Screening For The Hepatocyte Proteins Interacting With Hepatitis B Spliced Protein By Yeast Two-hybrid Assays

The hepatitis B spliced protein (HBSP) encoded by a2.2kb singly spliceddefective HBV genome has been shown to play an important role inhepatopathogenesis, yet the exact mechanisms remain to be fully elucidated. To screenfor hepatocellular proteins interacting with HBSP, a conventional yeast two-hybridassay was used.1166positive clones were preliminary obtained, and the false positiveand repeated clones were discarded according to the results of phenotype validationand transactivational ability detection. Finally,38positive clones were confirmed to beinteracted with HBSP in yeast cells including metallothionein-2(MT2), alcoholdehydrogenase1A (class I), alpha polypeptide (ADH1A), tRNA-splicing endonucleasesubunit34(TSEN34) and acetyl-CoA acyltransferase2(ACAA2). The interactionbetween HBSP and ADH1A was tested furtherly. pGEX-4T-1-HBSP expressingGST-HBSP fusion protein and pCMVTNT-ADH1A expressing ADH1A by in vitrotranslation were constructed, and HBSP-ADH1A interaction were validated by GSTpull-down assay. Huh7cells were infected with HBSP-expressed recombinantadenoviruses, and the interaction between HBSP and endogenous ADH1A were testedby co-immunoprecipitation. This study will lay foundation for the further study ofpathogenicity of HBSP.