Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid.
OBJECTIVE
To identify the pre-X region in hepatitis B virus (HBV) genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.


METHODS
The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for alpha-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics.


RESULTS
After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4-CD81 (TM4SF4).


CONCLUSIONS
The pre-X gene exists in HBV genome. Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.
Yang
Qian
Q
Department of Infectious Diseases, The Second Hospital of Xi'an Jiaotong University, Shaanxi Province, China.
Cheng
Jun
J
Dong
Jing
J
Zhang
Jian
J
Zhang
Shu-Lin
SL
D001483:Base Sequence; D016679:Genome, Viral; D006509:Hepatitis B / Q000453:epidemiology / Q000821:virology*; D006515:Hepatitis B virus / Q000235:genetics*; D022781:Hepatocytes / Q000821:virology*; D006801:Humans; D017720:Molecular Epidemiology; D008969:Molecular Sequence Data; D015534:Trans-Activators / Q000235:genetics*; D020798:Two-Hybrid System Techniques; D054334:Viral Regulatory and Accessory Proteins; D015003:Yeasts
D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein