Identification And Analysis Of A Novel Negative Regulatory Element In Hepatitis B Virus S-(+)-Strand

Hepatitis B virus which infects the livers of human,causing acute and chronic liver diseases,and even liver cirrhosis and primary hepatocellular carcinoma,is a dangerous pathogen for human health. One of the most important reasons that the anti-virus drugs have limited or no effects on virus infection is that they fail to quickly and effectively inhibit HBV replication and clear the virus.HBV replicates in infected hepatocyte nuclei continuously and it is difficult for the anti-virus drugs to clear them clearly. The robust replicating ability of HBV demonstrates the existence of a self-adjustion mechanism of the virus, which enables the continuous replication of the virus and the longlasting infection of the hosts. Therefore, the detection of the critical regulatory elements in HBV replication and transcription, will be helpful to further explore the molecular feature of HBV and provid new potential target for the development of anti-virus drugs.A regulatory DNA element can positively or negatively regulate the gene expression on transcription or posttranscription level by binding cellular or viral proteins. Till now, transcription from the minus strand has been analyzed extensively, while little is known about the transcriptional regulation of the complementary plus strand. The expression levels of the transcripts of plus strand (HBV asRNAs), which can negatively regulate the transcription from minus strand, are extremely low, suggesting that some"strategies"have been developed during HBV evolvement to inhibit the transcription from the S-(+)-strand, for example, the existence of the negative regulatory elements on HBV plus strand.In this study, we scanned the whole HBV genome for the potential regulatory elements located on the S-(+)-strand by inserting fragments of HBV DNA upstream a heterologous luciferase reporter vector in antisense orientation. We found that the reporter plasmid containing nt 509-1(3182)-2639 of HBV inhibitied luciferase activity and repressed the luciferase mRNA level in HepG2 cells. Serial deletional analysis delineated the sequence essential for the inhibitory effect to nt 453-250 of HBV genome. Repression mediated by this 203nt fragment of HBV sequence acted in both orientation- and position-independent manners since inhibition was also observed when this DNA element was inserted downstream of the polyA site of the luciferase gene in either sense or antisense orientation. The inhibitory effect of the negative element would not be affected by different promoter demonstrated that the inhibition had no promoter specificity. Furthermore, our results indicated that this negative regulatory element functioned in hepatocye-independent fashion, since it could exert the inhibitory effect in both hepatocyte and nonhepatocyte cell lines. Quantitative PCR using probes at different target sites of luciferase transcript, revealed that nt 453-250 of HBV greatly inhibited the transcription initiation. Using site-directed mutagesis method, three subelements, HBV nt 453-434,HBV nt 403-384,HBV nt 283-264 sequence, were identified in the negative regulatory element, which inhibited gene transcription. Alignment analysis revealed that HBV nt453-250 sequence and the three subelements had high similarity in other HBV gene subtypes and other viruses of Hepadnaviruses (WHV, DHBV).Thus, our results strongly suggested that nt 453-250 of HBV should act as a novel negative regulatory element, which had not been reported before. The negative regulatory element could inhibit the transcription of HBV plus strand and enhance the transcription and expression of HBV minus strand. The existence of the novel inhibitory element serves as a self-regulation of HBV and provides us new insights into the molecular behaviour of the virus and its host cells.