title: The topology of hepatitis B virus pregenomic RNA promotes its replication.
abstract: Previous analysis of hepatitis B virus (HBV) indicated base pairing between two cis-acting sequences, the 5' half of the upper stem of epsilon and phi, contributes to the synthesis of minus-strand DNA. Our goal was to identify other cis-acting sequences on the pregenomic RNA (pgRNA) involved in the synthesis of minus-strand DNA. We found that large portions of the pgRNA could be deleted or substituted without an appreciable decrease in the level of minus-strand DNA synthesized, indicating that most of the pgRNA is dispensable and that a specific size of the pgRNA is not required for this process. Our results indicated that the cis-acting sequences for the synthesis of minus-strand DNA are present near the 5' and 3' ends of the pgRNA. In addition, we found that the first-strand template switch could be directed to a new location when a 72-nucleotide (nt) fragment, which contained the cis-acting sequences present near the 3' end of the pgRNA, was introduced at that location. Within this 72-nt region, we uncovered two new cis-acting sequences, which flank the acceptor site. We show that one of these sequences, named omega and located 3' of the acceptor site, base pairs with phi to contribute to the synthesis of minus-strand DNA. Thus, base pairing between three cis-acting elements (5' half of the upper stem of epsilon, phi, and omega) are necessary for the synthesis of HBV minus-strand DNA. We propose that this topology of pgRNA facilitates first-strand template switch and/or the initiation of synthesis of minus-strand DNA.
authors: McArdle Laboratory for Cancer Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.
authors: Teresa M
authors: TM
authors: Abraham
authors: Daniel D
authors: DD
authors: Loeb
mesh_terms: D001483:Base Sequence; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics; D017353:Gene Deletion; D016679:Genome, Viral; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000235:genetics*; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D012367:RNA, Viral
references: Virology. 2002 Apr 10;295(2):348-59
references: 12033794
references: Virology. 2007 Mar 15;359(2):371-81
references: 17056086
references: J Virol. 2004 Jan;78(2):642-9
references: 14694095
references: Cell. 1980 Oct;21(3):801-9
references: 7438207
references: Cell. 1982 Jun;29(2):403-15
references: 6180831
references: Cell. 1986 Sep 12;46(6):865-72
references: 3019554
references: Nature. 1990 Apr 5;344(6266):552-5
references: 1690862
references: EMBO J. 1992 Sep;11(9):3413-20
references: 1380455
references: Cell. 1992 Nov 13;71(4):663-70
references: 1384989
references: J Virol. 1993 Jun;67(6):3254-63
references: 7684464
references: J Virol. 1993 Nov;67(11):6507-12
references: 7692081
references: J Virol. 1994 Jan;68(1):6-13
references: 7504742
references: J Virol. 1994 Jun;68(6):3536-43
references: 8189492
references: J Virol. 1994 Sep;68(9):5579-87
references: 7520092
references: J Virol. 1995 May;69(5):3067-73
references: 7707534
references: J Virol. 1996 Jan;70(1):585-9
references: 8523575
references: J Virol. 1996 May;70(5):2764-73
references: 8627750
references: Virology. 1997 Nov 24;238(2):363-71
references: 9400609
references: Mol Cell Biol. 1998 Nov;18(11):6265-72
references: 9774643
references: J Virol. 2006 May;80(9):4380-7
references: 16611897
references: Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1984-9
references: 12578983