HEPADNAVIRUS REVERSE TRANSCRIPTION INITIATES WITHIN THE STEM-LOOP OF THE RNA PACKAGING SIGNAL AND EMPLOYS A NOVEL STRAND TRANSFER

Replication of the hepadnavirus genome occurs by reverse transcription of an RNA pregenome and is mediated by the viral polymerase; the poly merase is also required for packaging of the pregenome through interac tion with the RNA packaging signal, epsilon. Previous work suggested t hat reverse transcription of minus-strand DNA initiates within the seq uence element DR1 (direct repent 1) and that disruption of DR1 activat es a cryptic initiation site in a downstream copy of epsilon. However, using active duck hepatitis B virus polymerase expressed in a yeast T y vector system, we demonstrate that synthesis of minus-strand DNAs wi th 5' ends at DR1 requires the stem-loop of epsilon, whereas the produ ction of DNAs mapping to epsilon does not require DR1. Mutations at ep silon that remove homology between epsilon and DR1 eliminate reverse t ranscripts with 5' ends in DR1, and restoring homolog at DR1 to a muta nt epsilon partially restores DNAs mapping to DR1. Insertions of one n ucleotide into the bulge region of the epsilon stem-loop increase the length of minus-strand DNA whose 5' ends map to DR1 by one nucleotide. Thus, very short minus-strand primers are initiated within epsilon, r ather than in DR1 as previously supposed; they are then transferred to a four-nucleotide homology in DR1. Transfer was also observed in vivo during replication of duck hepatitis B virus in avian cells; in this case, transfer is from the 5' copy of epsilon to the 3' copy of DR1. T his minus-strand transfer reaction is likely to be a general feature o f all hepadnaviruses.