Project 3. Integration

项目3. 集成

基本信息

批准号：
10653268
负责人：
Peter Cherepanov
金额：
$ 27.05万
依托单位：
UNIVERSITY OF PITTSBURGH AT PITTSBURGH
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：
2022-07-01 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10653268
关键词：
ATAC-seq Active Sites Affinity Architecture Binding Biochemical Biological Biological Assay CD4 Positive T Lymphocytes CRISPR/Cas technology Cells Chemicals Chromatin Chromosomes Code Color Complex Confocal Microscopy Creativeness Cryoelectron Microscopy DNA DNA Footprint DNA Integration Data Enzymes Epigenetic Process Fluorescence Fluorescence Microscopy Genes Genomic DNA Glass Goals HIV HIV-1 HIV-1 integrase Histone H3 Homo Human In Vitro Individual Integrase Integrase Inhibitors Integration Host Factors Knock-in Label Lentivirus Maps Mediating Medicine Microfluidics Microscopy Modeling Modification Molecular Weight Monoclonal Antibodies Mutagenesis Mutation Nucleoproteins Nucleosomes Nucleotides PWWP Domain Persons Polynucleosome Positioning Attribute Preparation Process Property Proteins Reader Reagent Recombinant Proteins Recombinants Resolution Reverse Transcription Role SIV Sheep Slide Specificity Streptavidin Structure T-Lymphocyte Techniques Testing Therapeutic Intervention Time Viral Viral Reverse Transcription Virus Diseases Virus Integration Visna-maedi virus Work deep sequencing experimental study fluorophore graphene histone methylation inhibitor insight integration site laser tweezer lentiviral integration mutant nanobodies novel optic trap optic tweezer particle single molecule suicidal trafficking transcriptional coactivator p75 viral DNA

项目摘要

Project 3 – Integration Retroviral replication requires integration of reverse-transcribed viral DNA into a host cell chromosome. This process is catalyzed by integrase (IN) in the context of the stable nucleoprotein complex, containing a multimer of IN assembled on viral DNA ends and termed the intasome. Characterization of in vitro-assembled intasomes from many retroviral species elucidated conserved as well as genus-specific features. Lentiviral intasomes harbor large IN homo-oligomers, containing as many as 16 subunits. However, the intasome represents only a small part of the pre-integration complex (PIC) that assembles and mediates intracellular trafficking of the intasome during virus infection. Furthermore, it is currently unclear how the HIV-1 PIC interfaces with its target, chromatinized host cell genomic DNA. This project aims to characterize the structure and properties of the HIV/lentiviral integration machinery using complementary top-down and bottom-up approaches. From the top- down, we will leverage several novel affinity reagents to enrich for PICs from cellular extracts and then study hemi-purified native HIV-1 PICs using advanced DNA footprinting and microscopy techniques. Experiments using HIV-1 PICs will reveal the function of the IN-binding partner LEDGF/p75 and its cognate epigenetic modification (H3K36me3) in integration in the context of chromatin. With the bottom-up approach, we will take advantage of the intasome from maedi-visna virus (MVV, an ovine lentivirus), which, unlike HIV/SIV intasomes, can be assembled in vitro as monodispersed preparations. This 0.6-MDa complex will allow us to characterize the dynamics and the structure of the complex between the lentiviral intasome and chromatin at high temporal and spatial resolutions. The proposed studies will shed light on the architecture of the lentiviral DNA integration apparatus and how it interfaces with host factors and chromosomes, while avoiding suicidal autointegration. Given that IN inhibitors are used worldwide to treat people living with HIV-1, our results will provide unprecedented details of the biological machine that is the target of these highly successful medicines and could reveal new ways to attack the PIC for therapeutic intervention.

项目 3 – 集成逆转录病毒复制需要将逆转录病毒DNA整合到宿主细胞染色体中。该过程由包含多聚体的稳定核蛋白复合物中的整合酶 (IN) 催化 IN 组装在病毒 DNA 末端，称为体外组装的 intasome 的表征。来自许多逆转录病毒物种的慢病毒嵌体阐明了保守的以及属特异性的特征。含有大量 IN 同源寡聚体，包含多达 16 个亚基，然而，整合体仅代表一个。预整合复合物 (PIC) 的一小部分，负责组装和介导细胞内转运此外，目前还不清楚HIV-1 PIC如何与其靶标相互作用。该项目旨在表征染色质化宿主细胞基因组 DNA 的结构和特性。 HIV/慢病毒整合机制采用互补的自上而下和自下而上的方法。接下来，我们将利用几种新型亲和试剂从细胞提取物中富集 PIC，然后进行研究使用先进的 DNA 足迹和显微镜实验半纯化的天然 HIV-1 PIC。使用 HIV-1 PIC 将揭示 IN 结合伴侣 LEDGF/p75 的功能及其同源表观遗传通过自下而上的方法，我们将在染色质的整合中进行修饰（H3K36me3）。利用 maedi-visna 病毒（MVV，一种绵羊慢病毒）的整合体的优势，与 HIV/SIV 整合体不同，可以在体外组装为单分散制剂，这种 0.6-MDa 复合物将使我们能够表征。慢病毒嵌体与染色质高时间复合体的动力学和结构拟议的研究将揭示慢病毒 DNA 整合的结构。装置以及它如何与宿主因子和染色体相互作用，同时避免自杀性自动整合。鉴于 IN 抑制剂在全世界范围内用于治疗 HIV-1 感染者，我们的结果将提供生物机器的前所未有的细节是这些非常成功的药物的目标，并且可以揭示了攻击 PIC 进行治疗干预的新方法。