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

项目摘要 HIV-1感染是全球死亡的主要原因之一。迫切需要 新的抗病毒疗法的发展源于疫苗的缺乏和药物的出现， 耐药菌株HIV-1逆转录酶（RT）是一种易出错的聚合酶， 臭名昭著的抗药性突变体的出现由于持续合成能力较低，RT在 某些RNA序列/结构和暂停与高突变相关， 重组率在RNA调控中保持相对较高保真度的机制 但在逆转录过程中开放阅读框中易于出错的区域的了解很少。 宿主解旋酶DHX 9/RNA解旋酶A（RHA）被募集到病毒体中并促进病毒的持续合成能力。 RT.初步数据表明在逆转录过程中动态RT：RHA相互作用， 揭示RHA提高了RT对结构化调控RNA区域的保真度。的总目标 这一建议是建立RHA介导的RT保真度调制的结构基础， 逆转录目标1将描述RHA依赖的保真度增强的热点， 通过对在宿主存在和不存在的情况下合成的cDNA进行测序， RHA。目的2将通过冷冻电镜表征RT：RHA相互作用，并通过基于细胞的 测定。这些研究将为深入了解宿主解旋酶介导的细胞凋亡机制提供新的思路。 逆转录过程中基因组RNA不同位置的RT暂停和保真度的调节 转录，并为开发新的抗病毒疗法提供结构基础。

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.