Structural Basis of Poxvirus Transcription: Transcribing and Capping Vaccinia Complexes (original) (raw)
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Structural Basis of Poxvirus Transcription: Vaccinia RNA Polymerase Complexes
Cell, 2019
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
Structure, 2014
Vaccinia virus capping enzyme is a heterodimer of D1 (844-aa) and D12 (287-aa) polypeptides that executes all three steps in m 7 GpppRNA synthesis. The D1 subunit comprises an N-terminal RNA triphosphatase (TPase)-guanylyltransferase (GTase) module and a C-terminal guanine-N7methyltransferase (MTase) module. The D12 subunit binds and allosterically stimulates the MTase module. Crystal structures of the complete D1•D12 heterodimer disclose the TPase and GTase as members of the triphosphate tunnel metalloenzyme and covalent nucleotidyltransferase superfamilies, respectively, albeit with distinctive active site features. An extensive TPase-GTase interface clamps the GTase nucleotidyltransferase and OB domains in a closed conformation around GTP. Mutagenesis confirms the importance of the TPase-GTase interface for GTase activity. The D1•D12 structure complements and rationalizes four decades of biochemical studies of this enzyme (the first capping enzyme to be purified and characterized) and provides new insights to the origins of the capping systems of other large DNA viruses.
Structure of the poxvirus decapping enzyme D9 reveals its mechanism of cap recognition and catalysis
2021
SUMMARYPoxviruses encode decapping enzymes that remove the protective 5’ cap from both host and viral mRNAs to commit transcripts for decay by the cellular exonuclease Xrn1. Decapping by these enzymes is critical for poxvirus pathogenicity by means of simultaneously suppressing host protein synthesis and limiting the accumulation of viral dsRNA, a trigger for antiviral responses. Here we present the first high resolution structural view of the vaccinia virus decapping enzyme D9. This Nudix enzyme contains a novel domain organization in which a three-helix bundle is inserted into the catalytic Nudix domain. The 5’ mRNA cap is positioned in a bipartite active site at the interface of the two domains. Specificity for the methylated guanosine cap is achieved by stacking between conserved aromatic residues in a manner similar to that observed in canonical cap binding proteins VP39, eIF4E, and CBP20 and distinct from eukaryotic decapping enzyme Dcp2.
Structure, 2003
The capsid shells of these viruses, however, exhibit striking architectural differences. Except for the single-Baylor College of Medicine Houston, Texas 77030 shelled cypoviruses such as the cytoplasmic polyhedrosis virus (CPV), all other viruses in the Reoviridae have 3 State Key Lab for Biocontrol Institute of Entomology additional protein shells, such as the double-shelled rice dwarf virus (RDV) (Lu et al., 1998), and triple-shelled Zhongshan University Guangzhou 510275 rotavirus (Shaw et al., 1993) and bluetongue virus (BTV) (Grimes et al., 1998). In addition to conferring host speci-China ficity and mediating cell entry, these additional layers are believed to play important structural roles in maintaining the stability of the thin inner shell and sequestering the Summary dsRNA genome (Lawton et al., 2000). The inner shells of the Reoviridae are more homogenous and can be The single-shelled cytoplasmic polyhedrosis virus divided into two major groups. Those in the first group (CPV) is a unique member of the Reoviridae. Despite have a smooth inner shell made up of 120 CSP molecules lacking protective outer shells, it exhibits striking capenclosed by one or two outer T ϭ 13 layers, as exemplisid stability and is capable of endogenous RNA tranfied by BTV, RDV, and rotavirus. Those in the second scription and processing. The 8 Å three-dimensional group also have an inner shell consisting of 120 CSP structure of CPV by electron cryomicroscopy reveals molecules, but this shell is decorated by turrets (the secondary structure elements present in the capsid mRNA capping complexes) on the icosahedral vertices proteins CSP, LPP, and TP, which have ␣ϩ folds. The and by molecular clamps (large protrusions) joining extensive nonequivalent interactions between CSP neighboring CSP molecules. In