STRUCTURE-FUNCTION STUDIES OF HIV-1 REVERSE TRANSCRIPTASE

工作总结：人类免疫缺陷病毒(HIV-1)具有很高的 在其基因组的某些部分内的突变率，允许快速 对药物产生抗药性的新型病毒的进化 治疗或可逃避宿主的免疫反应。这个项目 调查了最有可能的原因，不准确的DNA合成 HIV-1逆转录酶(RT)。大多数在试管中犯下的错误是 由模板和底物链的滑移引发，可能在 聚合酶解离，然后与DNA重新结合。按顺序 为了深入了解为什么HIV-1 RT对这些类型如此容易出错 在错误中，我们正在检查HIV-1RT的突变衍生物， 强调氨基酸被认为是模板-引物的重要组成部分 互动。我们完成了丙氨酸取代突变体的研究 对于拇指亚区α螺旋H的四个特定氨基酸和 一颗棕榈油残留物。都降低了DNA结合亲和力，对 AZTTP，降低了处理速度和/或改变了帧移保真度。他们也 改变了它们与环氧苯乙烷的相互作用能力 加合物，DNA主要和次要凹槽中相互作用的探针。 模拟表明，这五种氨基酸组成了一个小沟 结合轨道(MGBT)，与模板-引子刚从 活动站点。今年，我们开始了对RT突变体的研究， MGBT残基对丙氨酸以外的氨基酸的重要性探讨 疏水和氢键相互作用。我们还在检查变种人 随着氨基酸的变化更接近聚合酶活性部位。分析 这些酶的相互作用将增强我们对RT如何与 底物，并可能提供对艾滋病的高度变异性的见解 病毒。由于这些研究侧重于链导致的突变 它们还可能提供与几个人类相关的见解 以可能由链滑动导致的突变为特征的疾病 在DNA复制过程中。

Summary of Work: The human immunodeficiency virus (HIV-1) has high mutation rates within certain portions of its genome, permitting rapid evolution of new forms of the the virus that are resistant to drug treatments or can evade the host's immune response. This project investigates the most likely cause for this, inaccurate DNA synthesis by the HIV-1 reverse transcriptase (RT). Most mistakes made in vitro are initiated by slippage of the template and primer strands, perhaps when the polymerase dissociates and then reassociates with the DNA. In order to gain insight into why the HIV-1 RT is so error-prone for these types of mistakes, we are examining mutant derivatives of HIV-1 RT, with emphasis on amino acids believed to be important for template-primer interactions. We completed studies of mutants with alanine substituted for four specific amino acids in alpha helix H of the thumb subdomain and one palm residue. All have reduced DNA binding affinity, resistance to AZTTP, reduced processivity and/or altered framshift fidelity. They also are altered in their ability to interact with styrene oxide N2-guanine adducts, a probe for interactions in the major and minor grooves of DNA. Modeling suggests that these five amino acids comprise a minor groove binding track (MGBT) that contacts the template-primer just back from the active site. This year we began studies of RT mutants with changes of MGBT residues to amino acids other than alanine, to probe the importance of hydrophobic and H-bonding interactions. We are also examining mutants with amino acid changes nearer to the polymerase active site. Analysis of these enzymes will enhance our understanding of how RT interact with substrates and may provide insights into the hypermutability of the AIDS virus. Since these studies focus on mutations resulting from strand slippage, they may also provide insights relevant to several human diseases characterized by mutations that may result from strand slippage during DNA replication.