title: Annealing to sequences within the primer binding site loop promotes an HIV-1 RNA conformation favoring RNA dimerization and packaging.
abstract: The 5' untranslated region (5' UTR) of HIV-1 genomic RNA (gRNA) includes structural elements that regulate reverse transcription, transcription, translation, tRNA(Lys3) annealing to the gRNA, and gRNA dimerization and packaging into viruses. It has been reported that gRNA dimerization and packaging are regulated by changes in the conformation of the 5'-UTR RNA. In this study, we show that annealing of tRNA(Lys3) or a DNA oligomer complementary to sequences within the primer binding site (PBS) loop of the 5' UTR enhances its dimerization in vitro. Structural analysis of the 5'-UTR RNA using selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) shows that the annealing promotes a conformational change of the 5' UTR that has been previously reported to favor gRNA dimerization and packaging into virus. The model predicted by SHAPE analysis is supported by antisense experiments designed to test which annealed sequences will promote or inhibit gRNA dimerization. Based on reports showing that the gRNA dimerization favors its incorporation into viruses, we tested the ability of a mutant gRNA unable to anneal to tRNA(Lys3) to be incorporated into virions. We found a ∼60% decrease in mutant gRNA packaging compared with wild-type gRNA. Together, these data further support a model for viral assembly in which the initial annealing of tRNA(Lys3) to gRNA is cytoplasmic, which in turn aids in the promotion of gRNA dimerization and its incorporation into virions.
authors: Elias
authors: E
authors: Seif
authors: Meijuan
authors: M
authors: Niu
authors: Lawrence
authors: L
authors: Kleiman
mesh_terms: D020121:5' Untranslated Regions / Q000235:genetics; D020029:Base Pairing; D001483:Base Sequence; D001665:Binding Sites; D019281:Dimerization; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012357:RNA, Transfer, Lys / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000502:physiology*; D019065:Virus Assembly
chemical_list: D020121:5' Untranslated Regions; D012357:RNA, Transfer, Lys; D012367:RNA, Viral
keywords: 5′ untranslated region; RNA conformation; RNA encapsidation; tRNALys3; viral assembly
references: PLoS Pathog. 2009 Oct;5(10):e1000627
references: 19834549
references: Nucleic Acids Res. 2004 Feb 03;32(2):819-27
references: 14762209
references: Curr HIV Res. 2004 Apr;2(2):163-75
references: 15078180
references: J Virol. 2000 Nov;74(22):10796-800
references: 11044125
references: J Biol Chem. 2001 Aug 17;276(33):31247-56
references: 11384976
references: J Virol. 2008 Mar;82(6):3090-8
references: 18160437
references: J Biol Chem. 2003 Mar 28;278(13):11601-11
references: 12458192
references: PLoS Biol. 2008 Apr 29;6(4):e96
references: 18447581
references: Nucleic Acids Res. 2008 Aug;36(13):4352-63
references: 18586822
references: J Virol. 2009 Aug;83(16):8099-107
references: 19494012
references: Nat Protoc. 2009;4(10):1413-21
references: 19745823
references: Virus Res. 2012 Oct;169(1):63-71
references: 22820401
references: J Virol. 2007 Aug;81(15):7985-92
references: 17507464
references: J Virol. 2000 Dec;74(23):11413-7
references: 11070043
references: J Virol. 1990 May;64(5):1920-6
references: 2109098
references: Nat Protoc. 2006;1(3):1610-6
references: 17406453
references: Biochemistry. 1994 Nov 15;33(45):13464-74
references: 7947755
references: Nucleic Acids Res. 2005 Jul 1;33(Web Server issue):W577-81
references: 15980540
references: Science. 2011 Oct 14;334(6053):242-5
references: 21998393
references: Biochemistry. 2005 Jun 28;44(25):9058-66
references: 15966729
references: Nucleic Acids Res. 1999 Jan 15;27(2):446-54
references: 9862964
references: RNA. 2001 Jan;7(1):143-57
references: 11214176
references: RNA. 2006 Apr;12(4):542-6
references: 16489186
references: J Virol. 2011 Feb;85(4):1847-60
references: 21106734
references: J Biol Chem. 2002 May 31;277(22):19967-75
references: 11896057
references: J Biol Chem. 1996 Apr 12;271(15):9054-61
references: 8621554
references: Nucleic Acids Res. 2003 Jul 1;31(13):3406-15
references: 12824337
references: Cell. 1987 Feb 27;48(4):691-701
references: 3643816
references: J Virol. 2003 Apr;77(7):4060-9
references: 12634365
references: Nucleic Acids Res. 2011 Dec;39(22):e151
references: 21965531
references: J Virol. 1990 Oct;64(10):5230-4
references: 2204725
references: Cell. 1985 Jul;41(3):813-23
references: 2988790
references: J Mol Biol. 2012 Mar 30;417(3):224-39
references: 22306406
references: Nucleic Acids Res. 1991 May 11;19(9):2349-57
references: 1645868
references: J Virol. 2007 Apr;81(8):4002-11
references: 17267488
references: J Virol. 1994 Apr;68(4):2065-72
references: 7511167
references: J Biol Chem. 1993 Dec 5;268(34):25269-72
references: 7503978
references: Virus Res. 2012 Nov;169(2):340-8
references: 22698876
references: J Virol. 2004 Oct;78(19):10814-9
references: 15367648
references: Nucleic Acids Res. 2012 Jun;40(11):5012-22
references: 22328732
references: J Virol. 1989 Sep;63(9):4085-7
references: 2760989
references: Nat Methods. 2012 Jul;9(7):671-5
references: 22930834
references: Proc Natl Acad Sci U S A. 1994 May 24;91(11):4945-9
references: 8197162
references: J Virol. 1997 Dec;71(12):9075-86
references: 9371564