RESUMEN
Poxviruses use virus-encoded multisubunit RNA polymerases (vRNAPs) and RNA-processing factors to generate m7G-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.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Metiltransferasas/química , Complejos Multienzimáticos/química , Nucleotidiltransferasas/química , Monoéster Fosfórico Hidrolasas/química , Virus Vaccinia/ultraestructura , Proteínas Virales/química , Microscopía por Crioelectrón , Complejos Multienzimáticos/ultraestructura , ARN Mensajero/química , Imagen Individual de Molécula , Transcripción Genética , Virus Vaccinia/genética , Virus Vaccinia/metabolismoRESUMEN
Poxviruses encode a multisubunit DNA-dependent RNA polymerase (vRNAP) that carries out viral gene expression in the host cytoplasm. We report cryo-EM structures of core and complete vRNAP enzymes from Vaccinia virus at 2.8 Å resolution. The vRNAP core enzyme resembles eukaryotic RNA polymerase II (Pol II) but also reveals many virus-specific features, including the transcription factor Rap94. The complete enzyme additionally contains the transcription factor VETF, the mRNA processing factors VTF/CE and NPH-I, the viral core protein E11, and host tRNAGln. This complex can carry out the entire early transcription cycle. The structures show that Rap94 partially resembles the Pol II initiation factor TFIIB, that the vRNAP subunit Rpo30 resembles the Pol II elongation factor TFIIS, and that NPH-I resembles chromatin remodeling enzymes. Together with the accompanying paper (Hillen et al., 2019), these results provide the basis for unraveling the mechanisms of poxvirus transcription and RNA processing.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/química , Factores de Transcripción/química , Virus Vaccinia/ultraestructura , Proteínas Virales/química , Microscopía por Crioelectrón , Complejos Multienzimáticos/química , Complejos Multienzimáticos/ultraestructura , Imagen Individual de Molécula , Virus Vaccinia/genética , Virus Vaccinia/metabolismoRESUMEN
In eukaryotic cells, the process of gene expression is confined to the nucleus and enabled by multisubunit RNA polymerases (RNAPs). Many viruses make use of the host cellular gene expression apparatus during infection, and hence transfer their genome at least transiently to the host nucleus. However, poxviruses have evolved a different strategy to propagate. Their double-stranded DNA genome is transcribed in the host cytoplasm by a virus-encoded RNAP (vRNAP), which is evolutionarily related to eukaryotic RNA polymerase II. In this Review, we highlight recent high-resolution structures of the poxviral transcription apparatus in different phases of action. These structures, along with biochemical data, now allow the definition of a comprehensive model of poxviral gene expression and its regulation.
Asunto(s)
Poxviridae , Núcleo Celular/genética , Citoplasma/genética , ARN Polimerasas Dirigidas por ADN/química , Expresión Génica , Poxviridae/genética , ARN Polimerasa II/genética , Transcripción GenéticaRESUMEN
The functional and structural characterization of macromolecular complexes requires protocols for their native isolation. Here, we describe a protocol for this task based on the recombinant poxvirus Vaccinia expressing tagged proteins of interest in infected cells. Tagged proteins and their interactors can then be isolated via affinity chromatography. The procedure is illustrated for the Vaccinia virus encoded multi-subunit RNA polymerase. Our protocol also allows the expression and isolation of heterologous proteins and hence is suitable for a broader application. For complete details on the use and execution of this profile, please refer to Grimm et al. (2019).
Asunto(s)
ARN Polimerasas Dirigidas por ADN , Proteínas , Cromatografía de Afinidad/métodos , Indicadores y Reactivos , Sustancias Macromoleculares , Virus Vaccinia/genéticaRESUMEN
Members of the Poxviridae family are large double-stranded DNA viruses that replicate exclusively in the cytoplasm of their hosts. This goes in hand with a high level of independence from the host cell, which supports transcription and replication events only in the nucleus or in DNA-containing organelles. Consequently, virus specific, rather than cellular enzymes mediate most processes involving DNA replication and mRNA synthesis. Recent technological advances allowed a detailed functional and structural investigation of the transcription machinery of the prototypic poxvirus vaccinia. The DNA-dependent RNA polymerase (RNAP) at its core displays distinct similarities to eukaryotic RNAPs. Strong idiosyncrasies, however, are apparent for viral factors that are associated with the viral RNAP during mRNA production. We expect that future studies will unravel more key aspects of poxvirus gene expression, helping also the understanding of nuclear transcription mechanisms.
Asunto(s)
Poxviridae , Proteínas Virales , ARN Polimerasas Dirigidas por ADN , Poxviridae/genética , Transcripción Genética , Proteínas Virales/genética , Replicación ViralRESUMEN
Poxviruses express their genes in the cytoplasm of infected cells using a virus-encoded multi-subunit polymerase (vRNAP) and unique transcription factors. We present cryo-EM structures that uncover the complete transcription initiation phase of the poxvirus vaccinia. In the pre-initiation complex, the heterodimeric early transcription factor VETFs/l adopts an arc-like shape spanning the polymerase cleft and anchoring upstream and downstream promoter elements. VETFI emerges as a TBP-like protein that inserts asymmetrically into the DNA major groove, triggers DNA melting, ensures promoter recognition and enforces transcription directionality. The helicase VETFs fosters promoter melting and the phospho-peptide domain (PPD) of vRNAP subunit Rpo30 enables transcription initiation. An unprecedented upstream promoter scrunching mechanism assisted by the helicase NPH-I probably fosters promoter escape and transition into elongation. Our structures shed light on unique mechanisms of poxviral gene expression and aid the understanding of thus far unexplained universal principles in transcription.