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 ins ide the viral nucleocapsid by reverse transcription of an RNA intermed iate. Encapsidation of this RNA pregenome is mediated by the interacti on of the viral replication enzyme P with the structured 5'-proximal R NA element epsilon; replication was thought to start in the 3'-proxima l direct repeat DR1. However, recent data obtained with the duck hepa titis B virus indicated a novel, discontinuous mechanism of negative-s trand DNA synthesis, Here we demonstrate, using DNA transfection of co mplete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV ep silon whose primary sequence is not important for encapsidation serves as template for a short DNA primer that is subsequently transferred t o DR1. Apparently, P protein copies any template sequence that does n ot interfere with epsilon structure; however, altered primary sequence s can induce polymerase stuttering, resulting in extended primers cont aining more than one equivalent of the template sequence. The importan ce of the bulged structure is emphasized by the dependence of primer l ength on bulge size. Transfer specificity is in part controlled by seq uence complementarity. The strategy of using the 5' encapsidation sign al 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 mode rn retroviruses and primitive retroelements like the Mauriceville retr oplasmid.