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将其遗传物质复制两份的病毒。的一个可能原因 这是逆转录病毒RNA似乎在逆转录之前被破坏;因此，存在两个 病毒基因组RNA的拷贝允许通过模板转换成功逆转录。当 在非变性北方印迹上检测病毒体RNA，其以单一二聚体条带运行。但是，当热- 变性以破坏二聚体连接，病毒体RNA包括强度降低的单体条带， 二聚体和更快速迁移的RNA的涂片，表明多聚体化的RNA被广泛地 支离破碎我们的中心假设是，HIV-1基因组RNA的这种片段化是由于 - 一种或多种宿主酶，其缺失将导致具有更多完整基因组RNA的病毒体， 更高的感染滴度。这项提案的目的是确定与艾滋病毒有关的细胞基因， 1使用无偏遗传筛选的RNA切口，并测试这些是否代表一类新的限制因子。 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 基因组遗传筛选细胞因子，调节病毒感染加上基德实验室的专业知识， 文库制备和生物信息学，并包括两个目标：在目标1：我们将寻求确定细胞基因 使用全基因组CRISPR文库， 在慢病毒载体中编码，并将通过使用遗传耗竭和拯救来验证候选基因。 实验在目标2中：我们将评估病毒体RNA片段化的特性，并研究其对 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子， 阐明新的细胞RNA加工途径，如果所确定的基因以前没有被证明， 参与RNA质量控制，并可能提供新的见解病毒体RNA的结构和可及性。

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