Muriaux D, De Rocquigny H, Roques B P, Paoletti J
Unité de Biochimie, URA 147 CNRS, 39 rue Camille Desmoulins, Institut Gustave Roussy, 94805 Villejuif, France.
J Biol Chem. 1996 Dec 27;271(52):33686-92. doi: 10.1074/jbc.271.52.33686.
Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.
核衣壳蛋白7（NCp7），即人类免疫缺陷病毒1型（HIV-1）的核衣壳蛋白，已被证明能强烈增强逆转录病毒基因组RNA的二聚化。这一过程涉及两个逆转录病毒RNA单体亚基在其5'端附近的相互作用。最近，一个位于剪接供体位点上游的区域被确定为负责形成二聚体HIV-1 RNA的区域。该区域似乎局限于一个茎环结构内，环中有一个自身互补序列。在一项关于自发二聚体形成的体外研究中，我们报告称77-402 RNA转录本形成了两种热稳定性不同的独特二聚体：D37和D55。我们将D37鉴定为一种“亲吻”复合结构，它是通过两个单体之间的环-环相互作用形成的，而D55是一种双链结构，通过标准碱基配对涉及茎环的所有核苷酸。在本报告中，我们通过使用不同长度的RNA转录本进行体外二聚化测定和二聚体热解离，对NCp7在HIV-1Lai RNA二聚化过程中的作用进行了表征。我们的结果表明，核衣壳蛋白NCp7很可能通过与“亲吻”复合物相互作用来激活RNA二聚化，并将其转化为稳定的二聚体。此外，只有当HIV-1Lai RNA分子中存在240-280茎环结构并包含自身互补的G257CGCGC262序列时，这种由NCp7促进的转化才会发生。这项研究表明，在生理条件下，NCp7介导的RNA构象变化参与了HIV-1 RNA二聚体的成熟。
