Compensating Tat/TAR mutations in drug-resistant HIV-1

描述(由申请人提供)：抗逆转录病毒疗法(ART)的目标是在活跃于病毒生产的CD4+T细胞中复制HIV-1。虽然ART可以将病毒载量降低到无法检测的水平，但选择了一小部分具有抗药性的病毒基因组，并在药物存在的情况下继续复制。在这一人群中，抗逆转录病毒治疗的分子靶标逆转录酶(RT)和蛋白酶(PR)积累了导致耐药性的突变。与此同时，这些耐药突变是有害的，降低了RT和PR的活性，并导致复制能力较低的病毒持续存在(即，较不适合的病毒)。持续的病毒进化选择补偿突变，导致出现更适合的病毒。RT和PR基因的这种突变具有很好的特征，但也有迹象表明，其他病毒基因也可能出现代偿性突变。 其中一个候选基因是重要的病毒转录激活因子TAT。我们和其他人已经确定了增加病毒基因表达的天然TAT突变。我们推测，TAT或其病毒靶标TAR发生突变的病毒会增加多药耐药病毒的适应性，并将被选择存活。在这个探索性项目中，我们建议解决这样的假设，即在ART过程中选择了效力更高的TAT和/或TAR变体。我们将在RT和PR耐药突变的背景下测试包含这些选定的TAT和/或TAR变体的病毒的适合性，后续实验将解决潜在的生化机制。 在这笔赠款的任期内，我们的目标是： 1.对HIV-1耐药株的Tat基因和TAR元件进行测序和分析。 2.鉴定所选TAT和TAR变异体的复制适合性和功能。

DESCRIPTION (provided by applicant): Anti-Retroviral Therapy (ART) targets HIV-1 replication in CD4+ T cells that are active in virus production. Although ART can reduce the viral load to undetectable levels, a small population of drug-resistant viral genomes is selected and continues to replicate in the presence of the drugs. In this population, the molecular targets of antiretroviral therapy, reverse transcriptase (RT) and protease (PR), accumulate mutations that confer drug resistance. At the same time these drug-resistance mutations are deleterious, reducing the activity of RT and PR and resulting in the persistence of viruses that have lower replicative capacity (i.e., lesser "fitness"). Continuing viral evolution selects for compensating mutations resulting in the emergence of viruses that are fitter. Such mutations in the RT and PR genes are well characterized, but there are also indications that compensating mutations can arise in other viral genes. One such candidate gene is the essential viral transcription activator, Tat. We and others have identified natural Tat mutations that increase viral gene expression. We postulate that viruses with mutations in Tat, or in its viral target TAR, increase the fitness of multidrug-resistant viruses and will be selected for survival. In this exploratory project we propose to address the hypothesis that Tat and/or TAR variants of higher potency are selected during ART. We will test the fitness of viruses containing these selected Tat and/or TAR variants in the context of drug-resistance mutations in RT and PR, and subsequent experiments will address the underlying biochemical mechanisms. During the term of this grant our aims will be: 1. To sequence and analyze HIV-1 Tat genes and the TAR element of viruses isolated from HIV-1 drug-resistant patients. 2. To characterize the replicative fitness and function of selected Tat and TAR variants.