Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes

Abstract

Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m 7 G-capped mRNAs in the host cytoplasm. In the accompanying paper, we report structures of core and complete vRNAP complexes of the prototypic Vaccinia poxvirus (Grimm et al., 2019; in this issue of Cell). Here, we present the cryo-electron microscopy (cryo-EM) structures of Vaccinia vRNAP in the form of a transcribing elongation complex and in the form of a co-transcriptional capping complex that contains the viral capping enzyme (CE). The trifunctional CE forms two mobile modules that bind the polymerase surface around the RNA exit tunnel. RNA extends from the vRNAP active site through this tunnel and into the active site of the CE triphosphatase. Structural comparisons suggest that growing RNA triggers large-scale rearrangements on the surface of the transcription machinery during the transition from transcription initiation to RNA capping and elongation. Our structures unravel the basis for synthesis and co-transcriptional modification of poxvirus RNA.

Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes

“…The structures reveal similarities and differences between the viral cytoplasmic transcription apparatus and the nuclear RNA polymerase machinery. Our results form the basis for unraveling the molecular mechanisms of poxvirus gene transcription and RNA processing and enabled structure determination of functional vRNAP complexes reported in the accompanying paper (Hillen et al, 2019).…”

“…Although the function of the Rpo30 C-tail remains unknown, structural superposition with a Pol II elongation complex (Gnatt et al, 2001) shows that it would interfere with binding of the DNA-RNA hybrid and thus impair formation of a transcribing complex. In the accompanying paper (Hillen et al, 2019), we show that the DNA-RNA hybrid indeed binds at the expected position and would clash with the Rpo30 C-tail. This indicates that the Rpo30 C-tail must be displaced for transcription.…”

Transcriptome view of a killer: African swine fever virus

“…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].…”

“…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.…”

Transcripts of vaccinia virus postreplicative genes do not contain a 5’ methylguanosine cap

“…VACV-encoded RNA polymerase is a multisubunit enzyme (8) that exists in two distinct forms that are specific for transcription of early and postreplicative genes (9). The 5' ends of early and postreplicative VACV transcripts are traditionally considered to be protected by the 7-methygluanosine cap structure (m 7 G) (10,11), which is synthesized by the VACV mRNA capping enzyme (12,13). The promoter composition and transcription regulation of VACV intermediate and late genes differ from those of early genes.…”