Impact of NNRTI Resistance on RNase H & HIV Replication

描述(由申请人提供)：我们建议调查 非核苷类药物耐药基因(NNRTI-R)突变对HIV-1的影响 复制和逆转录酶功能。我们已经证明了4 HIV-1的NNRTI-R突变体(K103N、V106A、Y181C和P236L)改变RNaseH HIV-1逆转录酶活性。具有更广泛RNaseH缺失的NNRTI-R突变体 活动损害了细胞培养中的复制适合性，而且不太可能 出现在NNRTIs临床失败期间。这些研究表明 开发一种活性的、生物可用的RNaseH抑制剂将是有效的 临床上，NNRTI治疗与RNaseH抑制剂联合治疗可能是 预防艾滋病毒-1 NNRTI-R变种出现的有效战略。 对这些和其他NNRTI-R突变体的进一步研究可能会导致更好的 对(A)NNRTI-R选择的致病后果的理解 突变体，(B)RNaseH催化循环中的哪些步骤受到NNRTI-R的影响 突变(C)反转录中的哪些步骤受特定基因的影响 核糖核酸酶H异常及其对病毒复制适合性的贡献，(D) NNRTI耐药突变的积累如何影响HIV-1复制 治疗失败时的适合性，以及(E)RNaseH裂解是如何调节的 RT中的残留物。我们建议解决这些与临床相关的、致病的 有以下具体目的的问题：1.进一步描述影响 NNRTI-R突变体在RNaseH和逆转录上的表达。2.确定 有助于早期选择原发耐药突变的因素 Efavirenz失败。3.确定选择次要的因素 突变后来在Eefavirenz失败。4.识别和表征突变 调节NNRTI-R突变体的作用。为了实现这些目标 具体目标，我们将利用一系列广泛的实验方法， 从体外RT结构和功能的研究，到对RT的分析 HIV-1患者体内复制特性及生化功能研究 样本。

DESCRIPTION (provided by applicant): We are proposing to investigate the effects of non-nucleoside inhibitor-resistance (NNRTI-R) mutations on HIV-1 replication and reverse transcriptase function. We have demonstrated that 4 NNRTI-R mutants of HIV- 1 (K103N, V106A, Y181C, and P236L) alter RNase H activity of HIV-1 RT. NNRTI-R mutants with more extensive reductions in RNase H activity impair replication fitness in cell culture, and are less likely to appear during clinical failure of NNRTIs. These studies suggest that development of an active, bioavailable RNase H inhibitor will be effective clinically, and that combining NNRTI treatment with an RNase H inhibitor may be an effective strategy to prevent the emergence of NNRTI-R variants of HIV-1. Further study of these and other NNRTI-R mutants could lead to a better understanding of (a) the pathogenic consequences of selection for NNRTI-R mutants, (b) which steps in the RNase H catalytic cycle are affected by NNRTI-R mutations (c) which steps in reverse transcription are affected by specific RNase H abnormalities and their contribution to viral replication fitness, (d) how the accumulation of NNRTI-resistance mutations affects HIV-1 replication fitness during treatment failure, and (e) how RNase H cleavages are modulated by residues in RT. We propose to address these clinically relevant, pathogenic questions with the following specific aims: 1. Characterize further the effects of NNRTI-R mutants on RNase H and reverse transcription. 2. Determine the factors that contribute to selection for primary resistance mutations early in efavirenz failure. 3. Determine the factors which select for secondary mutations later in efavirenz failure. 4. Identify and characterize mutations that modulate the effects of NNRTI-R mutants. In order to accomplish these specific aims, we will utilize a broad array of experimental approaches, ranging from studies of RT structure and function in vitro, to analyses of the replication characteristics and biochemical function of HIV-1 from patient samples.