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Assembly and structure of vaccinia virus RNA-polymerase

痘苗病毒RNA聚合酶的组装和结构

基本信息

批准号：
315167842
负责人：
Professor Dr. Utz Fischer
金额：
--
依托单位：
Lehrstuhl für Biochemie I
依托单位国家：
德国
项目类别：
Research Grants
财政年份：
2016
资助国家：
德国
起止时间：
2015-12-31 至 2019-12-31
项目状态：
已结题

来源：
https://gepris.dfg.de/gepris/projekt/315167842?language=en
关键词：
Assembly structure vaccinia virus RNA

项目摘要

Poxviruses comprise a diverse family of complex DNA-genome viruses. While many members of this family are pathogenic to mammals, birds and insects, others have also great medical benefit. This is exemplified for vaccinia virus, which not only served as a vaccine against smallpox but is also used as a promising tool in viral anti-cancer therapies. Understanding the life cycle of vaccinia virus (and other poxviruses) is hence of major importance not only for basic research but also for applied biomedical research. A key feature that distinguishes the poxvirus family from other DNA viruses is their replication cycle, which is confined to the cytoplasm. This resulted in a high level of independence from the host cell, which supports transcription and replication events only in the nucleus (or in DNA-containing organelles). Accordingly, virus specific, rather than host cell enzymes mediate most processes involving DNA replication and mRNA synthesis. In this proposal, we aim to analyze the vaccinia virus RNA polymerase (vvRPO) a macromolecular machine ensuring viral gene expression in the cytosol. Although this enzyme has been studied in some details in the past years, neither its mode of assembly in vivo nor its spatio-temporal association with transcription and processing factors has been understood in detail. Furthermore, due to the lack of protocols that allow for the efficient purification of highly pure and native vvRPO, the structure of this enzyme has been elusive. To gain insight into the structure, assembly and composition of the vaccinia virus transcription system, we have recently generated virus strains expressing tagged subunits of RPO. These recombinant viruses enable the biochemical investigation of RPO assembly and its interaction with cellular and viral co-factors. Furthermore, they facilitate the native and large-scale purification of the polymerase and its functional and structural investigation.

痘病毒包括复杂DNA基因组病毒的不同家族。虽然该家族的许多成员对哺乳动物、鸟类和昆虫具有致病性，但其他成员也具有很大的医疗益处。牛痘病毒就是一个例子，牛痘病毒不仅可以作为天花疫苗，而且还可以作为一种有前途的病毒抗癌治疗工具。因此，了解牛痘病毒（和其他痘病毒）的生命周期不仅对基础研究而且对应用生物医学研究都具有重要意义。痘病毒家族区别于其他DNA病毒的一个关键特征是它们的复制周期，其局限于细胞质。这导致高度独立于宿主细胞，其仅在细胞核（或含DNA的细胞器）中支持转录和复制事件。因此，病毒特异性而不是宿主细胞酶介导涉及DNA复制和mRNA合成的大多数过程。在这个建议中，我们的目标是分析牛痘病毒RNA聚合酶（vvRPO），一个大分子机器，确保病毒基因在细胞质中的表达。虽然这种酶在过去的几年中已经研究了一些细节，但无论是其在体内的组装模式，还是其与转录和加工因子的时空关联，都没有被详细了解。此外，由于缺乏允许高效纯化高纯度和天然vvRPO的方案，该酶的结构一直难以捉摸。为了深入了解牛痘病毒转录系统的结构、组装和组成，我们最近产生了表达标记的RPO亚基的病毒株。这些重组病毒使得能够对RPO组装及其与细胞和病毒辅因子的相互作用进行生化研究。此外，它们促进了聚合酶的天然和大规模纯化及其功能和结构研究。