Berkowitz R D, Goff S P
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, New York 10032.
Virology. 1994 Jul;202(1):233-46. doi: 10.1006/viro.1994.1339.
We previously used RNA gel mobility shift assays to demonstrate specific binding of the HIV-1 gag precursor polyprotein and nucleocapsid (NC) protein to HIV-1 RNA and to map the binding elements in each species by mutagenesis. Here we report finer mapping of binding elements in the HIV-1 genomic RNA and NC protein, performed by analyzing the binding behavior of fragments of each species in the gel shift assay. With regard to the RNA, the strongest binding activity resided in a 120-nucleotide segment flanking the gag start codon, containing three potential stem-loop structures. Binding analysis of various combinations of these three potential stem-loop structures and their flanking sequences revealed that no one element could bind to the gag polyprotein or NC protein as well as the entire 120-nucleotide segment. Mutational analysis of the NC protein showed that two nonoverlapping regions exhibited specific binding for HIV-1 RNA. Each region includes a Cys-His box, though each box could not bind to HIV-1 RNA on its own. In construct lacking both boxes exhibited primarily nonspecific RNA-binding activity.
我们之前使用RNA凝胶迁移率变动分析来证明HIV-1前体gag多聚蛋白和核衣壳（NC）蛋白与HIV-1 RNA的特异性结合，并通过诱变来定位每个物种中的结合元件。在此，我们报告了通过分析凝胶迁移率变动分析中每个物种片段的结合行为，对HIV-1基因组RNA和NC蛋白中的结合元件进行更精细的定位。关于RNA，最强的结合活性存在于gag起始密码子侧翼的一个120个核苷酸的片段中，该片段包含三个潜在的茎环结构。对这三个潜在茎环结构及其侧翼序列的各种组合进行结合分析表明，没有一个元件能像整个120个核苷酸的片段那样与gag多聚蛋白或NC蛋白结合。对NC蛋白的突变分析表明，两个不重叠的区域对HIV-1 RNA表现出特异性结合。每个区域都包含一个半胱氨酸-组氨酸框，尽管每个框自身不能与HIV-1 RNA结合。在缺乏这两个框的构建体中，主要表现出非特异性RNA结合活性。
