OBJECTIVETo clone the unknown gene of hepatocyte protein interacting with hepatitis C virus core protein.

METHODSUsing the yeast dual hybrid system 3, bait plasmids of hepatitis C virus core were constructed. After identifying hepatitis C virus core protein that could stably expressed in AH109 yeast strains, we performed yeast two hybrid by mating AH109 with Y187 that transformed with liver cDNA library plasmids pACT2 and then plated on quadrople dropout (QDO) medium and assayed for alpha-gal activity. The genes of yeast colonies that could grow on QDO and had alpha-gal activity were sequenced.

RESULTSAmong the 30 positive colonies, we blasted the gene of the sixth colony; we coined human hepatitis C virus binding protein 6(Hu Hcbp6) with Genbank, realized that the Hu Hcbp6 shares as much as 98% homology with two cDNA without knowing functions. We have proved that Hu Hcbp6 could interact with hepatitis C virus core protein.

CONCLUSIONSHepatitis C virus core binding protein (Hu Hcbp 6 Genbank number: AY032594) was successfully cloned and identified. The study partly paved the way for investigating physiological function of the Hu Hcbp6.

Cloning, Molecular ; DNA, Complementary ; genetics ; DNA-Binding Proteins ; genetics ; Hepacivirus ; Humans ; Molecular Sequence Data ; Plasmids ; Sequence Analysis, DNA ; Transfection ; Two-Hybrid System Techniques ; Viral Core Proteins ; genetics ; metabolism ; Yeasts ; genetics

Hepatitis C virus (HCV) is a major causative agent in liver disease. In order to investigate if Korean type HCV core protein and its related mutants, S99Q and S116I, are cytopathic to liver, three types of transgenic mice were established. The expression of transgenes was confirmed by HCV specific RT-PCR and Western immunoblotting. The livers of all wild type core and S116I transgenic lineages remained largely histologically normal. However, the livers of the S99Q transgenic mice showed significant high level of cell dysplasia associated with the transgene expression in hepatocytes largely located around the central veins by in situ hybridization analysis. In conclusion, the mutant HCV core protein at S99Q may contribute to the progress of HCV induced liver disease.
Amino Acid Sequence ; Animals ; Base Sequence ; Gene Expression ; Genetic Vectors/genetics ; Hepatitis C/*pathology/virology ; Hepatitis, Viral, Animal/*pathology/virology ; Hepatocytes/pathology/virology ; Liver/pathology/*virology ; Mice ; Mice, Transgenic ; Molecular Sequence Data ; Mutation/genetics ; RNA, Messenger/chemistry/metabolism ; Research Support, Non-U.S. Gov't ; Transgenes ; Viral Core Proteins/analysis/*genetics/metabolism