Cytochrome C oxidase III interacts with hepatitis B virus X protein in vivo by yeast two-hybrid system.
OBJECTIVE
To screen and identify the proteins which interact with hepatitis B virus (HBV) X protein in hepatocytes by yeast two-hybrid system and to explore the effects of X protein in the development of hepatocellular carcinoma (HCC).


METHODS
With HBV X gene amplified by polymerase chain reaction (PCR), HBV X bait plasmid, named pAS2-1-X, was constructed by yeast-two hybridization system3 and verified by auto-sequencing assay. pAS2-1-X was transformed into the yeast AH109, and X-BD fusion protein expressed in the yeast cells was detected by Western blotting. The yeast cells cotransformed with pAS2-1-X and normal human liver cDNA library were grown in selective SC/-trp-leu-his-ade medium. The second screen was performed with beta-gal activity detection, and false positive clones were eliminated by segregation analysis, true positive clones were amplified, sequenced and analyzed with bioinformatics. Mating experiment was performed to confirm the binding of putative proteins to X protein in the yeast cells.


RESULTS
Bait plasmid pAS2-1-X was successfully constructed and pAS2-1-X correctly expressed BD-X fusion protein in yeast AH109. One hundred and three clones grew in the selective SC/-trp-leu-his-ade medium, and only one clone passed through beta-gal activity detection and segregation analysis. The inserted cDNA fragment showed high homology with Homo sapiens cytochrome C oxidase III (COXIII). Furthermore, mating experiment identified that the binding of COXIII to X protein was specific.


CONCLUSIONS
COXIII protein is a novel protein that can interact with X protein in vivo by yeast two-hybrid system, and may contribute to the development of HCC through the interaction with X protein.
Li
Dan
D
Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China. lidan2006@hotmail.com
Wang
Xiao-Zhong
XZ
Yu
Jie-Ping
JP
Chen
Zhi-Xin
ZX
Huang
Yue-Hong
YH
Tao
Qi-Min
QM
D006528:Carcinoma, Hepatocellular / Q000821:virology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D003576:Electron Transport Complex IV / Q000235:genetics / Q000378:metabolism*; D015723:Gene Library; D006515:Hepatitis B virus / Q000378:metabolism*; D006801:Humans; D008099:Liver / Q000201:enzymology; D008113:Liver Neoplasms / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D054334:Viral Regulatory and Accessory Proteins
D018076:DNA, Complementary; D011994:Recombinant Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D003576:Electron Transport Complex IV