addition, these viruses and LPP, the unique CSP protrusion domain, and the either have incomplete outer T ϭ 13 layers (e.g., orthoperfect inter-CSP surface complementarities may acreovirus [Dryden et al., 1993; Reinisch et al., 2000] and count for the enhanced capsid stability. The slanted aquareovirus [Shaw et al., 1996]) or completely lack any disposition of TP functional domains and the stacking outer protein layer (e.g., CPV [Hill et al., 1999; Xia et of channel constrictions suggest an iris diaphragmal., 2003; Zhang et al., 1999]). In these viruses, mRNA like mechanism for opening/closing capsid pores and transcription and posttranscriptional processing take turret channels in regulating the highly coordinated place in a series of well-coordinated steps, beginning steps of mRNA transcription, processing, and release. with mRNA transcription at the transcriptional enzyme complexes underneath the vertices of the inner shell, Introduction followed by 5Ј end mRNA capping and subsequent release through the multifunctional turret (Bartlett et al., RNA transcription is a fundamental process involving a 1974; Bellamy and Harvey, 1976; Furuichi, 1974; Furuichi series of well-coordinated processes catalyzed by multiet al., 1976; Reinisch et al., 2000; White and Zweerink, functional enzymes, often embedded in multicompo-1976; Xia et al., 2003; Yazaki and Miura, 1980; Zhang et nent macromolecular complexes. Double-stranded (ds) al., 1999). RNA viruses in the family Reoviridae are extreme exam-Having only a single shell, CPV is structurally the simples of such multifunctional RNA transcriptional maplest member of the Reoviridae. Despite lacking the chines. Their hosts include plants, insects, mammals, outer protective layers existing in other dsRNA viruses, and humans, and their structural proteins have little to CPV virions are resistant to chemical treatments, includno recognizable sequence homologies (reviewed by ing cations, high pH, trypsin, chymotrypsin, ribo-Mertens et al., 2000). Still, viruses in the nine genera of nuclease A, deoxyribonuclease, phospholipase, and this family all contain a characteristic segmented dsRNA SDS, and retain infectivity for weeks at Ϫ15ЊC to 25ЊC genome and a highly conserved dsRNA-dependent sin-(Mertens et al., 2000; Zhang et al., 2002). The relative gle-stranded RNA polymerase enclosed in a capsid shell simplicity and unusual stability of CPV make it an attracmade up of 120 molecules of the inner capsid shell tive system for studying the structural basis of RNA protein (CSP) (reviewed by Lawton et al., 2000; Nibert transcription and posttranscriptional processing. While and Schiff, 2001; Patton and Spencer, 2000). The Reovirits infection of silkworms can have a negative economic idae are all capable of endogenous mRNA transcription impact in Asia, CPV is also recognized as an emerging within an intact virus particle, using viral-encoded enbiocontrol agent, serving as an environmentally friendly zymes for transcription initiation, elongation, 5Ј capping, pesticide for fruit and vegetable farming (Mertens et al., 2000). Previous low resolution electron cryomicroscopy (cryoEM) structures showed that CPV shares similar *Correspondence: z.h.zhou@uth.tmc.edu
Proceedings of the National Academy of Sciences, 2011
The cytoplasmic polyhedrosis virus (CPV) from the family Reoviridae belongs to a subgroup of "turreted" reoviruses, in which the mRNA capping activity occurs in a pentameric turret. We report a full atomic model of CPV built from a 3D density map obtained using cryoelectron microscopy. The image data for the 3D reconstruction were acquired exclusively from a CCD camera. Our structure shows that the enzymatic domains of the pentameric turret of CPV are topologically conserved and that there are five unique channels connecting the guanylyltransferase and methyltransferase regions. This structural organization reveals how the channels guide nascent mRNA sequentially to guanylyltransferase, 7-Nmethyltransferase, and 2′-O-methyltransferase in the turret, undergoing the highly coordinated mRNA capping activity. Furthermore, by fitting the deduced amino acid sequence of the protein VP5 to 120 large protrusion proteins on the CPV capsid shell, we confirmed that this protrusion protein is encoded by CPV RNA segment 7.
