Assembly and structure of vaccinia virus RNA-polymerase

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Poxviruses comprise a diverse family of complex DNA-genome viruses. While many members of this family are pathogenic to mammals, birds and insects, others have also great medical benefit. This is exemplified for vaccinia virus, which not only served as a vaccine against smallpox but is also used as a promising tool in viral anti-cancer therapies. Understanding the life cycle of vaccinia virus (and other poxviruses) is hence of major importance not only for basic research but also for applied biomedical research. A key feature that distinguishes the poxvirus family from other DNA viruses is their replication cycle, which is confined to the cytoplasm. This resulted in 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, virus specific, rather than host cell enzymes mediate most processes involving DNA replication and mRNA synthesis. In this proposal, we aim to analyze the vaccinia virus RNA polymerase (vvRPO) a macromolecular machine ensuring viral gene expression in the cytosol. Although this enzyme has been studied in some details in the past years, neither its mode of assembly in vivo nor its spatio-temporal association with transcription and processing factors has been understood in detail. Furthermore, due to the lack of protocols that allow for the efficient purification of highly pure and native vvRPO, the structure of this enzyme has been elusive. To gain insight into the structure, assembly and composition of the vaccinia virus transcription system, we have recently generated virus strains expressing tagged subunits of RPO. These recombinant viruses enable the biochemical investigation of RPO assembly and its interaction with cellular and viral co-factors. Furthermore, they facilitate the native and large-scale purification of the polymerase and its functional and structural investigation.