Replication of DHBV genomes with mutations at the sites of initiation of minus- and plus-strand DNA synthesis

We have examined the consequences on duck hepatitis B virus DNA synthesis of deleting the 5′ and 3′ copies of the 12 base sequence, DR1, from the viral pregenome. With the wild-type virus, reverse transcription initiates at nt 2537 within the 3′ copy of DR1. When this sequence was deleted, initiation of reverse transcription was found at two other sites located closer to the 3′ end of the pregenome (nt 2576 and nt 2644). The 3-base motif UUA was the only sequence common to these sites as well as the wild-type initiation site in DR1. Deletion of the 5′ copy of DR1 did not alter minus strand synthesis, but led to aberrant priming of plus strand synthesis to generate predominantly linear rather than relaxed circular, double-stranded viral DNA, in agreement with the recent report by Loeb et al. (EMBO J. 10, 3533-3540, 1991). A mutant lacking only the 3′ copy of DR1 rapidly converted to wild type in transfected cells. This apparently occurred as a consequence of conversion of newly synthesized relaxed circular to covalently closed circular (CCC) DNA, which might then serve as a template for the synthesis of wild-type viral RNAs. A mutant lacking only the 5′ copy of DR1 did not exhibit this behavior. These results support the conclusion that amplified CCC DNA serves as transcriptional template.