Impact of NNRTI-resistance on RNase H & HIV replication

描述（由申请人提供）：项目总结：本申请的目的是了解复制适应性在多大程度上影响对非核苷类逆转录酶抑制剂（NNRTI）耐药的HIV变异体的出现，并描述耐药突变如何相互作用以影响复制适应性的潜在生化机制。如果病毒复制不受抑制，HIV的NNRTI耐药变异体（通常与其他NNRTI交叉耐药）会迅速发展。我们已经表明，在细胞培养中测得的HIV复制适应性在临床上不常见的NNRTI耐药突变体中降低，即使它们比最常见的突变体K103 N具有更高水平的耐药性。这些复制不良的突变体也减少了逆转录酶对RNase H的切割。两种突变体G190 S和A也减少了tRNA（Lys，3）的引发，提出了RNA引发缺陷是否也导致NNRTI抗性突变体的适应性降低的问题。我们还表明，核苷（nRTI）抗性突变L74 V补偿了K103 N + L100 I降低的适应性，但没有改善其降低的RNA酶H切割速率。其他人发表的研究表明，L74 V减少了RNA引发。因此，我们提出，L74 V可以通过改善逆转录中的步骤，如链转移和链置换合成，补偿RNA酶H切割缺陷，来补偿复制不良、高度NNRTI抗性变体的RNA酶H活性降低。我们假设，选择高度耐NNRTI的变异，提高了适应性的补偿nRTI耐药突变可能会导致早期病毒学失败。这些研究对更合理地设计nRTI-NNRTI联合治疗方案具有重要意义。在下一个融资期内，我们计划：1。评价临床样本中nRTI和NNRTI耐药突变的相关性。2.确定nRTI耐药突变对NNRTI耐药突变体的复制适应性和耐药性的影响。3.描述L74 V如何提高NNRTI抗性变体适应性的生化基础。相关性：拟议的研究将评估哪些因素导致艾滋病毒耐药株的发展，逆转录酶功能的变化如何影响艾滋病毒的复制，以及是否可以选择药物组合来影响耐药株的发展。

DESCRIPTION (provided by applicant): Project Summary: The goals of this application are to understand to what extent replication fitness influences the emergence of HIV variants resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs), and to delineate the underlying biochemical mechanisms for how drug resistance mutations interact to influence replication fitness. NNRTI-resistant variants of HIV, usually cross-resistant to other NNRTIs, develop rapidly if viral replication is not suppressed. We have shown that HIV replication fitness, as measured in cell culture, is reduced in NNRTI-resistant mutants that are infrequent clinically, even though they confer higher levels of resistance than the most common mutant, K103N. These poorly replicating mutants also reduce RNase H cleavage by reverse transcriptase. Two mutants, G190S and A, also reduce priming from tRNA(Lys,3), raising the question of whether defects in RNA priming also contribute to the reduced fitness of NNRTI- resistant mutants. We also have shown that the nucleoside (nRTI) resistance mutation L74V compensates for the reduced fitness of K103N+L100I, but does not improve its reduced RNase H cleavage rates. Published studies by others have shown that L74V reduces RNA priming. We therefore propose that L74V can compensate for the reduced RNase H activity of poorly replicating, highly NNRTI-resistant variants by improving steps in reverse transcription, such as strand transfer and strand displacement synthesis, that compensate for RNase H cleavage defects. We postulate that selection for highly NNRTI-resistant variants that have improved fitness from compensatory nRTI resistance mutations can lead to early virologic failure. These studies have important implications for the more rational design of nRTI-NNRTI combination regimens. During the next funding period, we plan to: 1. Evaluate the association of nRTI and NNRTI resistance mutations in clinical samples. 2. Determine the effects of nRTI resistance mutations on the replication fitness and drug resistance of NNRTI-resistant mutants. 3. Characterize the biochemical basis for how L74V improves the fitness of NNRTI-resistant variants. Relevance: The proposed studies will evaluate what factors lead to the development of drug resistant strains of HIV, how changes in reverse transcriptase function affect replication of HIV, and whether combinations of drugs can be chosen to influence which drug resistant strains develop.