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

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级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(TAT)蛋白与TAT反应RNA(TAR)元件结合而激活转录是病毒复制所必需的，被认为是抗病毒药物的潜在靶点。富含精氨酸的基序(RKKRRQRRR)介导TAT对TAR RNA的识别。然而，以前的研究表明，这一短暂的碱性拉伸不足以确定TAT的整个结合活性，因为该碱性区域以外的氨基酸也参与了这种相互作用。为了更好地了解调节Tat-TAR相互作用的功能和结构决定因素，我们使用了酵母三杂交系统的深度突变扫描。深度突变扫描是一种询问感兴趣蛋白质结构域中每个位置突变的适合度影响的方法。在这项试验中，TAT-TAR相互作用产生了一种功能转录因子，激活了HIS3报告基因的表达，使酵母细胞能够在组氨酸饥饿条件下生长。我们确定TAT蛋白中的一段40个氨基酸是TAR结合所必需的，并创建了一个TAT基因库，包含约106个不同的突变体，该区域平均有2.5个随机突变。每个TAT突变对TAR结合的影响决定了HIS3报告基因在每个转化子中的诱导水平，从而决定了它在组氨酸缺乏条件下的生长速度。

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.