REVERSE TRANSCRIPTASE AND DRUG RESISTANT HIV

人类免疫缺陷病毒（HIV-1），分离自患者， 用3 '-叠氮基-2'，3 '-双脱氧胸苷（AZT，齐多夫定）长期治疗， 对药物的敏感性可能会降低100倍。 这 这一发现引起了人们的极大关注，因为目前许多艾滋病患者 长期接受AZT治疗 类似的担忧也适用于患者 用其他抗病毒药物如双脱氧肌苷（DDL）治疗。 AZT 耐药性与编码序列中的特定突变有关， 病毒编码的逆转录酶 该提案重点关注两个 逆转录酶和耐药艾滋病毒相关领域。 一是 制备了含有氨基酸的重组HIV-1逆转录酶， 在位置215和219处的取代，并且已经观察到20倍的增加 在粗提物或部分纯化的 酵素 这表明耐药表型是由于 可以在酶功能中检测到，我们建议进行 对该突变蛋白进行了广泛的动力学研究和结构分析 和其他突变蛋白质，以确定抗AZT的机制。 分子水平。 AZTTP对聚合酶活性的影响将在 用模板和引物测量，所述模板和引物被设计成对应于不同的 病毒复制中逆转录酶催化的亚反应 周期 含AZT的核苷酸对RNase H活性的抑制将 也可以比较突变体和野生型酶。 为了确定 这些突变的生物学意义，我们将描述 通过直接DNA序列分析的儿科患者临床样本 在使用聚合酶链式反应进行前病毒DNA扩增之后。 每个 还将通过病毒培养试验对样本进行药物敏感性分析 耐药性的水平将与特定的突变有关 检测到 DNA序列分析和药物敏感性测定也将在 对服用过AZT的孕妇的样本进行了研究 和他们的婴儿来测试与以下疾病相关的突变的传播 从母亲到婴儿的抗药性。 这项研究的最终目的是 是了解HIV耐药性突变的分子机制 并提高我们监测耐药病毒感染的能力。

Human immunodeficiency virus (HIV-1), isolated from patients after long-term treatment with 3'-azido-2',3'-dideoxythymidine (AZT, zidovudine), may exhibit as much as 100-fold reduced sensitivity to the drug. This finding is of major concern since many AIDS patients are currently undergoing prolonged AZT therapy. Similar concerns apply to patients treated with other antiviral drugs such as dideoxyinosine (ddl). AZT resistance is associated with specific mutations in the coding sequence for the virus-encoded reverse transcriptase. This proposal focuses on two areas related to reverse transcriptase and drug-resistant HIV. First, we have prepared recombinant HIV-1 reverse transcriptase containing amino acid substitutions at positions 215 and 219 and have observed a 20-fold increase in the IC50 for AZTTP measured in crude extract or in partially purified enzyme. This suggests that the drug resistant phenotype is due to changes that can be detected in enzyme function and we propose to carry out extensive kinetic studies and structural analysis on this mutant protein and on other mutant proteins to define the mechanism of AZT resistance at the molecular level. Effects of AZTTP on polymerase activity will be measured with templates and primers designed to correspond to different subreactions catalyzed by reverse transcriptase in the viral replication cycle. Inhibition of RNase H activity by AZT-containing nucleotides will also be compared for mutant and wild type enzymes. In order to determine the biological significance of these mutations, we will characterize clinical samples from pediatric patients by direct DNA sequence analysis after proviral DNA amplification using the polymerase chain reaction. Each specimen will also be assayed for drug sensitivity by virus culture assay and the level of drug resistance will be related to the specific mutations detected. DNA sequence analysis and drug sensitivity assays will also be carried out on specimens from pregnant women who have been treated with AZT and their infants to test for transmission of mutations associated with drug resistance from mother to infant. The ultimate goal of this research is to understand molecular mechanisms of drug-resistance mutations in HIV and to improve our ability to monitor drug-resistant virus infections.