title: Role of the primer activation signal in tRNA annealing onto the HIV-1 genome studied by single-molecule FRET microscopy.
abstract: HIV-1 reverse transcription is primed by a cellular tRNAlys3 molecule that binds to the primer binding site (PBS) in the genomic RNA. An additional interaction between the tRNA molecule and the primer activation signal (PAS) is thought to regulate the initiation of reverse transcription. The mechanism of tRNA annealing onto the HIV-1 genome was examined using ensemble and single-molecule Förster Resonance Energy Transfer (FRET) assays, in which fluorescent donor and acceptor molecules were covalently attached to an RNA template mimicking the PBS region. The role of the viral nucleocapsid (NC) protein in tRNA annealing was studied. Both heat annealing and NC-mediated annealing of tRNAlys3 were found to change the FRET efficiency, and thus the conformation of the HIV-1 RNA template. The results are consistent with a model for tRNA annealing that involves an interaction between the tRNAlys3 molecule and the PAS sequence in the HIV-1 genome. The NC protein may stimulate the interaction of the tRNA molecule with the PAS, thereby regulating the initiation of reverse transcription.
authors: 1Department of Molecular Biology, Aarhus University, Aarhus, Denmark.
authors: Nancy
authors: N
authors: Beerens
authors: Mette D E
authors: MD
authors: Jepsen
authors: Volodymyr
authors: V
authors: Nechyporuk-Zloy
authors: Asger C
authors: AC
authors: Krüger
authors: Jean-Luc
authors: JL
authors: Darlix
authors: Jørgen
authors: J
authors: Kjems
authors: Victoria
authors: V
authors: Birkedal
mesh_terms: D031541:Fluorescence Resonance Energy Transfer; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D006801:Humans; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D048348:Reverse Transcription
chemical_list: D012357:RNA, Transfer, Lys; D012367:RNA, Viral
references: Proc Natl Acad Sci U S A. 2007 Mar 27;104(13):5261-7
references: 17372205
references: J Biol Chem. 2001 Jul 20;276(29):27721-30
references: 11353768
references: RNA. 2002 Jan;8(1):8-15
references: 11873759
references: Nucleic Acids Res. 2001 Jul 1;29(13):2757-65
references: 11433020
references: J Virol. 1997 Oct;71(10):7648-56
references: 9311847
references: J Virol. 1994 Feb;68(2):611-8
references: 7507181
references: Proc Natl Acad Sci U S A. 2002 Jun 25;99(13):8614-9
references: 12084921
references: J Biol Chem. 2001 Aug 17;276(33):31247-56
references: 11384976
references: Viruses. 2010 Jan;2(1):213-43
references: 21994608
references: Virus Res. 2012 Nov;169(2):324-39
references: 22721779
references: J Virol. 1989 Jan;63(1):319-27
references: 2908924
references: Biophys J. 2010 May 19;98(10):2265-72
references: 20483335
references: Eur Biophys J. 2010 Aug;39(9):1343-50
references: 20213319
references: J Mol Biol. 2007 Dec 7;374(4):1041-53
references: 18028945
references: Nucleic Acids Res. 1993 Mar 11;21(5):1171-8
references: 8464701
references: Retrovirology. 2009 Mar 04;6:24
references: 19261185
references: J Virol. 1999 Aug;73(8):6307-18
references: 10400722
references: J Virol. 1992 Apr;66(4):2464-72
references: 1548772
references: Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):459-64
references: 9892655
references: J Virol. 1997 Aug;71(8):5750-7
references: 9223461
references: J Biol Chem. 2006 Apr 28;281(17):11736-43
references: 16500895
references: J Virol. 1998 Nov;72(11):9353-8
references: 9765488
references: Nucleic Acids Res. 2006 Jan 30;34(2):593-605
references: 16449201
references: Biochemistry. 2010 Nov 2;49(43):9171-80
references: 20818825
references: RNA Biol. 2010 Nov-Dec;7(6):754-74
references: 21160280
references: J Virol. 2000 Oct;74(19):8980-8
references: 10982342
references: J Biol Chem. 1995 Oct 6;270(40):23867-74
references: 7559565
references: J Mol Biol. 1996 Nov 29;264(2):243-54
references: 8951374
references: J Virol. 1988 Oct;62(10):3622-30
references: 2458484
references: J Mol Biol. 1997 May 2;268(2):250-60
references: 9159468
references: J Mol Biol. 2011 Jul 22;410(4):565-81
references: 21762801
references: Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8936-41
references: 15175430
references: J Mol Biol. 2004 Apr 2;337(4):951-68
references: 15033363
references: Nat Struct Mol Biol. 2010 Dec;17(12):1453-60
references: 21102446
references: Nat Methods. 2008 Jun;5(6):507-16
references: 18511918
references: J Mol Biol. 1995 Mar 24;247(2):236-50
references: 7707372
references: Biochemistry. 1990 Aug 28;29(34):7786-9
references: 2261434
references: Nano Lett. 2011 Dec 14;11(12):5482-8
references: 22023515
references: J Virol. 2002 Mar;76(5):2329-39
references: 11836411
references: RNA. 2002 Mar;8(3):357-69
references: 12003495
references: Trends Biochem Sci. 1998 Aug;23(8):297-301
references: 9757830