Synthesis and biological activities of fluorescent acridine-containing HIV-1 nucleocapsid proteins for investigation of nucleic acid-NCp7 interactions.
Specific interactions between the 72-amino acid nucleocapsid protein NCp7 of the human immunodeficiency virus, type 1 and the genomic RNA are essential for virus replication. Studies on the mechanism of action of NCp7 require a direct visualization of its complexes with nucleic acids and the determination of binding affinities. To facilitate these investigations, fluorescent NCp7 derivatives were developed by introduction in the NCp7 sequence of a non-natural amino acid, (S)-beta-(9-acridinyl)alanine (Aca) obtained by a chiral synthetic method. Three fluorescent NCp7 derivatives were obtained by introducing this amino acid at different positions. As shown by NMR, the three-dimensional structure of NCp7 is not altered by introduction of Aca. The fluorescent peptides were found to be as potent as their precursors in interacting with nucleic acids and in promoting HIV-1 genomic RNA dimerization. Moreover, because of their fluorescent properties, these NCp7s can be used at submicromolar concentrations to directly visualize and quantify protein-nucleic acid interactions in solution or after gel electrophoresis. This could facilitate the development of new antiviral agents aimed at inhibiting the functions of NCp7 and studies on the intracellular traffic of NCp7 within the preintegration complex.
Dong
C Z
CZ
Département de Pharmacochimie Moléculaire et Structurale, INSERM U266-CNRS URA D 1500, UFR des Sciences Pharmaceutiques et Biologiques, Paris, France.
De Rocquigny
H
H
Rémy
E
E
Mellac
S
S
Fournié-Zaluski
M C
MC
Roques
B P
BP
D000166:Acridines / Q000737:chemistry*; D000409:Alanine / Q000031:analogs & derivatives* / Q000737:chemistry; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D002213:Capsid / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D036022:Capsid Proteins; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D015683:Gene Products, gag / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D015497:HIV-1 / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism; D011485:Protein Binding; D012367:RNA, Viral / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus
D000166:Acridines; D036022:Capsid Proteins; D005456:Fluorescent Dyes; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D009838:Oligodeoxyribonucleotides; D010455:Peptides; D012367:RNA, Viral; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C109302:(9-acridinyl)alanine; D000409:Alanine