Screening For Hepatocellular Proteins Interacted With Hepatitis B Doubly Spliced Protein (HBDSP) Encoded By2.2Kb Doubly Spliced Variant Of Hepatitis B Virus By Immunoprecipitation

Hepatitis B virus (HBV) is a small enveloped hepatotropic virus with a partiallydouble-stranded DNA genome of approximately3.2kb in length. There are about350million people infected with hepatitis B virus throughout the world, of which Asiaaccounted for75%. HBV is the main cause of chronic hepatitis, cirrhosis andhepatocellular carcinoma, leading to the deaths of one million people each year.The spliced variants of hepatitis B virus genomes are a group of the subgenomicDNA which is generated from the spliced3.5Kb pregenomic RNA (pgRNA) of HBVand reverse transcription.2.2kb HBV spliced variants accounted for more than80%and are composed of single-spliced and double-spliced types. It has been documentedthat2.2Kb HBV spliced variants may induce hepatocellular apoptosis and are closelyrelated to persistent infection of HBV. To investigate the pathogenesis of hepatitis Bdoubly spliced protein (HBDSP), which encoded by the double-spliced splicingvariants of HBV, immunoprecipitation was subjected to screen for the hepatocellularproteins interacting with HBDSP.In the first part of this study, immunoprecipitation and Matrix-assisted laserdesorption ionization time of flight mass spectrometry (MALDI-TOF-MS) methodswere used and four proteins were identified as partners interacting with HBDSP, asfollows, General transcription factor II I (GTF2I or TFII-I), Glucose-regulated protein78(GRP78), Heat shock protein,70kDa (HSP70), Potassium voltage-gated channel(KVβ.2). In the second part of this study, co-immunoprecipitation experiments wereprimarily used to verify interaction between HBDSP and three proteins of TFII-I,GRP78and KVβ.2. And the results confirmed the interaction between HBDSP andTFII-I in vivo, however, the interaction between HBDSP with GRP78and KVβ.2donot occur in vivo. Moreover, GST pull-down experiments was applied to furtherconfirm the interaction between HBDSP and TFII-I in vitro. TFII-I is a multifunctional transcription factor. It not only promotes chromatin remodeling, alsoplays an important role in the cell cycle. It is postulated that the interaction of HBDSPand TFII-I may affect the survival and proliferation of hepatoma cells. This study willprovide a clue to further explore of the pathogenicity of HBDSP.