Assembly and structure of vaccinia virus RNA-polymerase

痘病毒由一系列复杂的DNA-基因组病毒组成。虽然这个家族的许多成员对哺乳动物、鸟类和昆虫都有致病作用，但其他成员也有很大的医疗益处。痘苗病毒就是一个例子，它不仅是一种预防天花的疫苗，而且在病毒抗癌治疗中也是一种很有前途的工具。因此，了解痘苗病毒(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，它局限于细胞质。这导致了对宿主细胞的高度独立性，宿主细胞只在细胞核(或含DNA的细胞器)中支持转录和复制活动。因此，大多数涉及DNA复制和信使核糖核酸合成的过程都是由病毒特有的酶而不是宿主细胞酶来调节的。在这项提议中，我们的目标是分析痘苗病毒RNA聚合酶(VvRPO)，这是一种确保病毒基因在细胞质中表达的大分子机器。尽管在过去的几年里，人们已经对该酶进行了一些详细的研究，但对它在体内的组装方式以及它与转录和加工因子的时空关联都没有详细的了解。此外，由于缺乏有效纯化高纯度和天然vvRPO的方案，这种酶的结构一直难以捉摸。为了深入了解痘苗病毒转录系统的结构、组装和组成，我们最近产生了表达RPO标记亚单位的病毒株。这些重组病毒能够对RPO组装及其与细胞和病毒辅助因子的相互作用进行生化研究。此外，它们还促进了该聚合酶的天然和大规模纯化及其功能和结构研究。

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.