Impact of NNRTI-resistance on RNase H & HIV replication

项目概述：本申请的目标是了解复制适应度在多大程度上影响对非核苷逆转录酶抑制剂（NNRTIs）耐药的HIV变异的出现，并描述耐药突变如何相互作用以影响复制适应度的潜在生化机制。如果不抑制病毒复制，耐nnrti的HIV变体通常会对其他nnrti产生交叉耐药。我们已经证明，在细胞培养中测量的HIV复制适应度在临床不常见的nnrti抗性突变体中降低，尽管它们比最常见的突变体K103N具有更高的抗性水平。这些复制能力差的突变体也通过逆转录酶减少RNase H的切割。两个突变体G190S和A也减少了tRNA的引物（Lys,3），这就提出了RNA引物缺陷是否也导致NNRTI抗性突变体适应度降低的问题。我们还发现核苷（nRTI）抗性突变L74V补偿了K103N+L100I的适应度降低，但并没有提高其降低的RNase H切割率。其他人发表的研究表明，L74V减少了RNA的启动。因此，我们提出L74V可以通过改善逆转录的步骤，如链转移和链位移合成，来补偿RNase H切割缺陷，从而补偿低复制、高nnrti抗性变异的RNase H活性降低。我们假设，选择高度nnrti抗性的变异，从代偿性nRTI抗性突变中提高适应性，可能导致早期病毒学失败。这些研究对更合理地设计nRTI-NNRTI联合方案具有重要意义。在接下来的资助期内，我们计划：评估临床样本中nRTI和NNRTI耐药突变的关系。2. 确定nRTI耐药突变对nnrti耐药突变体复制适应度和耐药性的影响。3. 描述L74V如何提高nnrti抗性变异适应度的生化基础。相关性：拟议的研究将评估哪些因素导致耐药HIV菌株的发展，逆转录酶功能的变化如何影响HIV的复制，以及是否可以选择药物组合来影响耐药菌株的发展。

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.