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。在目前的资助期间 期间，我们已经证明了HIV-1的4个NNRTI-R突变体(K103N，V106A， Y181C和P236L)对HIV-1RT的核糖核酸酶H活性有特有的影响。 我们还证明了RNase有较大降低的NNRTI-R突变体 H活性损害了细胞培养中的复制适合性。变种人 核糖核酸酶H活性的更大异常似乎也会减少 在NNRTIs临床失败期间出现的可能性。核糖核酸酶H裂解 我们所描述的异常不仅影响非特异性RNaseH 降解病毒RNA基因组的切割，但也包括特定部位的 形成多聚尿路(PPT)的裂解，它是 正链合成。这些发现是相当出人意料的，因为NNRTI-R 突变位于聚合酶区域，远离RNaseH活性部位。 对这些突变体的进一步研究应有助于更好地理解(1) NNRTI-R突变体选择的致病后果，(2) 核糖核酸酶H在逆转录特定步骤上的裂解及其意义 对病毒复制适合性的贡献，以及(3)RNaseH裂解的方式 受RT聚合酶结构域中残基的调控。我们建议继续进行 这些重要问题有以下具体目的： 1.反向确定NNRTI-R突变体对特定步骤的影响 抄写。 2.确定NNRTI-R突变影响RNase H裂解的机制。 3.确定HIV-1临床分离株可以补偿的机制 因为NNRTI-R突变带来的复制适合性降低。 4.确定影响二级NNRTI-R选择的因素 在法韦伦治疗过程中的突变。 为了实现这些具体目标，我们将利用广泛的 实验方法，从RT结构和功能的研究在 体外培养，分析细胞的复制特性和生化功能 从患者样本中提取HIV-1病毒。

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.