2020
ABSTRACTBunyavirales is an order of segmented negative stranded RNA viruses comprising several life-threatening pathogens such as Lassa fever virus (Arenaviridae), Rift Valley Fever virus (Phenuiviridae) and La Crosse virus (LACV, Peribunyaviridae) against which neither specific treatment nor licenced vaccine is available. Replication and transcription of Bunyavirales genome constitute essential reactions of their viral cycle that are catalysed by the virally encoded RNA-dependent RNA polymerase or L protein. Here we describe the complete high-resolution cryo-EM structure of the full-length (FL) LACV-L protein. It reveals the presence of key C-terminal domains, notably the cap-binding domain that undergoes large movements related to its role in transcription initiation and a zinc-binding domain that displays a fold not previously observed. We capture the structure of LACV-L FL in two functionally relevant states, pre-initiation and elongation, that reveal large conformational change...
Site-specific RNA cleavage generates the 3' end of a poxvirus late mRNA
Proceedings of the National Academy of Sciences of the United States of America, 1992
The cowpox virus late mRNAs encoding the major protein of the A-type inclusions have 3' ends corresponding to a single site in the DNA template. The DNA sequence of the Alu I-Xba I fragment at this position encodes an RNA cis-acting signal, designated the AX element, which directs this RNA 3' end formation. In cells infected with vaccinia virus the AX element functions independently of either the nature of the promoter element or the RNA polymerase responsible for generating the primary RNA. At late times during virus replication, vaccinia virus induces or activates a site-specific endoribonuclease that cleaves primary RNAs within the AX element. The 3' end produced by RNA cleavage is then polyadenylylated to form the 3' end of the mature mRNA. Therefore, the poxviruses employ at least two mechanisms of RNA 3' end formation during the viral replication cycle. One mechanism, which is operative at early times in viral replication, involves the termination of transc...
Towards a structural understanding of RNA synthesis by negative strand RNA viral polymerases
Current opinion in structural biology, 2016
Negative strand RNA viruses (NSVs), which may have segmented (sNSV) or non-segmented genomes (nsNSV) are responsible for numerous serious human infections such as Influenza, Measles, Rabies, Ebola, Crimean Congo Haemorrhagic Fever and Lassa Fever. Their RNA-dependent RNA polymerases transcribe and replicate the nucleoprotein coated viral genome within the context of a ribonucleoprotein particle. We review the first high resolution crystal and cryo-EM structures of representative NSV polymerases. The heterotrimeric Influenza and single-chain La Crosse orthobunyavirus polymerase structures (sNSV) show how specific recognition of both genome ends is achieved and is required for polymerase activation and how the sNSV specific 'cap-snatching' mechanism of transcription priming works. Vesicular Stomatitis Virus (nsNSV) polymerase shows a similar core architecture but has different flexibly linked C-terminal domains which perform mRNA cap synthesis. These structures pave the way fo...
Cryo-EM structure of the respiratory syncytial virus RNA polymerase
Nature Communications, 2020
The respiratory syncytial virus (RSV) RNA polymerase, constituted of a 250 kDa large (L) protein and tetrameric phosphoprotein (P), catalyzes three distinct enzymatic activities — nucleotide polymerization, cap addition, and cap methylation. How RSV L and P coordinate these activities is poorly understood. Here, we present a 3.67 Å cryo-EM structure of the RSV polymerase (L:P) complex. The structure reveals that the RNA dependent RNA polymerase (RdRp) and capping (Cap) domains of L interact with the oligomerization domain (POD) and C-terminal domain (PCTD) of a tetramer of P. The density of the methyltransferase (MT) domain of L and the N-terminal domain of P (PNTD) is missing. Further analysis and comparison with other RNA polymerases at different stages suggest the structure we obtained is likely to be at an elongation-compatible stage. Together, these data provide enriched insights into the interrelationship, the inhibitors, and the evolutionary implications of the RSV polymerase.
Structure of a hexameric RNA packaging motor in a viral polymerase complex
Journal of Structural Biology, 2007
Packaging of the Cystovirus 8 genome into the polymerase complex is catalysed by the hexameric P4 packaging motor. The motor is located at the Wvefold vertices of the icosahedrally symmetric polymerase complex, and the symmetry mismatch between them may be critical for function. We have developed a novel image-processing approach for the analysis of symmetry-mismatched structures and applied it to cryo-electron microscopy images of P4 bound to the polymerase complex. This approach allowed us to solve the threedimensional structure of the P4 in situ to 15-Å resolution. The C-terminal face of P4 was observed to interact with the polymerase complex, supporting the current view on RNA translocation. We suggest that the symmetry mismatch between the two components may facilitate the ring opening required for RNA loading prior to its translocation.