Evidence of interactions between the nucleocapsid protein NCp7 and the reverse transcriptase of HIV-1.
The human immunodeficiency virus (HIV-1) nucleocapsid protein NCp7 containing two CX2CX4HX4C-type zinc fingers was proposed to be involved in reverse transcriptase (RT)-catalyzed proviral DNA synthesis through promotion of tRNA3Lys annealing to the RNA primer binding site, improvement of DNA strand transfers, and enhancement of RT processivity. The NCp7 structural characteristics are crucial because mutations altering the finger domain conformation led to noninfectious viruses characterized by defects in provirus integration. These findings prompted us to study a putative RT/NCp7 protein-protein interaction. Binding assays using far Western analysis or RT immobilized on beads clearly showed the formation of a complex between NCp7 and RT. The affinity of NCp7 for p66/p51RT was 0.60 microM with a 1:1 stoechiometry. This interaction was confirmed by chemical cross-linking and co-immunoprecipitation of the two proteins in a viral environment. Competition experiments using different NCp7 mutants showed that alteration of the finger structure disrupted RT recognition, giving insights into the loss of infectivity of corresponding HIV-1 mutants. Together with structural data on RT, these results suggest that the role of NCp7 could be to enhance RT processivity through stabilization of a p51-induced active form of the p66 subunit and open the way for designing new antiviral agents.
Druillennec
S
S
Département de Pharmacochimie Moléculaire et Structurale U 266 INSERM-UMR 8600 CNRS, UFR des Sciences Pharmaceutiques et Biologiques 4, 75270 Paris Cedex 06, France.
Caneparo
A
A
de Rocquigny
H
H
Roques
B P
BP
D000595:Amino Acid Sequence; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D054303:HIV Reverse Transcriptase / Q000378:metabolism*; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013388:Succinimides; D014764:Viral Proteins; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D013388:Succinimides; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; C035759:3,3'-dithiobis(sulfosuccinimidyl propionate); D054303:HIV Reverse Transcriptase