The SH3 binding motif of HCV [corrected] NS5A protein interacts with Bin1 and is important for apoptosis and infectivity.
OBJECTIVE
HCV nonstructural protein 5A (NS5A) has been implicated in regulating cell growth and interferon response. The NS5A protein contains proline-rich regions that are highly conserved among HCV genotypes and match Src homology 3 (SH3)-binding motifs (PxxP) found in various cellular signaling molecules.


METHODS
We screened for HCV NS5A interacting proteins by using the yeast 2-hybrid system and studied the functional consequence of this interaction.


RESULTS
Several independent clones containing SH3 domains were isolated along with Bin1, a tumor suppressor with pro-apoptotic properties, being the most frequently identified clone. The protein-protein interaction between NS5A and Bin1 was confirmed by in vitro binding, in vivo co-immunoprecipitation, and confocal microscopy. Deletion and mutation analyses indicated that the SH3 binding motif of HCV NS5A and SH3 domain of Bin1 are essential for interaction. Human hepatoma (HepG2) cells lacking expression of Bin1 undergo apoptosis upon infection with adeno-Bin1. Bin1-induced apoptosis was inhibited in HepG2 cells expressing wild-type NS5A but not NS5A mutant with mutations in the SH3 binding motif. Infectious HCV genome containing mutations in the SH3 binding motif was not infectious in chimpanzees.


CONCLUSIONS
Our results indicate that this interaction is implicated in productive HCV infection and may contribute to the pathogenesis of hepatocellular carcinoma. In addition, the NS5A PxxP motif may represent a novel target for antiviral development.
Nanda
Santosh K
SK
Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Herion
David
D
Liang
T Jake
TJ
D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D016174:Hepacivirus / Q000502:physiology*; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D002679:Pan troglodytes; D012367:RNA, Viral / Q000096:biosynthesis; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D014779:Virus Replication; D018909:src Homology Domains; D012324:RNA-Dependent RNA Polymerase
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D025521:Tumor Suppressor Proteins; D017361:Viral Nonstructural Proteins; C101410:BIN1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase