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

HIV-1 逆转录中 RHA:RT 相互作用的表征

基本信息

批准号：
10614580
负责人：
Kathleen A. Boris-Lawrie
金额：
$ 19.52万
依托单位：
UNIVERSITY OF MISSOURI-COLUMBIA
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2025-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10614580
关键词：
Address Affect Affinity Antiviral Therapy Binding Biological Assay Biophysics Cause of Death Cells Complement Complementary DNA Complex Cryoelectron Microscopy DNA biosynthesis Data Development Docking Double-Stranded RNA Double-Stranded RNA Binding Domain Drug resistance Elements Ensure Equilibrium Eukaryota Event Evolution Gel Genetic Recombination Genetic Variation Genome Genomic Segment Goals HIV-1 Immune Evasion Immune response Infection Investigation Length Location Maps Mediating Modeling Molecular Molecular Biology Molecular Virology Mutation Nucleotides Open Reading Frames Polymerase Prokaryotic Cells Qualifying RNA RNA Helicase RNA Sequences RNA chemical synthesis RNA helicase A RNA-Binding Proteins RNA-Directed DNA Polymerase Regulatory Element Resolution Retroviridae Reverse Transcription Ribonucleic Acid Regulatory Sequences Role Structure Transcription Process Vaccines Variant Viral Viral Genome Virion Virus Virus Replication Work genomic RNA genomic locus helicase improved insight monomer mutant novel preference pressure reconstruction recruit resistant strain structural biology targeted treatment transcriptome sequencing viral DNA viral RNA viral fitness viral genomics

项目摘要

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.

项目概要 HIV-1 感染是全球死亡的主要原因之一。新的抗病毒疗法的开发源于疫苗的缺乏和药物的出现 HIV-1 逆转录酶 (RT) 是一种容易出错的聚合酶。由于持续合成能力低，RT 出现了臭名昭著的耐药突变体。某些 RNA 序列/结构和暂停与高突变相关重组率保持 RNA 调控相对较高保真度的机制。但在逆转录过程中开放读码框中容易出错的区域我们知之甚少。宿主解旋酶 DHX9/RNA 解旋酶 A (RHA) 被招募到病毒粒子中并促进病毒粒子的持续合成 RT。初步数据表明逆转录过程中存在动态 RT：RHA 相互作用。揭示了 RHA 提高了 RT 对结构化调控 RNA 区域的保真度。该提案旨在为 RHA 介导的 RT 保真度调制建立结构基础目标 1 将表征 RHA 依赖性保真度增强的热点。通过对在宿主不存在和存在的情况下合成的 cDNA 进行测序来分析病毒基因组 RNA RHA。目标 2 将通过冷冻电镜表征 RT:RHA 相互作用，并通过基于细胞的方法进行验证拟议的研究将深入了解宿主解旋酶介导的机制。反向过程中基因组 RNA 不同位置的 RT 暂停和保真度调节转录，并为开发新型抗病毒疗法提供结构基础。