Poxvirus transcription: Structural and functional characterization of Vaccinia virus initiation and initially transcribing complexes

痘病毒具有复杂的DNA基因组，其仅在感染的宿主细胞的细胞质中表达和复制。对痘病毒复制和基因表达的了解主要是通过分析原型痘苗病毒（VV）获得的，所述原型痘苗病毒作为针对天花的有效疫苗并且作为病毒抗癌疗法中有前途的工具。VV的独特的生命周期需要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，病毒而不是宿主因子促进牛痘复制和mRNA合成。在上一个资助期，我们能够a）生物化学表征催化活性的牛痘RNA聚合酶（vRNAP）核心酶以及B）完整的vRNAP酶复合物，其促进整个早期转录周期，包括起始、延伸、RNA加帽和终止。此外，我们c）描述了两种聚合酶复合物在2.8 nm分辨率下的冷冻电镜结构，以及延伸/加帽vRNAP在2.9 nm分辨率下的冷冻电镜结构。因此，我们的研究使详细的结构洞察痘病毒转录机制，也转录耦合mRNA修饰的机制洞察。在这些结果的基础上，我们现在的目标是开发策略，以重建和结构表征vRNAP起始复合物（即vRNAP结合到早期启动子）和vRNAP早期转录复合物（即vRNAP执行从起始到延伸模式的过渡）。我们期望深入了解痘病毒基因表达策略的关键方面，这也可能有助于理解细胞转录事件的机制。

Poxviruses possess a complex DNA genome that is expressed and replicated exclusively in the cytoplasm of infected host cells. Insight into poxvirus replication and gene expression has been obtained mostly through the analysis of the prototypic Vaccinia virus (VV), which serves as a potent vaccine against smallpox and as a promising tool in viral anti-cancer therapies. The unique live cycle of VV entails 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, viral, rather than host factors facilitate Vaccinia replication and mRNA synthesis. In the previous funding period, we were able to a) biochemically characterize the catalytically active Vaccinia RNA polymerase (vRNAP) core enzyme as well as b) a complete vRNAP enzyme complex, which facilitates the entire early transcription cycle including initiation, elongation, RNA capping and termination. Furthermore, we c) described cryo-EM structures of both polymerase complexes at 2.8 Å resolution, as well as of the elongating/capping vRNAP at 2.9 Å resolution. Our studies hence enabled detailed structural insight into the poxvirus transcription machinery and also mechanistic insight into transcription-coupled mRNA modification. Building on these results we now aim to develop strategies to reconstitute and structurally characterize vRNAP initiation complexes (i.e. vRNAP bound to an early promoter) and vRNAP early transcribing complexes (i.e. vRNAP executing the transition from initiation into the elongation mode). We expect insight into key aspects of poxvirus gene expression strategies, which may also add to the understanding of mechanisms of cellular transcription events.