Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin β3

It has been suggested that nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays a role in the incapacitation of interferon by inactivation of RNA-dependent protein kinase PKR. In order to further investigate the role of NS5A, we tried to identify cellular proteins interacting with NS5A by using the yeast two-hybrid system. The karyopherin beta 3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta 3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta 3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta 3 gene. Human karyopherin beta 3 complemented the loss of the PSE1 and KAP123 functions, supporting growth of the double mutant cells. However, expression of NS5A hampered the growth of the double mutant cells supplemented with human karyopherin beta 3. On the other hand, expression of NS5A by itself had no effect on the grow-th of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta 3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.