| Acute and chronic liver diseases caused by hepatitis B virus (HBV) infection are very dangerous for human health, and no effective therapeutic methods and solutions have been found till now. HBV depends on its self-adjustion mechanism to ensure the continuous replication of the virus and the persistant infection of the hosts. Study of HBV expression regulation is one of the hotspots during Molecular biology field recently. Various of hepatocyte nuclear factors and regulatory elements display their function on transcriptional regulation by interacting with HBV promoters. In our previous studies for HBV antisense RNAs, we used polymerase chain reaction to scan the whole HBV genome for the potential regulatory elements located on the S-(+)-strand, and fragments of HBV DNA were inserted upstream a heterologous luciferase reporter vector in antisense orientation. We found that the reporter plasmid containing nt250-453 of HBV inhibitied luciferase activity and repressed the luciferase mRNA level in HepG2 cells. Serial deletion analysis delineated this sequence is essential for the inhibitory effect. Repression mediated by the 203 base pair of HBV sequence did not act in both orientation- or position-independent manners. All showed it satisfied the requirements as"silencer". So we presumed that the novel negative element nt250-453 of HBV should inhibit the transcription of HBV plus stand. It should also suppress some gene expression in HBV minus strand, such as HBV X protein (HBx). The existence of the novel inhibitory element serves as a self-regulation of HBV and provides us new insights into the molecular behaviour between the virus and its host cells, and also into the effective anti virus therapeutic methods.In this study, four pairs of plasmids containing the four HBV promoters sequences were constructed respectively. In each pair, one had the fragment nt250-453 of HBV inserting downstream the SV40 late poly A signal of plasmid pGL3control, while the other lacking the fragment was used as the control. The firefly luciferase activity and mRNA expression of firefly luciferase gene were both detected by dual-luciferase assay system and reverse transcription polymerase chain reaction to confirm the negative regulatory effect of the novel element on HBV promoters. The results suggested that this element could down-regulated the mRNA transcription of luciferase gene driven by the four promoters of HBV, and the effect on X promoter is more effective than others. So X promoter was chosen as the studying object for the next step. Then, in the following study, fragment nt250-453 of HBV were removed from plasmid LJ196 which expresses HBV pregenome RNA under the control of cytomegalovirus (CMV) immediate early promoter and does not express P, C, or envelope proteins. The mutated LJ196 and the expression plasmid for HBV P and C proteins, LJ96, were used to co-transfect HepG2 cell line to ensure the virus can replicate in the host cell. RT-PCR was performed to detect the X gene mRNA level, while Western blot for the HBx. The results showed that the expressions of HBV X gene mRNA and X protein were both increased as the sequence's removal from HBV genes, which provided a reverse proof of its negative regulatory effect on X promoter.Thus, our results strongly demonstrated that nt250-453 of HBV should acts as a novel negative regulatory element which could suppress the four HBV promoters activity in luciferase reporter plasmid. Since it made more effect on X promoter, it also inhibited the X promoter activity in the HBV replicating model. HBx coded by 0.8kb mRNA which transcribes under the control of X promoter is a trans-activator of transcription, which could activate transcription of many genes, and is related with tumor occurrence, and can regulate the replicate of HBV. Therefore, the repression of X promoter activity by the novel negative regulatory element should prompt us that it would be such element balancing virus and its hosts,which we should study in the future research. |
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