Poxvirus transcription: Structural and functional characterization of Vaccinia virus initiation and initially transcribing complexes

• Applicant: Professor Dr. Utz Fischer
• Subject Area: Biochemistry
• Term: from 2020 to 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 445237915

Poxviruses possess a complex DNA genome that is expressed and replicated exclusively in the cytoplasm of infected host cells. Insight into poxvirus replication and gene expression has been obtained mostly through the analysis of the prototypic Vaccinia virus (VV), which serves as a potent vaccine against smallpox and as a promising tool in viral anti-cancer therapies. The unique live cycle of VV entails 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, viral, rather than host factors facilitate Vaccinia replication and mRNA synthesis. In the previous funding period, we were able to a) biochemically characterize the catalytically active Vaccinia RNA polymerase (vRNAP) core enzyme as well as b) a complete vRNAP enzyme complex, which facilitates the entire early transcription cycle including initiation, elongation, RNA capping and termination. Furthermore, we c) described cryo-EM structures of both polymerase complexes at 2.8 Å resolution, as well as of the elongating/capping vRNAP at 2.9 Å resolution. Our studies hence enabled detailed structural insight into the poxvirus transcription machinery and also mechanistic insight into transcription-coupled mRNA modification. Building on these results we now aim to develop strategies to reconstitute and structurally characterize vRNAP initiation complexes (i.e. vRNAP bound to an early promoter) and vRNAP early transcribing complexes (i.e. vRNAP executing the transition from initiation into the elongation mode). We expect insight into key aspects of poxvirus gene expression strategies, which may also add to the understanding of mechanisms of cellular transcription events.

• DFG Programme: Research Grants