Systematic identification of hepatocellular proteins interacting with NS5A of the hepatitis C virus.
The hepatitis C virus is associated with the development of liver cirrhosis and hepatocellular carcinomas. Among the 10 polyproteins produced by the virus, no function has been clearly assigned to the non-structural 5A (NS5A) protein. This study was designed to identify the hepatocellular proteins that interact with NS5A of the HCV. Yeast two-hybrid experiments were performed with a human liver cDNA prey-library, using five different NS5A derivatives as baits, the full-length NS5A (NS5A-F, amino acid (aa) 1 approximately 447) and its four different derivatives, denoted as NS5A-A (aa 1 approximately 150), -B (aa 1 approximately 300), -C (aa 300 approximately 447) and D (aa 150 approximately 447). NS5A-F, NS5A-B and NS5A-C gave two, two and 10 candidate clones, respectively, including an AHNAK-related protein, the secreted frizzled-related protein 4 (SFRP4), the N-myc downstream regulated gene 1 (NDRG1), the cellular retinoic acid binding protein 1 (CRABP-1), ferritin heavy chain (FTH1), translokin, tumor-associated calcium signal transducer 2 (TACSTD2), phosphatidylinositol 4-kinase (PI4K) and centaurindelta 2 (CENTdelta2). However, NS5A-A produced no candidates and NS5A-D was not suitable as bait due to transcriptional activity. Based on an in vitro binding assay, CRABP-1, PI4K, CENTdelta2 and two unknown fusion proteins with maltose binding protein (MBP), were confirmed to interact with the glutathione S-transferase (GST)/NS5A fusion protein. Furthermore, the interactions of CRABP-1, PI4K and CENTdelta2 were not related to the PXXP motif (class II), as judged by a domain analysis. While their biological relevance is under investigation, the results contribute to a better understanding of the possible role of NS5A in hepatocellular signaling pathways.
Ahn
Jiwon
J
Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Daejeon, Korea.
Chung
Kyung-Sook
KS
Kim
Dong-Uk
DU
Won
Misun
M
Kim
Lila
L
Kim
Kyung-Shin
KS
Nam
Miyoung
M
Choi
Shin-Jung
SJ
Kim
Hyoung-Chin
HC
Yoon
Michung
M
Chae
Suhn-Kee
SK
Hoe
Kwang-Lae
KL
D016174:Hepacivirus / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D020798:Two-Hybrid System Techniques; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase