Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.
The nucleocapsid protein (NC) of human immunodeficiency virus type 1 (HIV-1) is formed of two highly conserved CCHC zinc fingers flanked by small basic domains. NC is required for the two obligatory strand transfers in viral DNA synthesis through its nucleic acid chaperoning properties. The first DNA strand transfer relies on NC's ability to bind and destabilize the secondary structure of complementary transactivation response region (cTAR) DNA, to inhibit self-priming, and to promote the annealing of cTAR to TAR RNA. To further investigate NC chaperone properties, our aim was to identify by fluorescence spectroscopy and gel electrophoresis, the NC structural determinants for cTAR binding and destabilization, and for the inhibition of self-primed DNA synthesis on a model system using a series of NC mutants and HIV-1 reverse transcriptase. NC destabilization and self-priming inhibition properties were found to be supported by the two fingers in their proper context and the basic (29)RAPRKKG(35) linker. The strict requirement of the native proximal finger suggests that its hydrophobic platform (Val13, Phe16, Thr24 and Ala25) is crucial for binding, destabilization and inhibition of self-priming. In contrast, only partial folding of the distal finger is required, probably for presenting the Trp37 residue in an appropriate orientation. Also, Trp37 and the hydrophobic residues of the proximal finger appear to be essential for the propagation of the melting from the cTAR ends up to the middle of the stem. Finally, both N-terminal and C-terminal basic domains contribute to cTAR binding but not to its destabilization.
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.
Clauss
Céline
C
Piémont
Etienne
E
Ficheux
Damien
D
Gorelick
Robert J
RJ
Roques
Bernard
B
Gabus
Caroline
C
Darlix
Jean-Luc
JL
de Rocquigny
Hugues
H
Mély
Yves
Y
D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics / Q000502:physiology; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016325:HIV Long Terminal Repeat / Q000235:genetics / Q000502:physiology*; D015497:HIV-1 / Q000235:genetics* / Q000502:physiology; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D017510:Protein Folding; D048348:Reverse Transcription / Q000235:genetics / Q000502:physiology*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers / Q000235:genetics / Q000502:physiology; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D004279:DNA, Viral; D004268:DNA-Binding Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus