Nucleosome assembly on pre-integrated DNA of HIV-1-based vectors: Histone composition and post-translational modifications

这项研究计划分析基于人类免疫缺陷病毒1型(HIV-1)和小鼠白血病病毒(MLV)的逆转录病毒载体的核小体形成的时间和未整合的逆转录病毒DNA的组蛋白组成。逆转录病毒载体已成为基础研究和临床基因治疗中基因传递的有效工具。基于载体的基因传递的早期步骤类似于各自野生型逆转录病毒的早期生命周期。因此，逆转录病毒载体不仅可以作为基因载体，还可以用于逆转录病毒生物学研究。人们已经认识到，非染色体非整合型逆转录病毒DNA在几乎所有细胞中转录水平都很低，而整合型逆转录病毒DNA在允许细胞中强势表达。沉默或转录基因表达减少是由宿主细胞作为对入侵病原体的保护而引起的，通过组蛋白修饰导致染色质状态不活跃而实现。这些转录沉默机制也限制了整合缺陷逆转录病毒载体的有效基因表达。这些载体可以用作在有丝分裂后细胞(例如视网膜细胞或神经元)中整合逆转录病毒载体的替代方案，或用于瞬时基因传递目的，例如用于传递CRISPR/Cas9技术的组件。拟议的博士后项目将系统地分析核小体组装到未整合的逆转录病毒DNA上所涉及的宿主细胞机制，核进入后核小体形成的时间以及组蛋白的组成及其翻译后修饰。这一知识将有助于更深入地了解逆转录病毒在整合前进入核后与宿主的相互作用，并有助于发现沉默的潜在机制。对非病毒DNA的分析也计划调查与病毒DNA相比，组蛋白装载和标记方面的任何相似或不同之处。这将解决是否存在潜在的共同的染色体外防御机制的问题。综上所述，这项奖学金提案的发现将揭示在预先整合的逆转录病毒DNA上形成核小体的原理，并将对整合缺陷的逆转录病毒载体和非病毒转染型DNA产生影响。此外，它可能定义新的、广泛使用的抗病毒防御机制。

This fellowship proposes to analyze the timing of nucleosome formation and the histone composition of unintegrated retroviral DNA of human immunodeficiency virus type 1(HIV-1)-based and murine leukemia virus (MLV)-based retroviral vectors. Retroviral vectors have become effective tools for gene delivery in basic research as well as in clinical gene therapy. The early steps of the vector-based gene delivery resembles the early life cycle of the respective wild-type retrovirus. Therefore, retroviral vectors can be utilized not only as gene delivery vehicles but also to study retrovirus biology. It has been recognized that extrachromosomal, unintegrated retroviral DNA is very poorly transcribed in virtually all cells, whereas the integrating version is robustly expressed in permissive cells. Silencing or decreased transcriptional gene expression is caused by the host cell as a protection against invading pathogens and is achieved by histone modifications that induce an inactive chromatin state. These transcriptional silencing mechanisms also limit efficient gene expression of integration-deficient retroviral vectors. These vectors can be used as an alternative to integrating retroviral vectors in post-mitotic cells, e.g. retina cells or neurons, or for transient gene delivery purposes, e.g. for the delivery of components of the CRISPR/Cas9 technology. The proposed postdoctoral project will systematically analyze the involved host cell machinery involved in nucleosome assembly onto unintegrated retroviral DNA, the timing of nucleosome formation after nuclear entry and the histone composition and their post-translational modifications. This knowledge will lead to a deeper understanding of retrovirus-host interactions after nuclear entry before integration, and will help to discover the underlying mechanisms for silencing. Analyses with non-viral transfected DNA are also planned to investigate any similarities or differences in histone loading and marking, as compared to viral DNA. This will address the question as to whether there is a potential common extrachromosomal defense mechanism. In conclusion, the findings of this fellowship proposal will reveal the principles of nucleosome formation onto pre-integrated retroviral DNA and will have also implications for integration-deficient retroviral vectors and non-viral transfected DNA. Furthermore, it may define new, broadly utilized anti-viral defense mechanisms.