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Hepadnaviruses are DNA viruses that are found in several mammalian and avian species. These viruses replicate their genome through reverse transcription of an RNA intermediate termed pregenomic RNA (pgRNA). pgRNA is reverse transcribed by the viral polymerase into a minus-strand DNA, followed by synthesis of the plus-strand DNA. There are multiple cis-acting sequences that contribute to the synthesis of minus-strand DNA for human hepatitis B virus (HBV). Less is known about the cis-acting sequences of avian hepadnaviruses that contribute to synthesis of minus-strand DNA. To identify cis-acting sequences of duck hepatitis B virus (DHBV) and heron hepatitis B virus (HHBV), we analyzed variants containing 200-nucleotide (nt) deletions. Most variants of DHBV synthesized minus-strand DNA to 50 to 100% of the wild-type (WT) level, while two variants synthesized less than 50%. For HHBV, most variants synthesized minus-strand DNA to less than 50% the WT level. These results differ from those for HBV, where most of the genome can be removed with little consequence. HBV contains a sequence, φ, that contributes to the synthesis of minus-strand DNA. It has been proposed that DHBV has an analogous sequence. We determined that the proposed φ sequence of DHBV does not contribute to the synthesis of minus-strand DNA. Finally, we found that the DR2 sequence present in all hepadnaviruses is important for synthesis of minus-strand DNA in both DHBV and HHBV but not in HBV. These differences in cis-acting sequences suggest that the individual hepadnaviruses have evolved differences in their mechanisms for synthesizing minus-strand DNA, more so than for other steps in replication.

cis -Acting Sequences That Contribute to Synthesis of Minus-Strand DNA Are Not Conserved between Hepadnaviruses