Modulation of cell growth by the hepatitis C virus nonstructural protein NS5A

Hepatitis C virus nonstructural protein, NS5A, is a phosphoprotein produced from the processing of the viral polyprotein precursor, NS5A associates with several cellular proteins in mammalian cells, and the biological consequences of this interaction are currently unknown. To this end, five stable NS5A-expressing murine and human cell lines were established, Tetracycline-regulated NIH3T3 cells and rat liver epithelial cells as well as the constitutive, NS5A-expressing, human Chang liver, HeLa, and NIH3T3 cells all exhibited cell growth retardation compared with the control cells. Cell cycle analysis by flow cytometry indicated that the NSSA-expressing human epitheloid tumor cells had a reduced S phase and an increase in the G(2)/M phase, which could be explained by a p53-dependent induction of p21(Waf1/Cip1) protein and mRNA levels. NS5A interacts with Cdk1 in vivo and in vitro, and a significant portion of the p21(Waf1/Cip1) was found to be in a complex with Cdk2 in the NS5A-expressing human hepatic cell line, Cdk1 and cyclin B1 proteins were also reduced in human Chang liver cells consistent with the increase in G(2)/M phase. Our results suggest that the NS5A protein causes growth inhibition and cell cycle perturbations by targeting the Cdk1/2-cyclin complexes.