Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

Abstract

Highlights d Isolation and characterization of two native Vaccinia virus transcription complexes d 2.8 Å cryo-EM structures of core and complete Vaccinia RNA polymerase (vRNAP) d The multisubunit core vRNAP resembles host Pol II with Vaccinia-specific features d Complete vRNAP integrates activities required for Vaccinia early transcription

Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes

“…To assemble vRNAP elongation complexes, we incubated vRNAP with a large excess of the pre-formed DNA-RNA scaffold after initial FLAG purification ( Figure S1A). After further purification by sucrose gradient centrifugation, two populations with distinct sedimentation coefficients could be observed, similar to the previously observed vRNAP complexes lacking nucleic acids (Figure S1B) (Grimm et al, 2019).…”

“…We purified Vaccinia vRNAP complexes as described (Grimm et al, 2019) and formed transcribing complexes on a DNA-RNA scaffold consisting of a double-stranded DNA with a mismatch bubble ( Figures S1A and 1C), a strategy previously used for the structural characterization of Pols I, II, and III (Hoffmann et al, 2015;Kettenberger et al, 2004;Neyer et al, 2016). The DNA fragment was derived from an early gene in the Vaccinia genome, encoding the largest subunit of vRNAP, Rpo147.…”

“…In the accompanying paper, we describe the purification and structural analysis of viral transcription complexes from human cells infected with a recombinant Vaccinia virus strain (Grimm et al, 2019). These studies revealed the structure of the eightsubunit core vRNAP enzyme and of the complete vRNAP complex with early viral transcription factors.…”

Transcriptome view of a killer: African swine fever virus

“…The exact origin of the NCLDV-RNAPs is still a subject of debate: it remains unclear whether they predate eukaryotic RNAPs, are derived from one of the Pols I, II, and III, or perhaps precede their divergence [21,24,27]. The transcription system of VACV is more extensively studied than ASFV [13]: the structure of VACV-RNAP has been solved by cryo-EM [28,29] and the mechanism and regulation of VACV transcription has been characterised using biochemical and NGS-based approaches [30][31][32][33][34]. ASFV utilises a more Pol II-like system compared with VACV, which lacks RNAP subunit RPB9, and TFIIB.…”

“…This diagrammatic model was drawn from PHYRE2 homology models [ 114 ] for ASFV homologs of Pol II subunits, mapped onto the Pol II structure using UCSF Chimera [ 115 ]. Homologous RNAP subunits are colour-coded and annotated, please note that the RPB3 and 11 subunits are fused in both ASFV and VACV, and ASFV-RPB7 contains an extended C-terminus, with no homology to characterised proteins [ 24 , 28 ]. Additionally, beyond canonical Pol II subunits, NCLDV-RNAPs also include, based on structurally characterised VACV-RNAP, a stably integrated mRNA capping enzyme (NP868R, highlighted in pale green) and termination factor (Q706L, in pale red) [ 28 , 29 ].…”