Assembly of the poxvirus transcription machinery and its activation upon infection

痘病毒只在宿主的细胞质中表达和复制它们的基因组。因此，病毒只能有限地接触到确保转录、RNA成熟和复制的细胞酶，因为这些酶隔离在细胞核中。因此，痘病毒的传播在很大程度上依赖于病毒编码的基因表达机制，该机制由多亚单位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粒子必须包含早期转录的所有成分，以便在进入细胞后立即开始基因表达。相关因子是如何组装、包装成病毒后代并在感染时被激活的，在很大程度上尚不清楚。在以前的研究中，我们已经建立了从牛痘病毒(一种典型的非致病性痘病毒)中分离纯化vRNAP复合体的策略。我们发现了一个稳定的超分子复合体(称为完整的vRNAP)，它将核心聚合酶与早期转录因子RAP94和VETF-S/L、封闭酶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，启动，延伸，mRNA封顶和体外终止。在目前的拨款建议中，我们将调查病毒感染周期中完整的vRNAP是如何组装的，以及在什么阶段组装，以及它是否构成新形成的病毒粒子中的“随时可用”单位。作为这些研究的一部分，我们还将询问病毒和宿主因素如何影响这些过程。我们期望深入了解痘病毒传播的关键方面及其在感染期间与宿主的相互作用。

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.