The Interaction Of Hepatitis B Spliced Protein HBSP And UXT And The Effects On NF-?B Pathway In Hepatocytes

Spliced variants of hepatitis B virus genomes are a group of subgenomic length DNA generated from 3.5 kb pregenomic RNA by splicing and reverse transcription.More than 80% of these spliced variants are 2.2 kb singly spliced variants of hepatitis B virus genome(spliced at the nucleotides position 2447 nt-489 nt, also called 2447nt-489 nt HBV spliced variant), which could encode Hepatitis B spliced protein(HBSP) and closely related with persistent infection and pathogenicity of HBV.Previously, we had dentified ubiquitously expressed transcript(UXT) as a hepatocyte protein interacted with HBSP by yeast two-hybrid screening. UXT mianly contains two isoforms, namely UXT-V1 and UXT-V2. The aim of this study was to further confirm the interaction between HBSP and UXT, and elucidate its biological significance.The first part of this study is to confirm the interactions of HBSP with UXT. To address these issues, the plasmids of pAcGFP-N1-UXT-V1 and pAcGFP-N1-UXT-V2(used for confocal microscopy) were constructed, and the co-immunoprecipitation, confocal microscopy and mammalian two hybrid screening were applied. The results demonstrated that HBSP could interact with UXT-V1 but not UXT-V2.The second part of this study aimed to explore the possible effects of the HBSP-UXT-V1 interaction on the NF-?B promoter activity. In doing so, hepatocytes were co-transfected with luciferase reporter plasmid harboring NF-?B promoter and HBSP expression vector, and treated with TNF-?.The results demonstrated that HBSP could enhance the activity of NF-?B promoter partly through interacting with UXT-V1.