Déméné H, Dong C Z, Ottmann M, Rouyez M C, Jullian N, Morellet N, Mely Y, Darlix J L, Fournié-Zaluski M C, Saragosti S
Département de Pharmacochimie Moléculaire et Structurale, U266 INSERM-URA D1500 CNRS, Faculté de Pharmacie, Université René Descartes, Paris, France.
Biochemistry. 1994 Oct 4;33(39):11707-16. doi: 10.1021/bi00205a006.
The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV-1), which has key functions in the virus life cycle, possesses two zinc fingers of the CX2CX4HX4C type characterized by three successive loops containing a tetrahedrally coordinated zinc atom. The replacement of any cysteine by a serine in either finger has been shown to result in the production of noninfectious viruses, probably by impairing the biological functions of NCp7. In order to more precisely elucidate the structural role of the zinc finger motif, His23 was replaced by Cys in the proximal finger of the peptide (13-64)NCp7 which retains NCp7 activities in vitro. The peptide Cys23(13-64)NCp7 was synthesized by solid phase and studied by 2D 1H NMR and molecular modeling. The His to Cys modification causes important structural modifications of the N-terminal zinc finger which impair the spatial proximity of the two zinc fingers as shown by the disappearance of several interresidue NOEs. The side chains of Val13, Lys14, Phe16, Thr24, Ala25, Trp37, Gln45, and Met46, which are thought to be involved in nucleic acid recognition, are no longer found clustered in the Cys23(13-64)NCp7 mutant as they are in the wild-type NCp7 structure. In vitro, Cys23(13-64)NCp7 is unable to tightly interact with the viral RNA or replication primer tRNA(Lys,3). The Cys23(NCp7) mutation was introduced into an infectious HIV-1 molecular clone, and virions produced upon DNA transfection into cells were analyzed for their viral protein and RNA compositions as well as for their infectivity. Results show that, while the Cys23(NCp7) mutation does not impair virion production, viruses contain a low amount of degraded viral RNA and are not infectious. These findings suggest that a bona fide conformation of the HIV-1 NCp7 is critical for the packaging of viral RNA, its stability in virions, and virus infectivity.
人类免疫缺陷病毒1型（HIV-1）的核衣壳蛋白NCp7在病毒生命周期中具有关键功能，它拥有两个CX2CX4HX4C型锌指结构，其特征是三个连续的环中含有一个四面体配位的锌原子。研究表明，任一锌指中的任何一个半胱氨酸被丝氨酸取代都会导致产生无感染性的病毒，这可能是因为损害了NCp7的生物学功能。为了更精确地阐明锌指基序的结构作用，在肽段（13-64）NCp7的近端锌指中将His23替换为Cys，该肽段在体外保留了NCp7的活性。通过固相合成法合成了肽段Cys23(13-64)NCp7，并通过二维1H NMR和分子模拟进行研究。His到Cys的修饰导致N端锌指发生重要的结构修饰，如几个残基间NOE的消失所示，这损害了两个锌指的空间接近性。在野生型NCp7结构中，被认为参与核酸识别的Val13、Lys14、Phe16、Thr24、Ala25、Trp37、Gln45和Met46的侧链，在Cys23(13-64)NCp7突变体中不再聚集在一起。在体外，Cys23(13-64)NCp7无法与病毒RNA或复制引物tRNA(Lys,3)紧密相互作用。将Cys23(NCp7)突变引入具有感染性的HIV-1分子克隆中，并对DNA转染细胞后产生的病毒粒子进行病毒蛋白和RNA组成以及感染性分析。结果表明，虽然Cys23(NCp7)突变不影响病毒粒子的产生，但病毒含有少量降解的病毒RNA且无感染性。这些发现表明，HIV-1 NCp7的真实构象对于病毒RNA的包装、其在病毒粒子中的稳定性以及病毒感染性至关重要。
