Determinants of Architecture on Retroviral Intasome Mechanics

逆转录病毒整合体力学结构的决定因素

• 批准号: 10651141
• 负责人: Richard Fishel
• 金额: $ 47.25万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-05 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10651141
• 关键词: Acquired Immunodeficiency Syndrome; Affect; Architecture; Binding; Biochemical; Biophysics; C-terminal; Catalytic Domain; Chromatin; Chromosomes; Complementary DNA; Complex; Costs and Benefits; DNA; DNA Binding; DNA Repair; DNA lesion; Disease; Exhibits; Experimental Models; Family; Genome; Genomics; Goals; HIV-1; Histones; Imaging Device; Imaging technology; In Vitro; Infection; Integrase; Kinetics; Knowledge; Length; Lentivirus; Lesion; Link; Long Terminal Repeats; Mechanics; Modeling; Mouse Mammary Tumor Virus; N-terminal; Nucleosomes; Peptides; Post-Translational Protein Processing; Process; Productivity; Proteins; Protocols documentation; Protomer; Publishing; Reporting; Retroviridae; Retrovirology; Reverse Transcription; Role; Rous sarcoma virus; Site; Spumavirus; Structure; Tail; Testing; Time; Viral; Viral Genome; Virion; Virus Assembly; Visna-maedi virus; Work; biophysical model; biophysical properties; cellular transduction; dimer; feature detection; flexibility; genomic RNA; imaging platform; in vivo; innovation; integration site; leukemia; mammary tumor virus; obligate intracellular parasite; prototype; single molecule; therapeutic target; tool; transforming virus; viral DNA

Retroviruses are obligate intracellular parasites that must integrate a copy of their viral genome (cDNA) into a host chromosome. Integration is accomplished by the retrovirus-encoded integrase (IN) that forms a catalytic complex with two viral cDNA long terminal repeat (LTR) ends, termed an intasome. Retroviral intasomes maintain a conserved intasome core that may be expanded into higher order IN multimer architectures. For example, the prototype foamy virus (PFV) intasome is a simple IN tetramer, while the mouse mammary tumor virus (MMTV) and Rous sarcoma virus (RSV) intasomes are IN octamers. Even higher IN multimers have been reported for the lentiviruses that include HIV-1 and Maedi-Visna virus (MVV). While numerous biochemical and cellular studies have detailed retroviral integration, the assembly mechanics and cost-benefit of different multimeric IN architecture on intasome biophysical properties is a substantial knowledge gap in retrovirology. Our previous work detailed the dynamic target search, integration kinetics, DNA lesion interactions, IN domain requirements and nucleosome targeting by PFV intasomes. Real-time single molecule studies were also performed with MMTV intasomes. Several important differences were identified between the PFV tetramer and MMTV octamer intasomes including distinct target search and strand transfer kinetics as well as the ability of MMTV to form multivalent complexes on a target DNA. These observations have prompted several key questions: What are the contributors that determine IN multimeric architecture? What are the factors of IN multimeric architecture that influence target search and strand transfer? How does intasome architecture influence chromatin DNA binding and target site selection? The PFV, MMTV, RSV and MVV intasomes are convenient biophysical models for probing intasome architecture since they naturally exist as an IN tetramer, octamer or 16-mer with published structures and assembly protocols. We have found that swapping the non-conserved peptides that link the signature conserved retroviral IN protein N-terminal domain (NTD), catalytic core domain (CCD) and C-terminal domain (CTD), converts them into active intasomes with a multimeric architecture of that often mimics the donor intasome. How and why these non- conserved linker peptides influence intasome architecture is unknown. We propose to utilize multiple highly quantitative single molecule imaging tools to understand the contributions of IN multimeric architecture on retroviral mechanics with the following Specific Aims: 1.) examine IN-multimer assembly and integrase activities that distinguish intasome architectures, 2.) determine the role of intasome architecture on the dynamic interactions with defined duplex and chromatin target DNA, and 3.) determine the role of intasome architecture on targeting host chromatin features in vivo. These studies will interrogate the contributors to IN multimer architecture and intasome dynamics with the goal of identifying new retroviral mechanics and therapeutic targets.

逆转录病毒是强制性的细胞内寄生虫，必须将其病毒基因组（cDNA）的副本整合到宿主染色体中。整合是通过逆转录病毒编码的整合酶（IN）完成的，该集成酶（IN）形成具有两个病毒cDNA长末期重复（LTR）末端的催化络合物，称为中型。逆转录病毒式插入保持一个保守的内核核心，该核心可以在多聚体架构中扩展到更高阶段。例如，原型泡沫病毒（PFV）内体在四聚体中很简单，而小鼠乳腺肿瘤病毒（MMTV）和Rous肉瘤病毒（RSV）intasomes在Octamers中。据报道，包括HIV-1和MAEDI-VISNA病毒（MVV）的慢病毒中，多聚体的较高。尽管大量的生化和细胞研究详细介绍了逆转录病毒的整合，但在逆转录病毒学中，多聚体的组装力学和成本效益是构造中不同的多聚体的成本效益。我们以前的工作详细介绍了域需求和核体靶向PFV intasomes的动态靶标搜索，集成动力学，DNA病变相互作用。实时单分子研究还使用MMTV诱导症进行。在PFV四聚体和MMTV八聚体插入体之间发现了几个重要差异，包括不同的靶搜索和链传递动力学以及MMTV在靶DNA上形成多价复合物的能力。这些观察结果提出了几个关键问题：在多聚体架构中确定的贡献者是什么？在多聚体架构中影响目标搜索和链传输的因素是什么？ Intasome架构如何影响染色质DNA结合和靶位点选择？ PFV，MMTV，RSV和MVV intasomes是方便的生物物理模型，用于探测Intasome架构，因为它们自然地作为四聚体，Octamer或16-MER具有具有已发表结构和组装协议的16-MER。我们已经发现，与蛋白质N末端结构域（NTD），催化核心结构域（CCD）（CCD）和C端结构域（CTD）中的特征保守逆转录病毒联系起来的非保守肽（CTD）将其转换为具有模拟供体的多中性体系结构的活性实力。这些非保守的接头肽如何以及为什么影响Intasome架构是未知的。我们建议利用多种高度定量的单分子成像工具来理解多聚体架构对逆转录病毒力学的贡献，具有以下具体目的：1。1.）检查区分Intasome体系结构的整合体和整合酶活性，2。）2。）确定Intasement在定义的duplex架构中的动态架构的作用。染色质特征在体内。这些研究将询问多聚体架构和Intasome动态的贡献者，以识别新的逆转录病毒力学和治疗靶标。