Project 3. Integration

项目3. 集成

• 批准号: 10506954
• 负责人: Peter Cherepanov
• 金额: $ 24.49万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 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/10506954
• 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; Color; Complex; Confocal Microscopy; 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; Light; 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; Role; SIV; Sheep; Slide; Specificity; Streptavidin; Structure; T-Lymphocyte; Techniques; Testing; Therapeutic Intervention; Time; Viral; Virus Diseases; Virus Integration; Visna-maedi virus; Work; deep sequencing; experimental study; fluorophore; graphene; inhibitor; insight; integration site; laser tweezer; lentiviral integration; mutant; nanobodies; novel; optical traps; particle; single molecule; suicidal; trafficking; transcriptional coactivator p75; viral DNA

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 -一体化 逆转录病毒复制需要将逆转录的病毒DNA整合到宿主细胞染色体中。这 该过程是由整合酶（IN）在稳定的核蛋白复合物中催化的，该复合物含有一个多聚体 IN组装在病毒DNA末端，称为整合体。体外组装的整合体的表征 从许多逆转录病毒物种中阐明了保守的以及属特异性特征。慢病毒包涵体 具有大的IN同源寡聚体，含有多达16个亚基。然而，intasome只代表一个 前整合复合物（PIC）的一小部分，组装并介导细胞内转运的 在病毒感染过程中。此外，目前还不清楚HIV-1 PIC如何与其靶标相互作用， 染色质化的宿主细胞基因组DNA。本项目的目的是表征的结构和性能的 使用互补的自上而下和自下而上方法的HIV/慢病毒整合机制。从头开始- 接下来，我们将利用几种新型的亲和试剂从细胞提取物中富集PIC，然后研究 使用先进的DNA足迹法和显微镜技术半纯化的天然HIV-1 PIC。实验 使用HIV-1 PIC将揭示IN结合伴侣LEDGF/p75及其同源表观遗传的功能， 修饰（H3 K36 me 3）在染色质整合中的作用。采用自下而上的方法，我们将 来自Maedi-visna病毒（MVV，一种绵羊慢病毒）的整合体的优点，与HIV/SIV整合体不同， 可以在体外组装成单分散制剂。这个0.6-MDa的复合物将使我们能够表征 慢病毒整合体与染色质复合物在高时相的动力学和结构 空间分辨率。这些研究将有助于阐明慢病毒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，同时避免自杀性自整合。 鉴于IN抑制剂在世界范围内用于治疗HIV-1感染者，我们的研究结果将提供 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。