Comparison Of The Different Activities Of Genotype B And C Hepatitis B Virus Regulatory Elements

Although safe and effective vaccine for hepatitis B virus (HBV) has been available fornearly three decades, infection by this virus remains a burden of globle health. Analyses of thedivergence of HBV genomic sequences have led to the identification of ten HBVgenotypes (Ato J). Genotype B and C are prevalent in China, a country with high prevalence of HBV.Accumulating evidences have shown that there are significant clinical and virologicaldifferences between genotype B and C. However, the specific mechanism of the differercesbetween the two genotypes has not been fully elucidated.The genome of HBV is3.2kb in length, which habors six regulatory elements includingfour promoters, namely Core, S1, S2and X, and two enhancers namely enhancerI andenhancerII. In view of the essential roles these regulatory elements play in HBV replicationand transcription, we tried to find out mechanisms of the differences between genotype B andC through camparison activities of their regulatory elements.In the first part of this study, the consensus sequences of genotype B and C HBV genomewere identified by homology analysis. Then the regulatory elements were amplified fromclinically isolated HBV stains with genotype B and C respectively. The luciferase reporterplasmids harboring HBV regulatory elements of genotype B and C consensus weresuccessfully constructed by site-directed mutagenesis, and this laid a foundation for furtherstudy.In the second part, the activities of genotype B and C HBV regulatory elements werecompared, and the results showed that there are no significant diferences in Xp-EnhI, Xp, andBCp activities, while Cp-EnhII, Cp and SpI activities of genotype B are higher than genotypeC, and SpII activity of genotype B is lower than that of genotype C. Further comparison ofSpII activities after exchanged of its3' half sequence of two genotypes showed that both5'and3'half of SpII are essential for its fuction. In the third part of this study, we first comfirmed our presumption that the activity ofgenotype B EnhII is higher than that of genotype C, and then after conducting severalgenotype B consensus to C site mutations, we found out that1635-1640and1654are the twosites which responsible for efficient EnhII activity between genotype B and C. EMSA wasperformed to comfirmed our presumption that there are nuclear proteins that bind to1635-1640site. Futrthermore, DNA pull donwn was conducted to screen these nuclearproteins, and PARP1, SFPQ, NonO, Ku70and Ku80were identified as five candidates. Theresults of our research indicated that the interaction between transcriptional factors and1635-1640site and its effects on EnhII may be one of the mechanisms involved in genotype Band C HBVdifferences.