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.

逆转录病毒是专性细胞内寄生虫，其必须将其病毒基因组（cDNA）的拷贝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，该整合酶与两个病毒cDNA长末端重复序列（LTR）末端形成催化复合物，称为整合体。逆转录病毒整合体保持保守的整合体核心，其可以扩展成更高阶的IN多聚体结构。例如，原型泡沫病毒（PFV）整合体是简单的IN四聚体，而小鼠乳腺肿瘤病毒（MMTV）和劳斯肉瘤病毒（RSV）整合体是IN八聚体。对于包括HIV-1和Maedi-Visna病毒（MVV）的慢病毒，已经报道了甚至更高的IN多聚体。虽然许多生物化学和细胞研究已经详细描述了逆转录病毒整合，但不同多聚体IN结构对整合体生物物理性质的组装机制和成本效益是逆转录病毒学中的一个重大知识缺口。我们以前的工作详细的动态目标搜索，整合动力学，DNA损伤的相互作用，IN结构域的要求和核小体靶向PFV整合体。实时单分子研究也用MMTV整合体进行。在PFV四聚体和MMTV八聚体整合体之间鉴定了几个重要的差异，包括不同的靶搜索和链转移动力学以及MMTV在靶DNA上形成多价复合物的能力。这些观察结果引发了几个关键问题：决定IN多聚体结构的贡献者是什么？IN多聚体结构中影响靶点搜索和链转移的因素是什么？整合体结构如何影响染色质DNA结合和靶位点选择？PFV、MMTV、RSV和MVV整合体是用于探测整合体结构的方便的生物物理模型，因为它们天然地以IN四聚体、八聚体或16聚体的形式存在，具有公开的结构和组装方案。我们已经发现，交换连接签名保守的逆转录病毒IN蛋白N-末端结构域（NTD）、催化核心结构域（CCD）和C-末端结构域（CTD）的非保守肽，将它们转化为具有通常模拟供体整合体的多聚体结构的活性整合体。这些非保守的接头肽如何以及为什么影响整合体结构尚不清楚。我们提出利用多种高定量单分子成像工具来理解IN多聚体结构对逆转录病毒机制的贡献，具体目的如下：1.检查区分整合体结构的IN-多聚体组装和整合酶活性，2.）确定整合体结构对与限定的双链体和染色质靶DNA的动态相互作用的作用，以及3.）确定整合体结构在体内靶向宿主染色质特征上的作用。这些研究将询问IN多聚体结构和整合体动力学的贡献者，目的是确定新的逆转录病毒机制和治疗靶点。