TRANSFER OF THE MINUS-STRAND OF DNA DURING HEPADNAVIRUS REPLICATION IS NOT INVARIABLE BUT PREFERS A SPECIFIC LOCATION

The current model for replication of duck hepatitis B virus has reverse transcription initiating and copying a UUAC motif within the encapsidation signal, epsilon, near the 5' end of the RNA template. This results in synthesis of four nucleotides of DNA. This short minus-strand DNA product is then transferred to a complementary position, at DR1, near the 3' end of the RNA template. Elongation of minus-strand DNA then ensues. We have examined the transfer of minus-strand DNA during replication of duck hepatitis B virus in cell culture. The initial aim of this work was to examine the effect of mutations at DR1 on the transfer process. We found that when mutations were introduced into the UUAC motif overlapping DR1, the 5' end of minus-DNA no longer mapped to position 2537 but was shifted two or four nucleotides. Mismatches were predicted to exist at the new sites of elongation. Elongation from nucleotide 2537 could be restored in these mutants by making compensatory changes in the UUAC motif within epsilon. This finding led us to examine limitations in the shifting of the site of transfer. When the UUAC motif in epsilon was changed to six different tetranucleotide motifs surrounding position 2537, transfer of minus-strand DNA shifted predictably, albeit inefficiently. Also, when multiple UUAC motifs were introduced near DR1, the UUAC motif at nucleotide 2537 was used preferentially. Overall, our findings confirm the current minus-strand DNA transfer model and demonstrate a marked preference for the site of the transfer.