Characterization of RHA:RT interactions in HIV-1 reverse transcription

项目摘要 艾滋病毒-1感染是全球主要死亡原因之一。迫切需要的是 新的抗病毒疗法的开发是由于疫苗的缺乏和药物的出现- 耐药菌株。HIV-1逆转录酶(RT)是一种容易出错的聚合酶，负责 臭名昭著的抗药性突变体的出现。由于处理速度较低，RT会暂停 某些RNA序列/结构和停顿与高度突变和 重组率。在RNA调节中保持相对高保真度的机制 但在逆转录过程中容易出错的开放阅读框中的区域却知之甚少。 宿主解旋酶DHX9/RNA解旋酶A(RHA)被招募到病毒粒子中，促进病毒的加工 RT.初步数据表明，RT：RHA在逆转录和 揭示RHA提高了RT在结构调控RNA区域的保真度。的总目标是 该建议旨在为RHA介导的RT保真度调制在 逆转录。目标1将描述依赖于RHA的保真度增强热点 无宿主和宿主存在条件下合成的病毒基因组RNA测序 啊哈。AIM 2将通过冷冻-EM表征RT：RHA的相互作用，并通过基于细胞的验证 化验。拟议的研究将为深入了解宿主解旋酶介导的机制提供帮助。 反转过程中基因组RNA不同位置RT停顿和保真度的调节 转录，并为新的抗病毒疗法的开发提供结构基础。

Project Summary HIV-1 infection is one of the leading causes of death globally. The urgent need for the development of new antiviral therapies arises from the lack of vaccine and emerging of drug- resistant strains. HIV-1 reverse transcriptase (RT) is an error-prone polymerase responsible for the emergence of notorious drug resistant mutants. Due to the low processivity, RT pauses on certain RNA sequences/structures and the pausing is correlated with high mutation and recombination rate. The mechanism that maintains fidelity relatively high in the RNA regulatory regions but error-prone in open reading frames during reverse transcription is poorly understood. Host helicase DHX9/RNA helicase A (RHA) is recruited into virions and promotes processivity of RT. Preliminary data suggest dynamic RT: RHA interactions during reverse transcription and reveal that RHA improves RT’s fidelity on structured regulatory RNA regions. The overall goal of this proposal is to establish the structural basis for RHA-mediated RT fidelity modulation during reverse transcription. Aim 1 will characterize the RHA-dependent fidelity enhanced hotspots in the viral genomic RNA by sequencing the cDNA synthesized in the absence and presence of host RHA. Aim 2 will characterize the RT:RHA interactions by cryo-EM, and validate by cell-based assays. The proposed studies will offer insights into the mechanism of the host helicase mediated modulation of RT pausing and fidelity at various locations of the genomic RNA during reverse transcription, and provide structural basis for the development of novel antiviral therapies.