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：
10403061
负责人：
Kathleen A. Boris-Lawrie
金额：
$ 26.25万
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 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10403061
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 response Infection Investigation Length Location Maps Mediating Modeling Molecular Molecular Biology Molecular Virology Mutation Nucleotides Open Reading Frames Polymerase Prokaryotic Cells 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 Genome Virion Virus Virus Replication Work base genomic RNA 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是由于缺乏疫苗和药物的出现 HIV-1逆转录酶（RT）是负责错误的聚合酶臭名昭著的耐药突变体的出现。由于加工率低，RT停止了某些RNA序列/结构和暂停与高突变相关，重组率。在RNA调节中保持忠诚度相对较高的机制逆转录过程中的开放阅读帧中的区域却容易出错。宿主解旋酶DHX9/RNA解旋酶A（RHA）被招募到病毒中，并促进 RT。初步数据建议动态RT：逆转录过程中的RHA相互作用和揭示RHA改善了RT对结构化调节RNA区域的保真度。总体目标该提议是为RHA介导的RT保真度调制的结构基础逆转录。 AIM 1将表征RHA依赖性的保真度增强的热点通过在不存在和存在宿主存在下对cDNA进行测序，病毒基因组RNA RHA。 AIM 2将通过低温EM来表征RT：RHA相互作用，并通过基于细胞的验证测定。拟议的研究将提供有关宿主解旋介导的机制的见解在反向期间，基因组RNA的各个位置的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。