A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis.
Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative-strand DNA synthesis, common to mammalian and avian hepadnaviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid.
Nassal
M
M
Zentrum für Molekulare Biologie, Universität Heidelberg, Germany.
Rieger
A
A
D001483:Base Sequence; D005075:Biological Evolution; D002460:Cell Line; D017931:DNA Primers; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis*; D015611:Hepatitis B Virus, Duck / Q000235:genetics / Q000378:metabolism; D006515:Hepatitis B virus / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008957:Models, Genetic; D008961:Models, Structural; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D018741:Replication Origin; D019098:Telomerase / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase; D019098:Telomerase