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 逆转录病毒是唯一封装其遗传物质副本的病毒。一个可能的原因 这就是逆转录病毒RNA在逆转录之前似乎受损。因此，存在两个 病毒基因组RNA的副本允许通过模板切换成功进行逆转录。什么时候 在非污染的北印迹上检查了病毒RNA，它作为单个二聚体带运行。但是，当热量 变性以破坏二聚体链接，Virion RNA包括与强度降低的单体带 二聚体和更快速迁移的RNA的涂片，表明封装的RNA广泛 分散。我们的核心假设是，这种HIV-1基因组RNA的碎片归因于 一种或多种宿主酶，其缺失将导致具有更完整的基因组RNA和 更高的传染性滴度。该提案的目的是确定与HIV相关的细胞基因 1使用公正的遗传筛选进行1个RNA划分，并测试它们是否代表了一类新的限制因素。 拟议的工作将逆转录病毒RNA的Telesnitsky Lab Lab专业知识与TAI LAB的专业知识结合在一起 基因组遗传筛选，用于调节病毒感染以及专业的KIDD LAB专业知识的细胞因子 图书馆的准备和生物信息学，由两个目的组成：在AIM 1：我们将寻求识别细胞基因 使用全基因组CRISPR库观察到封装的HIV-1 RNA损坏所必需的 在慢病毒载体中编码，并将通过遗传耗竭和救援来验证候选基因 实验。在AIM 2中：我们将评估病毒RNA碎片的特性，并检查其后果 病毒复制。如果成功，该项目可能会揭示新的细胞限制因素，可能会有所帮助 如果以前没有证明已鉴定的基因 参与RNA质量控制，并可能提供有关病毒RNA结构和可访问性的新见解。

项目成果

SARS-CoV-2 remodels the Golgi apparatus to facilitate viral assembly and secretion.

• DOI: 10.1101/2022.03.04.483074
• 发表时间: 2024-03-15
• 期刊: bioRxiv : the preprint server for biology
• 作者: Zhang, Jianchao;Kennedy, Andrew;Wang, Yanzhuang
• 通讯作者: Wang, Yanzhuang

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