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

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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.