REVERSE TRANSCRIPTASE AND DRUG RESISTANT HIV IN CHILDREN

人类免疫缺陷病毒(HIV-1)，从患者体内分离出来 长期使用3‘-叠氮-2’，3‘-双脱氧胸腺嘧啶核苷(AZT，齐多夫定)， 对这种药物的敏感度可能会降低100倍。这 由于许多艾滋病患者目前 接受长期的AZT治疗。类似的担忧也适用于患者 使用其他抗病毒药物，如双脱氧肌苷(DDL)。AZT 抗药性与编码序列中的特定突变有关 病毒编码的逆转录酶。这项建议集中在两个方面 与逆转录酶和抗药性艾滋病毒相关的领域。首先，我们 已制备含有氨基酸的重组HIV-1逆转录酶 第215位和第219位的替换，并观察到20倍的增加 AZTTP粗提物或部分纯化样品的半数抑制浓度 酵素。这表明耐药表型是由于变化引起的。 这可以在酶的功能中检测到，我们建议进行 该突变蛋白的广泛动力学研究和结构分析 和其他突变蛋白，以确定AZT的抗性机制 分子水平。AZTTP对聚合酶活性的影响 使用设计为与不同的不同 病毒复制中逆转录酶催化的亚反应 周而复始。含AZT的核苷酸对核糖核酸酶H活性的抑制作用 也可以比较突变型和野生型酶。为了确定 这些突变的生物学意义，我们将描述 儿科患者临床标本DNA直接测序分析 用聚合酶链式反应进行前病毒DNA扩增。每个人 标本的药物敏感性也将通过病毒培养试验进行检测。 而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分析也将 对服用AZT的孕妇标本进行的研究 和他们的婴儿进行相关突变的传播检测 从母亲到婴儿的耐药性。这项研究的最终目的是 是为了了解艾滋病毒耐药突变的分子机制 并提高我们监测抗药性病毒感染的能力。

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.