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，仅在受感染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洞察主要是通过对原型痘苗病毒(VV)的分析获得的，VV是一种有效的天花疫苗，也是一种有望用于病毒抗癌治疗的工具。VV独特的生命周期需要高度独立于宿主细胞，宿主细胞只支持细胞核(或含DNA的细胞器)的转录和复制活动。因此，病毒而不是宿主因素促进了痘苗病毒的复制和mRNA的合成。在之前的资助期间，我们能够a)对具有催化活性的Vaccinia RNA聚合酶(VRNAP)核心酶进行生化表征，以及b)完整的vRNAP酶复合体，它促进了整个早期转录周期，包括起始、延伸、RNA封顶和终止。此外，我们c)描述了两个聚合酶复合体在2.8？分辨率下的冷冻-EM结构，以及在2.9？分辨率下延长/封顶vRNAP的结构。因此，我们的研究使我们能够对痘病毒转录机制进行详细的结构洞察，并从机制上洞察转录偶联的mRNA修饰。在这些结果的基础上，我们现在的目标是开发策略来重组和结构表征vRNAP起始复合体(即与早期启动子结合的vRNAP)和vRNAP早期转录复合体(即vRNAP执行从起始到延伸模式的转变)。我们期待对痘病毒基因表达策略的关键方面的洞察，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.