Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7.
Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strong-stop DNA ((-)ssDNA) is transferred to the 3'end of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the 10C.A(44) mismatch or T(40) bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity.
Beltz
Hervé
H
Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR 7034 CNRS, Faculté de Pharmacie, Université Louis Pasteur, Strasbourg 1, 74, Route du Rhin, 67401 Illkirch Cedex, France.
Azoulay
Joel
J
Bernacchi
Serena
S
Clamme
Jean-Pierre
JP
Ficheux
Damien
D
Roques
Bernard
B
Darlix
Jean-Luc
JL
Mély
Yves
Y
D000595:Amino Acid Sequence; D020029:Base Pairing; D001483:Base Sequence; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D004279:DNA, Viral / Q000737:chemistry* / Q000378:metabolism; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D015683:Gene Products, gag / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry; D020218:Response Elements / Q000235:genetics*; D012235:Rhodamines / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013816:Thermodynamics; D015533:Transcriptional Activation; D014764:Viral Proteins; D016662:Virus Integration; D054301:gag Gene Products, Human Immunodeficiency Virus
C106141:6-carboxytetramethylrhodamine; D036022:Capsid Proteins; D004279:DNA, Viral; D005452:Fluoresceins; D005456:Fluorescent Dyes; D015683:Gene Products, gag; D018832:Molecular Chaperones; C071884:NCP7 protein, Human immunodeficiency virus 1; D010446:Peptide Fragments; D012235:Rhodamines; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C024098:6-carboxyfluorescein