Paillart J C, Westhof E, Ehresmann C, Ehresmann B, Marquet R
Unité Propre de Recherche du CNRS no 9002, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
J Mol Biol. 1997 Jul 4;270(1):36-49. doi: 10.1006/jmbi.1997.1096.
Retroviruses encapsidate two molecules of genomic RNA that are noncovalently linked close to their 5' ends in a region called the dimer linkage structure (DLS). The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) constitutes the essential part of the DLS in vitro and is crucial for efficient HIV-1 replication in cell culture. We previously identified the DIS as a hairpin structure, located upstream of the major splice donor site, that contains in the loop a six-nucleotide self-complementary sequence preceded and followed by two and one purines, respectively. Two RNA monomers form a kissing loop complex via intermolecular interactions of the six nucleotide self-complementary sequence. Here, we introduced compensatory mutations in the self-complementary sequence and/or a mutation in the flanking purines. We determined the kinetics of dimerization, the thermal stabilities and the apparent equilibrium dissociation constants of wild-type and mutant dimers and used chemical probing to obtain structural information. Our results demonstrate the importance of the 5'-flanking purine and of the two central bases of the self-complementary sequence in the dimerization process. The experimental data are rationalized by triple interactions between these residues in the deep groove of the kissing helix and are incorporated into a three-dimensional model of the kissing loop dimer. In addition, chemical probing and molecular modeling favor the existence of a non-canonical interaction between the conserved adenine residues at the first and last positions in the DIS loop. Furthermore, we show that destabilization of the kissing loop complex at the DIS can be compensated by interactions involving sequences located downstream of the splice donor site of the HIV-1 genomic RNA.
逆转录病毒将两个基因组RNA分子包装在一起，这两个分子在靠近其5'端的一个称为二聚体连接结构（DLS）的区域非共价连接。1型人类免疫缺陷病毒（HIV-1）的二聚化起始位点（DIS）在体外构成DLS的关键部分，对HIV-1在细胞培养中的有效复制至关重要。我们之前将DIS鉴定为一种发夹结构，位于主要剪接供体位点的上游，其环中包含一个六核苷酸的自我互补序列，该序列前后分别有两个和一个嘌呤。两个RNA单体通过六核苷酸自我互补序列的分子间相互作用形成一个亲吻环复合物。在此，我们在自我互补序列中引入了补偿性突变和/或在侧翼嘌呤中引入了一个突变。我们测定了野生型和突变型二聚体的二聚化动力学、热稳定性和表观平衡解离常数，并使用化学探针来获取结构信息。我们的结果证明了5'侧翼嘌呤和自我互补序列的两个中心碱基在二聚化过程中的重要性。这些残基在亲吻螺旋深沟中的三重相互作用使实验数据合理化，并被纳入亲吻环二聚体的三维模型中。此外，化学探针和分子建模支持DIS环中第一个和最后一个位置的保守腺嘌呤残基之间存在非经典相互作用。此外，我们表明，DIS处亲吻环复合物的不稳定可以通过涉及HIV-1基因组RNA剪接供体位点下游序列的相互作用来补偿。
