Assembly of the poxvirus transcription machinery and its activation upon infection

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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 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.