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

基于 HIV-1 的载体预整合 DNA 上的核小体组装：组蛋白组成和翻译后修饰

• 批准号: 411422933
• 负责人: Dr. Franziska Geis
• 金额: --
• 依托单位: Department of Biochemistry and Molecular Biophysics
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/411422933?language=en
• 关键词: Nucleosome; assembly; pre; integrated; DNA

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.

本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时间和未整合逆转录病毒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。基于载体的基因递送的早期步骤类似于相应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用作基因传递载体，而且可用于研究逆转录病毒生物学。已经认识到，染色体外的、未整合的逆转录病毒DNA在几乎所有细胞中转录非常差，而整合的版本在允许细胞中稳健表达。沉默或降低转录基因表达是由宿主细胞引起的，作为对入侵病原体的保护，并通过诱导非活性染色质状态的组蛋白修饰来实现。这些转录沉默机制也限制了整合缺陷型逆转录病毒载体的有效基因表达。这些载体可以用作将逆转录病毒载体整合到有丝分裂后细胞（例如视网膜细胞或神经元）中的替代方案，或者用于瞬时基因递送目的，例如用于递送CRISPR/Cas9技术的组分。拟进行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，核小体进入核后形成的时间以及组蛋白的组成及其翻译后修饰。这一知识将导致更深入地了解逆转录病毒-宿主相互作用后，核进入前整合，并将有助于发现沉默的潜在机制。还计划对非病毒转染的DNA进行分析，以研究与病毒DNA相比，组蛋白加载和标记的任何相似性或差异。这将解决是否存在潜在的共同染色体外防御机制的问题。总之，这项研究金提案的结果将揭示核小体形成到预整合的逆转录病毒DNA的原则，也将有整合缺陷的逆转录病毒载体和非病毒转染的DNA的影响。此外，它可以定义新的，广泛使用的抗病毒防御机制。

项目成果

Unintegrated HIV-1 DNAs are loaded with core and linker histones and transcriptionally silenced

• DOI: 10.1073/pnas.1912638116
• 发表时间: 2019-11-19
• 期刊: PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
• 影响因子: 11.1
• 作者: Geis, Franziska K.;Goff, Stephen P.
• 通讯作者: Goff, Stephen P.