OBJECTIVE To study the suppressive effect of HCV nonstructural 5A (NS5A) protein on tumor necrosis factor alpha (TNF alpha) mediated apoptosis of HepG2 cells. METHODS NS5A gene fragment was amplified by reverse transcription and nested polymerase chain reaction from serum samples positive for anti-HCV. The PCR product was directly cloned using TA cloning kit, and 2 independent clones were isolated, digested and sequenced. Then we constructed HCV NS5A expression plasmid (pcDNA3.1-NS5A), stably transfected into HepG2 cells with lipofectamine. Successful transfection of NS5A gene and expression of NS5A protein were confirmed by Western blot. Transfected cells were incubated with TNFalpha for 48 h, then labeled with Annexin V and visualized by fluorescence microscopy. To examine the effects of NS5A protein on the apoptotic signaling pathway, caspase-3 cleavages and release of cytochrome C were investigated in the transfectant treated with TNF alpha for 48 h and the cell cytosol was subjected to SDS-PAGE and Western blot analysis. RESULTS The stable transfectant of HepG2 cells lines for HCV NS5A protein expression was achieved. The NS5A protein blocked the activation of caspase-3 and the release of cytochrome C in the TNF alpha treated cells. CONCLUSIONS HCV NS5A protein inhibits TNF alpha mediated apoptosis of HepG2 cells in vitro.