Impact of NNRTI-resistance on RNase H & HIV replication

描述(由申请人提供)：项目摘要：本申请的目标是了解复制适合性在多大程度上影响对非核苷类逆转录酶抑制剂(NNRTI)耐药的艾滋病毒变异的出现，并描绘耐药突变如何相互作用影响复制适合性的潜在生化机制。艾滋病毒的NNRTI耐药变种通常与其他NNRTI交叉耐药，如果病毒复制得不到抑制，它们就会迅速发展。我们已经证明，在细胞培养中测量的HIV复制适合性在临床上不常见的NNRTI耐药突变株中降低，尽管它们提供的耐药性水平高于最常见的突变株K103N。这些复制能力差的突变体还会通过逆转录酶减少RNaseH的裂解。两个突变体G190S和A也减少了tRNA(Lys，3)的引发，这提出了一个问题，即RNA引发的缺陷是否也导致抗NNRTI突变体的适合度降低。我们还发现，核苷(NRTI)抗性突变L74V补偿了K103N+L100I降低的适合度，但不能提高其降低的RNase H裂解率。其他人发表的研究表明，L74V减少了RNA的启动。因此，我们认为L74V可以通过改进逆转录中的步骤，如链转移和链置换合成，来弥补复制不良、高度抗NNRTI的变异体RNase H活性的降低，从而弥补RNase H裂解缺陷。我们推测，从代偿性NRTI耐药突变中选择对NNRTI高度耐药的变异体可以导致早期病毒学失败。这些研究对设计更合理的NRTI-NNRTI联合方案具有重要意义。在下一个资助期间，我们计划：1.评估临床样本中NRTI和NNRTI耐药突变的相关性。2.确定NRTI耐药突变对NNRTI耐药突变株复制适合度和耐药性的影响。3.鉴定L74V如何提高NNRTI抗性变异体适合性的生化基础。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.