Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin beta3

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 beta3 gene was isolated from a human liver cell library as a protein interacting with NS5A. The protein-protein interaction between NS5A and karyopherin beta3 was confirmed by in vitro binding assay and an in vivo coimmunoprecipitation method. The effect of NS5A on the karyopherin beta3 activity was investigated using a yeast cell line containing mutations in both PSE1 and KAP123, genes that are homologous to the human karyopherin beta3 gene. Human karyopherin beta3 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 beta3. On the other hand, expression of NS5A by itself had no effect on the growth of the double mutant expressing wild-type yeast PSE1. This indicates that NS5A may inhibit karyopherin beta3 function via protein-protein interaction. The role of NS5A in HCV replication is discussed.     

GPS2 Is Required for the Association of NS5A with VAP-A and Hepatitis C Virus Replication

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a component of the replication complex associated with various cellular proteins. It has been reported that G protein pathway suppressor 2 (GPS2) is a potential NS5A-binding factor, as identified in a yeast two-hybrid screens of human cDNA library using viral proteins as baits [1]. In this study, we demonstrated the interaction between GPS2 and NS5A in mammalian cells by coimmunoprecipitation analysis and found that both exogenously and endogenously expressed GPS2 interacted with NS5A of genotype 1b and 2a. Mutagenesis study demonstrated that Domain I of NS5A and coiled-coil domain of GPS2 are responsible for the interaction. Knockdown of GPS2 in hepatoma cell lines suppressed the replication of HCV RNA, which can be rescued by the expression of an RNAi-resistant GPS2. Furthermore, overexpression of GPS2 enhanced the association of NS5A with a proviral cellular factor, human vesicle-associated membrane protein-associated protein A (VAP-A), while knockdown of GPS2 disrupted interaction between VAP-A and NS5A. Taken together, our results suggest that GPS2 acts as a bridge between NS5A and VAP-A and is required for efficient HCV replication.