Abstract
Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5′ and 3′ ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5′ ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5′ end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.
Highlights
Hepadnaviruses are small DNA viruses that replicate their genomes by reverse transcription of an RNA intermediate [1,2]
During early stages of infection, additional closed circular (CCC) DNA is produced from newly synthesized relaxed circular (RC) DNA present in cytoplasmic core particles by an intracellular amplification pathway [8,9]
Model 1 predicts that r5 is present in CCC DNA whereas model 2 predicts that r5 is removed prior to CCC DNA formation (Figure 1B,C)
Summary
Hepadnaviruses are small DNA viruses that replicate their genomes by reverse transcription of an RNA intermediate [1,2]. The first (model 1) predicts that the reverse transcriptase performs a cleavage-ligation reaction to synthesize the minus strand of CCC DNA, which could serve as a template for the repair of plus strand DNA.
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