title: The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus.
abstract: For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.
authors.affiliation: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
authors.forename: Kathleen M
authors.initials: KM
authors.lastname: Haines
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Loeb
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012313:RNA / Q000235:genetics*; D016914:Ribonuclease H / Q000378:metabolism; D014779:Virus Replication
chemical_list: D004279:DNA, Viral; C034477:RNA primers; D012313:RNA; D016914:Ribonuclease H