genes compared to control interferon-treated cells not expressing HCV proteins. We found that the viral protease NS3-4A interacted with SMURF2, a negative regulator of TGF-signaling. In cells expressing HCV subgenomic replicon or NS3-4A, TGF-stimulation induced an increased expression of SMAD-dependent genes compared to control cells. This enhanced signal-ing was suppressed by SMURF2 overexpression and mimicked by SMURF2 silencing. In addition, NS3-4A expression resulted in an increased and prolonged TGF-induced phosphorylation of SMAD2/3 that was abrogated by SMURF2 overexpression. Neither NS3-4A protease activity nor SMURF2 ubiquitin-ligase activity was required to affect TGF-signaling. Therefore, by targeting SMURF2, NS3-4A appears to block the negative regulation of TGF-signaling, increasing the responsiveness of cells to TGF-. Verga-Gérard, A., Porcherot, M., Meyniel-Schicklin, L., André, P., Lotteau, V., and Perrin-Cocon, L. Hepatitis C virus/human interactome identifies SMURF2 and the viral protease as critical elements for the control of TGF-signaling. FASEB J. 27, 4027-4040 (2013). www.fasebj.org