Removal Of The Redundant Sequence From The Relaxed Circular DNA Of Hepatitis B Virus During The Formation Of Covalently Closed Circular DNA

Hepatitis B virus infection represents a global public health problem.A major challenge facing current therapy for chronic Hepatitis B virusinfection is that neither the long-acting interferon, nor the nucleosideanalogues can clear HBV cccDNA in vivo thoroughly. Development ofnew strategies aiming at the clearance of cccDNA thus becomes imperativeunder this circumstance. A prerequisite for the development of such newstrategies is the clarification of the mechanism of the formation of HBVcccDNA. Comparison of the structure of the HBV rcDNA with that of theHBV cccDNA indicates that an indispensable step during the formation ofHBV cccDNA is the removal of the redundant sequence at the5' or the3'end of the minus strand DNA of the HBV rcDNA. In the present study, weinvestigated the mechanisms by which the redundant sequence is removed. Objective:To clarify how the redundant sequence (5'r or3'r) is removed fromthe minus-strand DNA in the conversion of HBV rcDNA into HBVcccDNA, and explores the molecular mechanism of HBV cccDNAformation.Methods:1,The HBV DNA expression plasmid with C1826A mutation wasconstructed, and the stable cell line replicating the HBV DNA carrying theC1826A mutation was constructed.2,Methods for specific amplification of HBV rcDNA plus-strandDNA and HBV cccDNA were established.3,A single-site mutation detection method based on PCR technologywas developed and evaluated.4,The percentage of the C1826A genotype in the plus-strand DNA ofHBV rcDNA was determined by TA cloning of plus-stand DNA fragmentand the mutation detection method above.5,The percentage of the C1826A genotype in the cccDNA wasdetermined as above.6,The question that which copy of the redundant sequence wasremoved was answered through the analysis of the data obtained above. Results:1,The stable cell line can replicate HBV DNA. Sequence analysis ofthe DNA fragment amplified from the genomic DNA of the cells confirmedthe C1826A mutation.2,The plus-strand DNA fragment containing nt1826can be amplifiedspecifically by using the two-step method. The first step was a singleprimer extension reaction using a reverse primer with exogenous sequence,and the second step was a PCR step using the exogenous sequence and anHBV forward sequence as primes.3,HBV cccDNA can be amplified specifically by using the twoprimers which anneal to the two sequences separated by the two gaps in theplus-and minus-strand DNA.4,Eighty-three percent to ninety-nine percent of the plus-strand DNAof HBV rcDNA contain an1826C, while1%-17%contain an1826A.5,Forty-three percent to sixty-nine percent of the HBV cccDNAcontain an1826C, while31%-57%contain an1826A.Conclusions:1,The stable cell line transfected with HBV DNA containing C1826Amutation can replicate HBV DNA.2,Methods for specific amplification of the plus-strand DNA of HBVrcDNA and cccDNA were established. 3, A method based on PCR technology, which can effectivelydistinguish specific nucleotides, was developled.4,In the reverse transcription process of pgRNA to HBV rcDNA, themajority of plus-strand DNA are synthesized using5'r as the template.5,During the formation of HBV cccDNA, at least the majority of the5'r, rather than the3'r, in the minus-strand DNA of rcDNA are removed.