title: [Location of the binding position for HBV X protein functional interaction with cytochrome C oxidase III].
abstract: OBJECTIVE
  To identify the binding site position of the hepatitis B virus (HBV) X protein (HBx) functional interaction with the cytochrome C oxidase subunit III (COX III, a key regulator of mitochondrial function) by using a yeast two-hybrid system.
  
  
  METHODS
  Two fragments of HBx mutants (X1 1-72aa and X2 1-117aa) were amplified by PCR and inserted into the bait plasmid pAS2-1.The resultant mutant plasmids were transfected into yeast cells using the lithium acetate-method.PCR and gene sequencing were used to confirm that the mutant fragments were expressed properly in yeast cells.Western blotting was used to verify that the mutant proteins were translated accurately in the yeast cells.Filter assay was used to exclude autoactivated mutants.Hybridization in solid medium and beta-gal activity detection were used to determine the precise position of the binding site for HBx and COX III interaction.
  
  
  RESULTS
  The two mutant plasmids containing HBx 1-72aa and 1-117aa respectively were successfully constructed and the mutants were both properly expressed and translated in yeast cells; no autoactivated mutants were detected throughout the experimental process.The binding site of HBx and COX III was found to be encompass the amino acids 72 through 117 of HBx.
  
  
  CONCLUSIONS
  Amino acids 72 through 117 of HBx are the key domain of the HBx functional interaction With COX III; this domain may represent a useful target for molecular-based therapies to treat HBV-related diseases.
authors.affiliation: Department of Digestive Disease, Union Hospital of Fujian Medical University, Fuzhou 350001, China.
authors.forename: Dan
authors.initials: D
authors.lastname: Li
authors.forename: Zhixin
authors.initials: Z
authors.lastname: Chen
authors.forename: Yun
authors.initials: Y
authors.lastname: Chen
authors.forename: Na
authors.initials: N
authors.lastname: Lin
authors.forename: Xiaozhong
authors.initials: X
authors.lastname: Wang
mesh_terms: D003576:Electron Transport Complex IV / Q000378:metabolism*; D006515:Hepatitis B virus / Q000378:metabolism*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D015534:Trans-Activators / Q000378:metabolism*; D014162:Transfection; D020798:Two-Hybrid System Techniques; D054334:Viral Regulatory and Accessory Proteins
chemical_list: D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D003576:Electron Transport Complex IV