Structural and Functional Investigation of Poxviral Intermediate Transcription Complexes

Throughout history, poxviruses have posed a serious threat to human and animal health. The most prominent member, Variola virus, caused smallpox, a devastating disease responsible for millions of deaths before its global eradication in 1980 through vaccination. The vaccine strain, Vaccinia virus, belongs to the Orthopoxvirus genus and has become a central model for studying poxviral biology. Poxviruses are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of host cells and therefore encode their own transcriptional apparatus, including a multi-subunit DNA-dependent RNA polymerase (RNAP) and numerous transcription and RNA processing factors. Poxviral gene expression occurs in three temporally regulated phases: early, intermediate and late. While the mechanisms and factors governing early transcription are well understood, the molecular basis of intermediate transcription has remained largely unexplored. In this work, a strategy to biochemically isolate and structurally analyze poxviral intermediate transcription complexes was developed. Viral DNA replication was blocked using cytosine β-D-arabinofuranoside (AraC) to enrich products of early gene transcription. Under these conditions, a distinct viral RNA polymerase complex, termed vRNAPAraC, was isolated, consisting of all polymerase subunits, the capping enzyme (CE), and sub-stoichiometric amounts of the heterodimeric Vaccinia virus intermediate transcription factor 3 (VITF-3). By adding intermediate promoter scaffold to AraC treated cell lysate, the Vaccinia intermediate pre-initiation complex (iPIC) was formed and could be investigated by biochemical and structural means. The cryogenic electron microscopy structure revealed VITF-3 bound to the upstream promoter element, while vRNAPAraC engaged the melted DNA at the transcription start site. VITF 3, which displays homology to the eukaryotic TATA-box binding protein (TBP) and transcription factor IIB, formed a closed ring around the promoter and thus contacts the promoter DNA in a highly unconventional manner. Phylogenetic analysis based on protein sequence homology revealed that both VITF-3 subunits are conserved exclusively among Chordopoxvirinae that infect vertebrate hosts, whereas no homologs were detected in Entomopoxvirinae, which infect insects. Biochemical assays demonstrated that both vRNAPAraC and VITF-3 are required and sufficient for specific transcription from intermediate promoters in vitro. Because VITF 3 binds DNA only in the presence of vRNAPAraC, these findings support a cooperative promoter-binding mechanism in which the vRNAP-associated CE acts as molecular clamp loader that positions VITF-3 on the promoter. Moreover, both components are essential for promoter recognition and transcription initiation, which is followed by co-transcriptional m7G capping of nascent RNA and subsequent elongation. Overall, these findings provide structural and mechanistic insight into intermediate transcription in Vaccinia virus, resolving long-standing questions about the transcription factors involved and revealing a thus far unknown mode of promoter recognition and initiation. The results further support an evolutionary connection between viral and cellular transcription systems, suggesting that the TBP fold of viral transcription factors may have originated from ancestral eukaryotic proteins.