MPI Campus Seminar: Structural Basis of Transcription by Cytoplasmic Large DNA Viruses

Poxviruses belong to a family of double-stranded DNA viruses with exceptionally large genomes, which includes prominent pathogens such as Variola virus, the causative agent of smallpox, and the African Swine Fever Virus. Unlike most other viruses, these viruses encode a large multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the cytoplasm. Sequence comparisons suggested that this enzyme is related to cellular RNA polymerases, but its structure and mechanism have remained elusive. We have determined cryo-EM structures of viral transcription complexes purified directly from cells infected with Vaccinia virus, the prototype poxvirus. These structures show that the core vRNAP complex resembles eukaryotic RNA polymerase II (Pol II), but also reveal many virus-specific features. The larger complete vRNAP complex additionally contains viral transcription factors and a host tRNA molecule. This machinery can carry out the entire early viral transcription cycle, but its structure suggests that rearrangements must occur upon nucleic acid binding. To gain functional insights into this process, we further determined structures of actively transcribing vRNAP and of a co- transcriptional capping complex that contains the viral mRNA capping enzyme (CE). These structures reveal the mechanism of viral RNA synthesis and co-transcriptional transcript modification and suggest that large-scale rearrangements of the viral transcription machinery are triggered by the growing RNA during the transition from initiation to RNA capping and elongation. Together, these structures provide the first snapshots of a viral multisubunit RNA polymerase and its interactions with nucleic acids as well as virus-specific transcription factors and host factors. This provides the framework for a mechanistic understanding of gene expression by large cytoplasmic DNA viruses and may help the development of therapeutic interventions.