Subelement Characterization And Function Analysis Of The Negative Regulatory Element In Hepatitis B Virus

Being as one of the hepadnavirus family member, hepatitis B virus (HBV), a widespread human pathogen causing a variety of clinical manifestations ranging from acute or fulminant hepatitis, various forms of chronic infection, liver cirrhosis to hepatocarcinoma, poses great danger to the human health.Self-replication of HBV can be efficiently regulated at several steps such as replication, expression, transcription and post-transcription, et al. HBV generates the viral chromosome (cccDNA) in the nuclei of the infected hepatocytes, in which the virus replicates continuously and causes the persistent infection. It is difficult for the anti-virus drugs to clear them clearly. Till now, transcription from the minus strand has been analyzed extensively, while little is known about the transcriptional regulation of the complementary plus strand. Previous study in our lab had identified a pieces of HBV sequence nt250-453 as a novel negative regulatory elment.Based on the previous study, this study is to identify the active subelments in HBV nt250-453 negative regulatory elment and to analyze its fuction in HBV replication.PARTâ… CHARACTERIZATION OF THE SUBELEMENTS IN THE NEGATIVE REGULATORY ELEMENT ON HEPATITIS B VIRUSObjectives: To identify the functional subelements in HBV nt250-453 negative regulatory element.Methods: The recombinant luciferase gene reporter plasmids containing the sequence of HBV nt453-434, nt403-384 and nt283-264 were constructed, respectively. The recombinant plasmids and the pRL-TK plasmid were cotransfected into HepG2 cells. The luciferase activities of recombinant plasmids were detected and the levels of mRNA of luciferase gene were determined using reverse transcription PCR.Results: Compared to the pGL3-control group, the relative luciferase activity of the three recombinant plasmids containing single subelement was decreased to 79.66%, 80.79% and 78.36%, respectively. When the recombinant plasmids containing two subelements, relative luciferase activity was reduced to 55.83%, 56.27% and 61.44%, respectively. While recombinant plasmids containing three subelements, relative luciferase activity was decreased to the lowest level of 38.83%. The mRNA levels of luciferase gene were in accordance with the luciferase activity.Conclusions: Three key subelements, nt453-434, nt403-384 and nt283-264 sequence, were successfully identified in the negative regulatory element on hepatitis B virus.PARTâ…¡FUNCTIONAL ANALYSIS OF THE NEGATIVE REGULATORY ELEMENT IN HEPATITIS B VIRUSObjectives: To analyze the function of the nt250-453 negative regulatory element in the process of HBV replication.Methods: The mutant plasmid which had been deleted the sequence of HBV nt250-453 and the helper plasmid were cotransfected into HepG2 cells. The intracellular total DNA were extracted and HBV replicative intermediates were detected by Southern blot assay.Results: HBV replicative intermediates were detected by Southern blot assay. The hybridization signal of HBV relaxed circle DNA in mutant plasmid group was similar to the control group.Conclusions: HBV replicative intermediates were not influenced by the HBV nt250-453 negative regulatory element.