Protein-Protein and Protein-RNA Interaction Assays to Determine Similarity of INI1/SMARCB1 and TAR RNA in Binding to HIV-1 Integrase.
Protein-Protein and Protein-RNA Interaction Assays to Determine Similarity of INI1/SMARCB1 and TAR RNA in Binding to HIV-1 Integrase.
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蛋白质-蛋白质和蛋白质-RNA 相互作用测定，以确定 INI1/SMARCB1 和 TAR RNA 在与 HIV-1 整合酶结合方面的相似性。
DOI：
10.1007/978-1-0716-2895-9_8
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Kalpana,GanjamV
中科院分区：
文献类型：
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作者：
Kalpana,GanjamV
INI1/SMARCB1是一种与HIV-1整合酶（IN）相互作用并影响病毒复制多个阶段的宿主蛋白。IN是一种负责整合的病毒酶，它也与HIV-1基因组RNA结合。我们实验室最近的研究表明，HIV-1基因组INI1和TAR RNA区域的IN相互作用Rpt1 （Repeat 1）结构域与IN c -末端结构域（CTD）的相同残基和表面结合。通过一系列的分析，我们发现INI1- rpt1和TAR RNA在结构上相互模仿，并且不能与INI1结合的IN突变体也不能与RNA结合并产生形态上有缺陷的病毒粒子。通过α法检测in - ctd突变体与INI1-Rpt1和TAR RNA的结合，建立了INI1-Rpt1和TAR RNA与in结合的相似性。在这里，我描述了比较INI1-Rpt1蛋白和HIV-1 TAR RNA与IN- ctd结合的α测定方法，并描述了一种三组分测定方法来证明TAR RNA和INI1-Rpt1与IN结合的竞争。
INI1/SMARCB1 is a host protein that interacts with HIV-1 integrase (IN) and influences multiple stages of viral replication. IN is a viral enzyme responsible for integration, and it also binds to HIV-1 genomic RNA. Recent studies from our laboratory demonstrated that IN-interacting Rpt1 (Repeat 1) domain of INI1 and TAR RNA region of HIV-1 genome both bind to the same residues and surface of IN C-terminal domain (CTD). Based on a series of analyses, we found that INI1-Rpt1 and TAR RNA structurally mimic each other and that IN mutants defective for binding to INI1 are also defective for binding to RNA and produce morphologically defective virions. The similarity of INI1-Rpt1 and TAR RNA in binding to IN was established by testing the binding of IN-CTD mutants with INI1-Rpt1 and TAR RNA using the Alpha assay. Here, I describe Alpha assay methods to compare the binding of INI1-Rpt1 protein and HIV-1 TAR RNA to IN-CTD and describe a three-component assay to demonstrate the competition between TAR RNA and INI1-Rpt1 to bind to IN.