Morellet N, de Rocquigny H, Mély Y, Jullian N, Déméné H, Ottmann M, Gérard D, Darlix J L, Fournie-Zaluski M C, Roques B P
Département de Pharmacochimie Moléculaire et Structurale INSERM U266, CNRS URA D 1500, Paris, France.
J Mol Biol. 1994 Jan 7;235(1):287-301. doi: 10.1016/s0022-2836(05)80033-6.
The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Lys33 and Trp37, Cys18 and Lys33 disappeared in the D-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by D-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis substituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles.
人类免疫缺陷病毒1型（HIV-1）的核衣壳蛋白NCp7是一种由72个氨基酸组成的肽，含有两个CX2CX4HX4C型锌指结构，通过短碱性序列29RAPRKKG35相连。研究表明，NCp7在体外可激活病毒RNA二聚化以及复制引物tRNA（Lys，3）与逆转录起始位点的退火。为了阐明锌指在NCp7各种功能中可能的结构作用，通过二维核磁共振实验完成了整个蛋白质的完整序列特异性1H NMR归属。此外，为了表征肽接头在NCp7折叠中的作用，采用核磁共振和荧光技术研究了Pro31构型反转的合成类似物。在D-Pro31（12-53）NCp7中，一些涉及折叠锌指和间隔区氨基酸质子的长程NOE消失，如Ala25和Trp37、Phe16和Trp37、Arg32和Trp37、Lys33和Trp37、Cys18和Lys33，证实了在（13-51）NCp7中观察到的两个CCHC框在空间上的接近。碘化物荧光猝灭实验也证实了这一点。除了两个短序列Tyr56至Gly58和Tyr64至Gly66外，NCp7的N端和C端部分表现出较大的灵活性，这两个短序列似乎在无规卷曲和螺旋构象之间振荡。在体外和体内研究了NCp7结构特征的生物学相关性。在活性（13-64）NCp7肽中用D-Pro31取代Pro31导致体外二聚化严重降低。此外，用Leu取代Pro31的定点诱变导致形成无感染性和不成熟的病毒颗粒。这些结果表明，肽接头诱导的锌指在空间上的接近在HIV-1感染性颗粒的基因组RNA包装和形态发生中起关键作用。
