HIV-1 Genomic RNA Integrity

HIV-1 基因组 RNA 完整性

• 批准号: 10241263
• 负责人: Jeffrey M Kidd
• 金额: $ 23.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-18 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10241263
• 关键词: Applications Grants; Attenuated; Bioinformatics; Biological; Biological Assay; CRISPR interference; CRISPR library; CRISPR screen; Candidate Disease Gene; Cells; Complex; Enzymes; Exploratory/Developmental Grant; Genes; Genetic; Genetic Materials; Genetic Screening; Genome; Genomics; Goals; HIV Genome; HIV-1; High-Throughput Nucleotide Sequencing; Informatics; Lead; Lentivirus Vector; Libraries; Light; Logic; Mediating; Methodology; Methods; Molecular Profiling; Monitor; Northern Blotting; Nucleic Acids; Pathway interactions; Phenotype; Population; Preparation; Primates; Procedures; Property; Quality Control; RNA; RNA Processing; RNA Viruses; RNA-Directed DNA Polymerase; Records; Reproducibility; Research; Retroviridae; Reverse Transcriptase Polymerase Chain Reaction; Reverse Transcription; Running; Structure; Testing; Time; Virion; Virus; Virus Diseases; Virus Replication; Work; base; design; dimer; experience; experimental study; genome integrity; genomic RNA; innovative technologies; insight; mindfulness; monomer; mutant; novel; novel strategies; success; technology development; vector; viral genomics; whole genome

HIV-1 genomic RNA integrity Retroviruses are the only viruses that encapsidate two copies of their genetic material. One possible reason for this is that retroviral RNA appears to be damaged prior to reverse transcription; therefore, the presence of two copies of viral genomic RNA allows for successful reverse transcription through template switching. When virion RNA is examined on non-denaturing northern blots, it runs as a single dimer band. However, when heat- denatured to disrupt the dimer linkage, virion RNA includes a monomer band of reduced intensity compared to the dimer and also a smear of more rapidly-migrating RNAs, suggesting encapsidated RNA is extensively fragmented. Our central hypothesis is that this fragmentation of HIV-1 genomic RNA is due to the action of one or more host enzymes, the absence of which would lead to virions with more intact genomic RNAs and higher infectious titers. The objective of this proposal is to identify cellular genes that are associated with HIV- 1 RNA nicking using unbiased genetic screens and to test if these represent a new class of restriction factors. The proposed work combines Telesnitsky lab expertise in retroviral RNA with Tai lab expertise in whole- genome genetic screens for cellular factors that modulate viral infection plus Kidd lab expertise in specialized library preparation and bioinformatics, and consists of two aims: In Aim 1: we will seek to identify cellular genes that are required for observed damage to encapsidated HIV-1 RNAs using a whole-genome CRISPR library encoded in a lentiviral vector, and will validate candidate genes by using genetic depletion and rescue experiments. In Aim 2: we will assess properties of virion RNA fragmentation and examine its ramifications for viral replication. If successful, this project may reveal a new class of cellular restriction factors, may help illuminate novel cellular RNA processing pathways if the identified genes have not previously been shown to participate in RNA quality control, and may provide new insight into virion RNA structures and accessibility.

HIV-1基因组RNA完整性 逆转录病毒是唯一一种封装了两份遗传物质的病毒。一个可能的原因是 这是逆转录病毒RNA似乎在逆转录之前被破坏；因此，两种逆转录病毒的存在 病毒基因组RNA的拷贝允许通过模板切换成功地进行逆转录。什么时候 病毒粒子RNA在非变性的Northern blotts上被检测，它以单一的二聚体条带形式运行。然而，当热度- 经过变性以破坏二聚体连接，病毒粒子RNA包括一条强度降低的单体带 二聚体和更快速迁移的RNA的涂片，表明包裹的RNA广泛存在 支离破碎。我们的中心假设是，HIV-1基因组RNA的这种片断是由于 一种或多种宿主酶，缺乏这些酶将导致病毒粒子具有更完整的基因组RNA和 更高的感染性滴度。这项提议的目的是识别与艾滋病毒相关的细胞基因- 1使用无偏见的基因筛查RNA，并测试这些是否代表一类新的限制因素。 拟议的工作结合了Telesnitsky实验室在逆转录病毒RNA方面的专业知识和TAI实验室在整个- 基因组遗传筛选调节病毒感染的细胞因子，加上Kidd实验室在专业领域的专业知识 文库准备和生物信息学，由两个目标组成：目标1：我们将寻求识别细胞基因 使用全基因组CRISPR文库观察到对封装的HIV-1 RNA的损伤所需的 编码在慢病毒载体中，并将通过基因耗尽和拯救来验证候选基因 实验。在目标2中：我们将评估病毒粒子RNA片段的特性，并检查其分支 病毒复制。如果成功，这个项目可能会揭示一类新的细胞限制因素，可能会有所帮助 阐明新的细胞RNA加工途径，如果识别的基因以前没有被证明 参与RNA质量控制，并可能对病毒RNA的结构和可及性提供新的见解。

项目成果

Directed evolution of potent neutralizing nanobodies against SARS-CoV-2 using CDR-swapping mutagenesis.

• DOI: 10.1016/j.chembiol.2021.05.019
• 发表时间: 2021-09-16
• 期刊: Cell chemical biology
• 影响因子: 8.6
• 作者: Zupancic JM;Desai AA;Schardt JS;Pornnoppadol G;Makowski EK;Smith MD;Kennedy AA;Garcia de Mattos Barbosa M;Cascalho M;Lanigan TM;Tai AW;Tessier PM
• 通讯作者: Tessier PM