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 - 集成 逆转录病毒复制需要将反转录的病毒DNA整合到宿主细胞染色体中。这 在稳定的核蛋白质复合物的背景下，整合酶（in）催化过程，其中包含多二聚体 在病毒DNA末端组装中，并称为中心体。体外组装的插入体的表征 来自许多逆转录病毒物种，阐明了配置和属特异性特征。慢病毒插入 在同性恋者中藏有大的含量，其中包含多达16个亚基。但是，富塔仅代表 聚集并介导细胞内贩运的前集成络合物（PIC）的一小部分 病毒感染过程中的插阿达体。此外，目前尚不清楚HIV-1 PIC与目标的接口如何 染色式化宿主细胞基因组DNA。该项目旨在表征 艾滋病毒/慢病毒集成机械使用完整的自上而下和自下而上的方法。从顶部 向下，我们将利用几种新型的亲和力试剂来丰富细胞提取物的照片，然后研究 使用晚期DNA足迹和显微镜技术，半纯化的天然HIV-1图片。实验 使用HIV-1图片将揭示内结合伙伴Ledgf/p75及其同源表观遗传学的功能 在染色质的背景下整合中的修改（H3K36me3）。通过自下而上的方法，我们将采取 来自Maedi-Visna病毒（MVV，卵巢慢病毒）的内tas体的优势，该病毒与HIV/SIV Intomes不同， 可以作为单分散制剂在体外组装。这个0.6-MDA综合体将使我们能够表征 高暂时性的慢病毒内塔和染色质之间复合物的动力学和结构 和空间决议。拟议的研究将阐明慢病毒DNA整合的结构 设备及其如何与宿主因子和染色体互动，同时避免自杀自动化。 鉴于在抑制剂中，全世界都用于治疗HIV-1的人，我们的结果将提供 生物机器的前所未有的细节，这是这些非常成功的药物的目标，可以 揭示攻击PIC进行治疗干预的新方法。