Melting of Model HIV-1 Stem-Loop 1 RNA Dimers Monitored by 2-Aminopurine Fluorescence

Abstract Viral maturation of HIV-1 involves refolding of its genomic RNA, which is believed to include a rearrangement of the SL1 stem-loop from a metastable conformation called kissing loop dimer (KD) to a stable one termed extended dimer (ED). To investigate this rearrangement in vitro we have studied the thermal melting of the RNA dimers formed by slightly modified 23-nucleotide SL1 RNA of HIV-1 Mal. Local structural changes in the RNA dimers during the melting were monitored by changes in the fluorescence of 2-aminopurine (2AP) incorporated in predetermined positions of RNA. We have shown that the stem regions of both preformed KD and ED melt in the temperature interval from 75°C to 90°C. Kissing loop interface of the KD RNA is found to be disrupted at lower temperatures from 20°C to 55°C, at which the stem regions remain intact. Conversion of the preformed KD to ED overcoming the kinetic barrier occurs between 55°C and 65°C. The melting of “loop-loop” regions in both preformed and newly formed EDs takes place around 70°C. Our finding that thermoinduced KD-to-ED conversion is preceded by transient dissociation of loop-loop interface disagrees with a common idea of strand exchange without disruption of loop-loop-contact.

[1]  Ivan Anishchenko,et al.  Computational Model of the HIV-1 Subtype A V3 Loop: Study on the Conformational Mobility for Structure-Based Anti-AIDS Drug Design , 2009, Journal of biomolecular structure & dynamics.

[2]  Katharine T. Briggs,et al.  Dissecting structural transitions in the HIV-1 dimerization initiation site RNA using 2-aminopurine fluorescence. , 2009, Methods.

[3]  A. Andrianov,et al.  Immunophilins and HIV-1 V3 Loop For Structure-Based Anti-AIDS Drug Design , 2009, Journal of biomolecular structure & dynamics.

[4]  A. Beniaminov,et al.  2-Aminopurine Fluorescence: Discrimination Between Specific and Unspecific Ligand Binding to the Kissing-Loop Dimer of the HIV-1 RNA , 2008, Journal of biomolecular structure & dynamics.

[5]  G. M. Wilson,et al.  Site-specific variations in RNA folding thermodynamics visualized by 2-aminopurine fluorescence. , 2007, Biochemistry.

[6]  Long Yang,et al.  HIV-1 viral RNA is selected in the form of monomers that dimerize in a three-step protease-dependent process; the DIS of stem-loop 1 initiates viral RNA dimerization. , 2007, Journal of molecular biology.

[7]  Y. Tor,et al.  Fluorescent HIV-1 Dimerization Initiation Site: design, properties, and use for ligand discovery. , 2007, Journal of the American Chemical Society.

[8]  T. James,et al.  Nucleocapsid protein-mediated maturation of dimer initiation complex of full-length SL1 stemloop of HIV-1: sequence effects and mechanism of RNA refolding , 2007, Nucleic acids research.

[9]  Philippe Dumas,et al.  Polymorphism of bulged-out residues in HIV-1 RNA DIS kissing complex and structure comparison with solution studies. , 2006, Journal of molecular biology.

[10]  N. Piganeau,et al.  Stabilities of HIV-1 DIS type RNA loop–loop interactions in vitro and in vivo , 2006, Nucleic acids research.

[11]  Fabien Kieken,et al.  A new NMR solution structure of the SL1 HIV-1Lai loop–loop dimer , 2006, Nucleic acids research.

[12]  J. Langowski,et al.  Mechanism of Hairpin-Duplex Conversion for the HIV-1 Dimerization Initiation Site* , 2005, Journal of Biological Chemistry.

[13]  Gota Kawai,et al.  Solution RNA structures of the HIV-1 dimerization initiation site in the kissing-loop and extended-duplex dimers. , 2005, Journal of biochemistry.

[14]  Roland Marquet,et al.  Dimerization of retroviral RNA genomes: an inseparable pair , 2004, Nature Reviews Microbiology.

[15]  V. Zhurkin,et al.  Formation of an intramolecular triple-stranded DNA structure monitored by fluorescence of 2-aminopurine or 6-methylisoxanthopterin. , 2004, Nucleic acids research.

[16]  N. Windbichler,et al.  Kissing complex-mediated dimerisation of HIV-1 RNA: coupling extended duplex formation to ribozyme cleavage. , 2003, Nucleic acids research.

[17]  J. Marino,et al.  Mechanism of nucleocapsid protein catalyzed structural isomerization of the dimerization initiation site of HIV-1. , 2002, Biochemistry.

[18]  M. Wainberg,et al.  Role of Stem B, Loop B, and Nucleotides next to the Primer Binding Site and the Kissing-Loop Domain in Human Immunodeficiency Virus Type 1 Replication and Genomic-RNA Dimerization , 2001, Journal of Virology.

[19]  T. Huynh-Dinh,et al.  The HIV‐1Lai RNA dimerization , 2000 .

[20]  Thomas L. James,et al.  Structure of the dimer a initiation complex of HIV-1 genomic RNA , 1998, Nature Structural Biology.

[21]  E. Westhof,et al.  Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA. , 1997, Journal of molecular biology.

[22]  T. Parslow,et al.  Mutant human immunodeficiency virus type 1 genomes with defects in RNA dimerization or encapsidation , 1997, Journal of virology.

[23]  D. Muriaux,et al.  A kissing complex together with a stable dimer is involved in the HIV-1Lai RNA dimerization process in vitro. , 1996, Biochemistry.

[24]  M. Laughrea,et al.  Kissing-loop model of HIV-1 genome dimerization: HIV-1 RNAs can assume alternative dimeric forms, and all sequences upstream or downstream of hairpin 248-271 are dispensable for dimer formation. , 1996, Biochemistry.

[25]  M Laughrea,et al.  A 19-nucleotide sequence upstream of the 5' major splice donor is part of the dimerization domain of human immunodeficiency virus 1 genomic RNA. , 1994, Biochemistry.

[26]  Christoph Flamm,et al.  Determination of thermodynamic parameters for HIV DIS type loop-loop kissing complexes. , 2004, Nucleic acids research.

[27]  G. Kawai,et al.  Structural requirement for the two-step dimerization of human immunodeficiency virus type 1 genome. , 2000, RNA.