HIV Reverse Transcriptase-Mediated Mutagenesis

描述（由申请人提供）：HIV-1具有高突变率，这有助于其逃避宿主免疫系统的能力，限制了抗逆转录病毒药物的疗效，并驱动 抗药性的出现。耐药性突变以及其他病毒突变主要归因于逆转录酶（RT）的易错性。RT介导的突变的有意增加通过将突变率增加到不能维持病毒群体存活的水平来降低病毒感染性。效价 通过增加RT介导的错误可以降低HIV-1的感染性，这导致了发现可能增加HIV-1突变率的小分子的倡议。组建了一个跨学科的合作团队，以1）进行发现研究，以确定增加RT介导错误的新小分子，2）使用分子分析来确定小分子增加HIV突变率并导致病毒灭绝的机制，以及3）使用生物化学方法评估RT介导突变的机制。通过初步研究，我们已经确定了四种增加RT介导突变的小分子。为了阐明驱动这种增加的结构-活性关系并优化这种活性，我们将首先进行发现研究，以确定可以增加RT介导的错误的小分子。将在细胞培养物中评估这些分子的抗病毒和致突变活性。其次，我们将研究小分子诱导突变和使用细胞培养方法导致HIV-1病毒灭绝的机制。在这里，我们将检查我们已经发现的小分子以及我们确定的任何铅分子。第三，我们将使用生物化学方法研究作用机制，以阐明增加RT介导的突变的机制基础。这些研究的成功完成将为RT介导的病毒诱变机制及其对病毒复制和灭绝的影响提供更深入的了解。

DESCRIPTION (provided by applicant): HIV-1 has a high mutation rate, which contributes to its ability to evade the host immune system, limits the efficacy of antiretroviral drugs and drives the emergence of drug resistance. Drug resistance conferring mutations as well as other viral mutations are primarily attributed to the error-prone nature of reverse transcriptase (RT). An intentional increase in RT-mediated mutations decreases virus infectivity by increasing the mutation rate to a level that is not able to maintain survival of the virus population. The potency by which HIV-1 infectivity can be decreased by increasing RT-mediated errors has led to an initiative to discover small molecules that may increase the HIV-1 mutation rate. An interdisciplinary collaborative team has been assembled to 1) conduct discovery studies to identify new small molecules that increase RT-mediated errors, 2) use molecular analyses to identify the mechanism(s) by which small molecules increase the HIV mutation rate and result in virus extinction, and 3) to assess the mechanism of RT-mediated mutation using biochemical methods. Through preliminary studies, we have identified four small molecules that increase RT-mediated mutations. In order to elucidate the structure-activity relationship driving this increase and to optimize this activity, we will first pursue discovery studies to identify small molecules that can increase RT-mediated errors. The antiviral and mutagenic activities of these molecules will be assessed in cell culture. Second, we will examine the mechanism by which small molecules induce mutations and cause virus extinction in HIV-1 using cell culture methodologies. Here we will examine small molecules that we have already discovered as well as any lead molecules that we identify. Third, we will investigate the mechanism of action using biochemical methods to elucidate the mechanistic basis for increased RT-mediated mutation. Successful completion of these studies will provide deeper insight into the mechanisms of RT-mediated viral mutagenesis and its impact on viral replication and extinction.