Impact of NNRTI Resistance on RNase H & HIV Replication

描述（由申请人提供）：我们建议调查 非核苷酸耐药（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，4 HIV- 1的NNRTI-R突变体（K103 N、V106 A、Y181 C和P236 L）改变RNA酶H RNA酶H更广泛降低的HIV-1 RT. NNRTI-R突变体的活性 活性损害细胞培养中的复制适应性，并且不太可能 出现在非核苷类逆转录酶抑制剂临床失效期间。这些研究表明 开发一种活性的、生物可利用的RNase H抑制剂将是有效的 在临床上，NNRTI治疗与RNase H抑制剂的组合可能 预防HIV-1的NNRTI-R变体出现的有效策略。 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 理解（a）选择NNRTI-R的致病后果 突变体，（B）RNase H催化循环中的步骤受NNRTI-R影响 （c）逆转录中的哪些步骤受到特定突变的影响， RNase H异常及其对病毒复制适应性的贡献，（d） NNRTI耐药突变的积累如何影响HIV-1复制 治疗失败期间的适应性，以及（e）RNase H切割如何被调节 我们建议解决这些临床相关的，致病的， 具体目标如下：1.进一步描述影响 NNRTI-R突变体对RNase H和逆转录的影响。2.确定 有助于选择早期原发性耐药突变的因素 依法韦仑失败。3.确定选择中学的因素 突变后，依法韦仑失败。4.识别和表征突变 调节NNRTI-R突变体的作用。为了完成这些 具体目标，我们将利用广泛的实验方法， 范围从体外RT结构和功能的研究，到 HIV-1复制特性及生化功能研究 样品

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.