Structural and functional analysis of the intermediate and late poxviral transcription machinery

痘病毒仅在宿主细胞质中表达和复制其基因组。因此，病毒只能有限地获得用于转录、RNA成熟和DNA复制的细胞酶，这些酶大多被隔离在细胞核中。因此，痘病毒的繁殖关键取决于由多亚基RNA聚合酶（vRNAP）以及病毒特异性转录和mRNA加工/成熟因子组成的病毒编码的基因表达机制。痘病毒转录在早期、中期和晚期在时间上分开，并且依赖于将vRNAP引导至相应启动子元件的专用因子。我们最近建立了一种从牛痘病毒（原型和可遗传操纵的痘病毒）中纯化早期vRNAP复合物的策略，这使得能够对早期转录进行全面的结构观察。这些研究将多亚基vRNAP鉴定为与病毒转录和mRNA加工因子一起作用的Pol II样酶，从而实现病毒早期基因表达的独特模式。在这项资助提案中，我们的目标是建立一个生化系统，允许重建中期和晚期vRNAP转录复合物，并作为主要目标，其功能和结构表征。从这些结果中，我们不仅期望对痘病毒的转录系统有一个全面的了解，而且还期望了解它与真核转录系统的关系以及共同进化。

Poxviruses express and replicate their genomes exclusively in the cytoplasm of their hosts. Consequently, the virus has only limited access to cellular enzymes for transcription, RNA maturation and DNA replication, which are mostly sequestered in the nucleus. Poxvirus propagation therefore critically depends on a virus-encoded gene expression machinery consisting of a multi-subunit RNA polymerase (vRNAP) as well as virus-specific transcription and mRNA processing/maturation factors. Poxviral transcription is temporally separated in early, intermediate and late phases and depends on dedicated factors that direct vRNAP to the respective promoter elements. We have recently established a purification strategy for early vRNAP complexes from Vaccinia, the prototypic and genetically manipulable poxvirus, which enabled a comprehensive structural view on early transcription. These studies identified the multi-subunit vRNAP as a Pol II-like enzyme acting together with viral transcription and mRNA processing factors enabling a unique mode of viral early gene expression. In this grant proposal, we aim to establish a biochemical system allowing the reconstitution of intermediate and late vRNAP transcription complexes and, as the primary goal, their functional and structural characterization. From the results we expect not only general insight into the poxviral transcription system, but also into its relation to eukaryotic transcription systems and co-evolution.