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。在目前的资金 在此期间，我们已经证明了HIV-1的4种NNRTI-R突变体（K103 N，V106 A， Y181 C和P236 L）对HIV-1 RT的RNase H活性具有特征性影响。 我们还证明了RNase降低更大的NNRTI-R突变体， H活性损害细胞培养中的复制适应性。突变体 RNA酶H活性的更大异常似乎也降低了 在NNRTI临床失败期间出现的可能性。RNase H切割 我们所描述的异常不仅影响非特异性RNase H， 裂解，降解病毒RNA基因组，但也位点特异性 裂解形成聚嘌呤段（PPT），它作为引物， 正链合成这些发现是相当出乎意料的，因为NNRTI-R 突变位于聚合酶结构域中，远离RNase H活性位点。 对这些突变体的进一步研究将导致更好地理解（1） 选择NNRTI-R突变体的致病性后果，（2） RNA酶H在逆转录中特定步骤上的切割及其应用 对病毒复制适应性的贡献，以及（3）RNase H切割是如何 由RT的聚合酶结构域中的残基调节。我们建议追求 这些重要问题的具体目标如下： 1.确定NNRTI-R突变体对特定步骤的影响 转录。 2.确定NNRTI-R突变影响RNase H切割的机制。 3.确定HIV- 1临床分离株的补偿机制 因为NNRTI-R突变降低了复制适应性。 4.确定有助于选择二级NNRTI-R的因素 在依法韦仑治疗期间发生突变。 为了实现这些具体目标，我们将利用广泛的 实验方法，从RT结构和功能的研究， 体外培养，分析其复制特性和生化功能 HIV- 1病毒的样本。

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. During the current funding period, we have demonstrated that 4 NNRTI-R mutants of HIV-1 (K103N, V106A, Y181C, and P236L) have characteristic effects on RNase H activity of HIV-1 RT. We have also demonstrated that NNRTI-R mutants with greater reductions in RNase H activity impair replication fitness in cell culture. Mutants that have greater abnormalities in RNase H activity also appear to have a reduced likelihood of appearing during clinical failure of NNRTIs. The RNase H cleavage abnormalities that we have described affect not only non-specific RNase H cleavages that degrade the viral RNA genome, but also the site-specific cleavages that form the polypurine tract (PPT), which serves as the primer for plus-strand synthesis. These findings are quite unexpected, since NNRTI-R mutations are in the polymerase domain, remote from the RNase H active site. Further study of these mutants should lead to a better understanding of (1) the pathogenic consequences of selection for NNRTI-R mutants, (2) the effects of RNase H cleavage on specific steps in reverse transcription and their contribution to viral replication fitness, and (3) how RNase H cleavages are modulated by residues in the polymerase domain of RT. We propose to pursue these important questions with the following specific aims: 1. Determine the effects of NNRTI-R mutants on specific steps in reverse transcription. 2. Determine the mechanisms by which NNRTI-R mutations affect RNase H cleavage. 3. Identify the mechanisms by which clinical isolates of HIV- 1 can compensate for the reduced replication fitness conferred by NNRTI-R mutations. 4. Determine the factors that contribute to selection for secondary NNRTI-R mutations during therapy with efavirenz. 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.