Determinants of Architecture on Retroviral Intasome Mechanics

逆转录病毒是专性的细胞内寄生虫，必须将其病毒基因组(CDNA)的副本整合到宿主染色体中。整合是由逆转录病毒编码的整合酶(IN)完成的，它与病毒的两个长末端重复序列(LTR)形成催化复合体，称为内切体。逆转录病毒内含体维持着一个保守的内切体核心，该核心可以在多聚体结构中扩展到更高的顺序。例如，泡沫状病毒(PFV)的原型是一个简单的IN四聚体，而小鼠乳腺肿瘤病毒(MMTV)和劳斯肉瘤病毒(RSV)的内含体是IN八聚体。据报道，包括HIV-1和Maedi-Visna病毒(MVV)在内的慢病毒具有更高的IN多聚体。虽然许多生化和细胞研究都有详细的逆转录病毒整合，但不同多聚体IN的组装机制和成本效益对内合体生物物理特性的影响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介绍了动态靶标搜索、整合动力学、DNA损伤相互作用、IN结构域要求和PFV内切体靶向核小体。实时单分子研究也用MMTV插入体进行。在PFV四聚体和MMTV八聚体之间发现了几个重要的差异，包括不同的靶标搜索和链转移动力学，以及MMTV在靶DNA上形成多价复合体的能力。这些观察引发了几个关键问题：决定多层体系结构的因素是什么？影响目标搜索和链转移的IN多聚体结构的因素是什么？端粒体结构如何影响染色质DNA结合和靶点选择？由于PFV、MMTV、RSV和MVV四聚体以IN四聚体、八聚体或16-聚体的形式存在，其结构和组装协议已公布，因此它们是探测连接体结构的方便的生物物理模型。我们发现，交换连接特征保守的逆转录病毒IN蛋白N末端结构域(NTD)、催化核心域(CCD)和C末端结构域(CTD)的非保守肽，可以将它们转化为具有多聚体结构的活性内聚体，这种多聚体结构通常模仿供体内聚体。目前尚不清楚这些非保守的连接肽是如何以及为什么影响内涵体结构的。我们建议利用多种高定量的单分子成像工具来了解IN多聚体结构对逆转录病毒机制的贡献，具体目的如下：1)检查区分内切酶结构的In-多聚体组装和整合酶活性，2.确定端粒体结构在与已定义的双链和染色质靶DNA动态相互作用中的作用，以及3.确定内含体结构在体内靶向宿主染色质特征方面的作用。这些研究将询问IN多聚体结构和连接体动力学的贡献者，目的是确定新的逆转录病毒机制和治疗靶点。

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.