Assembly of the poxvirus transcription machinery and its activation upon infection

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确保转录、RNA成熟和复制的细胞酶，因为这些酶被隔离在细胞核中。因此，痘病毒的繁殖关键取决于病毒编码的基因表达机制，该机制由多亚基RNA聚合酶（vRNAP）、转录和mRNA加工/成熟因子组成。这种病毒因子驱动的繁殖策略进一步要求病毒体必须含有用于以“随时可用”方式进行早期转录的所有组分，从而允许基因表达在进入细胞后立即开始。相关因子是如何组装、包装成病毒后代并在感染后被激活的，这在很大程度上是未知的。在以前的研究中，我们已经建立了一个纯化策略vRNAP复合物从牛痘，一个原型nonpathogenicpoxvirus。我们发现了一种稳定的超分子复合物（称为“完整的vRNAP”），它将核心聚合酶与早期转录因子Rap 94和VETF-s/l、加帽酶D1/12和终止因子NPH-I结合在一起。该单位足以执行整个早期转录周期，包括早期启动子识别、起始、延伸、mRNA加帽和体外终止。在本拨款提案中，我们将研究如何以及在病毒感染周期的哪个阶段组装完整的vRNAP，以及它是否构成纳入新形成的病毒体的“随时可用”单元。作为这些研究的一部分，我们还将询问这些过程如何受到病毒和宿主因素的影响。我们期望深入了解痘病毒传播的关键方面及其在感染过程中与宿主的相互作用。

Poxviruses express and replicate their genomes exclusively in the cytoplasm of their hosts. Consequently, the virus has only limited access to cellular enzymes ensuring transcription, RNA maturation and replication, as these are sequestered in the nucleus. Poxvirus propagation therefore critically depends on a virus-encoded gene expression machinery consisting of a multi-subunitRNA polymerase (vRNAP), transcription and mRNA processing/maturation factors. This virus-factor driven propagation strategy further entails that virions must contain all components for early transcription in a “ready-to-go” fashion, allowing gene expression to start immediately upon cell entry. How the relevant factors are assembled, packaged into viral progenies and activated upon infection is largely unknow. In previous studies we have established a purification strategy for vRNAP complexes from Vaccinia, a prototypic nonpathogenicpoxvirus. We discovered a stable supramolecular complex (termed “complete vRNAP”) that unites the core polymerase with the early transcription factors Rap94 and VETF-s/l, the capping enzyme D1/12 and the termination factor NPH-I. This unit is sufficient for the execution of the entire early transcription cycle, including early promotor recognition, initiation, elongation, mRNA capping and termination in vitro. In the present grant proposal, we will investigate how and at what stage in the viral infection cycle the complete vRNAP is assembled and whether it constitutes the “ready-to-go” unit incorporated into newly formed virions. As part of these studies, we will also ask how these processes are influenced by viral and host factors. We expect insight into key aspects of poxvirus propagation and its interplay with the host during infection.