Dayton E T, Konings D A, Powell D M, Shapiro B A, Butini L, Maizel J V, Dayton A I
Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
J Virol. 1992 Feb;66(2):1139-51. doi: 10.1128/JVI.66.2.1139-1151.1992.
The significance and location of sequence-specific information in the CAR/RRE, the target sequence for the Rev protein of the human immunodeficiency virus type 1 (HIV-1), have been controversial. We present here a comprehensive experimental and computational approach combining mutational analysis, phylogenetic comparison, and thermodynamic structure calculations with a systematic strategy for distinguishing sequence-specific information from secondary structural information. A target sequence analog was designed to have a secondary structure identical to that of the wild type but a sequence that differs from that of the wild type at every position. This analog was inactive. By exchanging fragments between the wild-type sequence and the inactive analog, we were able to detect an unexpectedly extensive distribution of sequence specificity throughout the CAR/RRE. The analysis enabled us to identify a critically important sequence-specific region, region IIb in the Rev-binding domain, strongly supports a proposed base-pairing interaction in this location, and places forceful constraints on mechanisms of Rev action. The generalized approach presented can be applied to other systems.
人免疫缺陷病毒1型（HIV-1）Rev蛋白的靶序列，即CAR/RRE中序列特异性信息的重要性和位置，一直存在争议。我们在此展示了一种综合的实验和计算方法，将突变分析、系统发育比较和热力学结构计算与一种区分序列特异性信息和二级结构信息的系统策略相结合。设计了一个靶序列类似物，使其具有与野生型相同的二级结构，但在每个位置的序列都与野生型不同。该类似物无活性。通过在野生型序列和无活性类似物之间交换片段，我们能够检测到CAR/RRE中序列特异性的意外广泛分布。该分析使我们能够识别出一个至关重要的序列特异性区域，即Rev结合域中的IIb区域，有力地支持了该位置提出的碱基配对相互作用，并对Rev作用机制施加了严格限制。所提出的通用方法可应用于其他系统。
