Rist Manuela J, Marino John P
Center for Advanced Research in Biotechnology of the University of Maryland, Rockville 20850, USA.
Biochemistry. 2002 Dec 17;41(50):14762-70. doi: 10.1021/bi0267240.
Dimerization of two homologous strands of genomic RNA is an essential feature of retroviral replication. In the human immunodeficiency virus type 1 (HIV-1), a conserved stem-loop sequence, the dimerization initiation site (DIS), has been identified as the domain primarily responsible for initiation of this aspect of viral assembly. The DIS loop contains an autocomplementary hexanucleotide sequence flanked by highly conserved 5' and 3' purines and can form a homodimer through a loop-loop kissing interaction. In a structural rearrangement activated by the HIV-1 nucleocapsid protein (NCp7) and considered to be associated with viral particle maturation, the DIS dimer converts from an intermediate kissing to an extended duplex isoform. Using 2-aminopurine (2-AP) labeled sequences derived from the DIS(Mal) variant and fluorescence methods, the two DIS dimer isoforms have been unambiguously distinguished, allowing a detailed examination of the kinetics of this RNA structural isomerization and a characterization of the role of NCp7 in the reaction. In the presence of divalent cations, the DIS kissing dimer is found to be kinetically trapped and converts to the extended duplex isoform only upon addition of NCp7. NCp7 is demonstrated to act catalytically in inducing the structural isomerization by accelerating the rate of strand exchange between the two hairpin stem helices, without disruption of the loop-loop helix. Observation of an apparent maximum conversion rate for NCp7-activated DIS isomerization, however, requires protein concentrations in excess of the 2:1 stoichiometry estimated for high-affinity NCp7 binding to the DIS kissing dimer, indicating that transient interactions with additional NCp7(s) may be required for catalysis.
基因组RNA两条同源链的二聚化是逆转录病毒复制的一个基本特征。在1型人类免疫缺陷病毒（HIV-1）中，一个保守的茎环序列，即二聚化起始位点（DIS），已被确定为主要负责病毒组装这一方面起始的结构域。DIS环包含一个自身互补的六核苷酸序列，两侧是高度保守的5'和3'嘌呤，并且可以通过环-环亲吻相互作用形成同源二聚体。在由HIV-1核衣壳蛋白（NCp7）激活并被认为与病毒颗粒成熟相关的结构重排过程中，DIS二聚体从中间亲吻构象转变为延伸双链体异构体。使用源自DIS（Mal）变体的2-氨基嘌呤（2-AP）标记序列和荧光方法，已明确区分出两种DIS二聚体异构体，从而能够详细研究这种RNA结构异构化的动力学，并表征NCp7在反应中的作用。在二价阳离子存在的情况下，发现DIS亲吻二聚体在动力学上被捕获，并且仅在添加NCp7后才转变为延伸双链体异构体。已证明NCp7通过加速两个发夹茎螺旋之间的链交换速率来催化诱导结构异构化，而不会破坏环-环螺旋。然而，观察到NCp7激活的DIS异构化存在明显的最大转化率，这需要蛋白质浓度超过针对高亲和力NCp7与DIS亲吻二聚体结合估计的2:1化学计量比，这表明催化作用可能需要与额外的NCp7进行瞬时相互作用。
