DEEP MUTATIONAL SCANNING TO ANALYZE THE HIV-1 TAT-TAR INTERACTION BY THE YEAST T

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 通过将人类免疫缺陷病毒1型（HIV-1）反式激活因子（达特）蛋白与Tat应答RNA（TAR）元件结合而进行的转录激活对于病毒复制是必不可少的，并且被认为是抗病毒药物的潜在靶标。富含精氨酸的基序（RKKRRQRRR）介导达特对TAR RNA的识别。然而，以前的研究表明，这个短的基本伸展不足以确定达特的整个结合活性，因为这个基本区域以外的氨基酸也有助于这种相互作用。为了更好地了解介导Tat-TAR相互作用的功能和结构决定因素，我们使用酵母三杂交系统进行深度突变扫描。深度突变扫描是一种查询目标蛋白质结构域中每个位置突变的适应性效应的方法。在该测定中，Tat-TAR相互作用产生了激活HIS 3报告基因表达的功能性转录因子，使酵母细胞在组氨酸饥饿条件下生长。我们确定了达特蛋白中的40个氨基酸的片段是TAR结合所必需的，并创建了达特基因文库，包括~106个不同的突变体，经工程改造在该区域平均具有2.5个随机突变。每个达特突变对TAR结合的影响决定了每个转化体中HIS 3报告基因的诱导水平，因此决定了其在组氨酸缺陷条件下的生长速率。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Transcription activation by binding of the Human Immunodeficiency Virus type-1 (HIV-1) transactivator (Tat) protein to the Tat-responsive RNA (TAR) element is essential for viral replication and is considered a potential target for antiviral drugs. An arginine rich motif (RKKRRQRRR) mediates TAR RNA recognition by Tat. However, previous studies suggest that this short basic stretch is insufficient to determine the entire binding activity of Tat, since amino acids outside of this basic region also contribute to this interaction. To gain a better understanding of the functional and structural determinants that mediate Tat-TAR interaction, we used deep mutational scanning with a yeast three-hybrid system. Deep mutational scanning is an approach that queries the fitness effects of mutations in every position in a protein domain of interest. In this assay, Tat-TAR interaction creates a functional transcription factor that activates the expression of the HIS3 reporter gene, allowing yeast cells to grow under histidine starvation conditions. We determined that a segment of 40 amino acids within the Tat protein is necessary for TAR binding and created a library of Tat genes, encompassing ~106 different mutants, engineered to have an average of 2.5 random mutations in this region. The effect of each Tat mutation on TAR binding determines the induction level of the HIS3 reporter gene in each tranformant and hence, its growth rate under histidine deficient conditions.