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 polymerase is also required for packaging of the pregenome through interaction with the RNA packaging signal, epsilon. Previous work suggested that reverse transcription of minus-strand DNA initiates within the sequence element DR1 (direct repeat 1) and that disruption of DR1 activates a cryptic initiation site in a downstream copy of epsilon. However, using active duck hepatitis B virus polymerase expressed in a yeast Ty vector system, we demonstrate that synthesis of minus-strand DNAs with 5' ends at DR1 requires the stem-loop of epsilon, whereas the production of DNAs mapping to epsilon does not require DR1. Mutations at epsilon that remove homology between epsilon and DR1 eliminate reverse transcripts with 5' ends in DR1, and restoring homology at DR1 to a mutant epsilon partially restores DNAs mapping to DR1. Insertions of one nucleotide 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, rather 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. This minus-strand transfer reaction is likely to be a general feature of all hepadnaviruses.
Tavis
J E
JE
Department of Microbiology and Immunology, University of California San Francisco 94143-0502.
Perri
S
S
Ganem
D
D
D000818:Animals; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004261:DNA Replication; D004279:DNA, Viral / Q000235:genetics; D015870:Gene Expression; D005814:Genes, Viral; D015611:Hepatitis B Virus, Duck / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014158:Transcription, Genetic; D014779:Virus Replication
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral