Atomic model of a cypovirus built from cryo-EM structure provides insight into the mechanism of mRNA capping (original) (raw)
Related papers
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
Visualization of protein-RNA interactions in cytoplasmic polyhedrosis virus
Journal of virology, 1999
Unlike the multiple-shelled organization of other Reoviridae members, cytoplasmic polyhedrosis virus (CPV) has a single-shelled capsid. The three-dimensional structures of full and empty CPV by electron cryomicroscopy show identical outer shells but differ inside. The outer surface reveals a T=1 icosahedral shell decorated with spikes at its icosahedral vertices. The internal space of the empty CPV is unoccupied except for 12 mushroom-shaped densities attributed to the transcriptional enzyme complexes. The ordered double-stranded RNA inside the full capsid forms spherical shells spaced 25 A apart. The RNA-protein interactions suggest a mechanism for RNA transcription and release.
Conformational changes accompany activation of reovirus RNA-dependent RNA transcription
Journal of Structural Biology, 2008
Many critical biologic processes involve dynamic interactions between proteins and nucleic acids. Such dynamic processes are often difficult to delineate by conventional static methods. For example, while a variety of nucleic acid polymerase structures have been determined at atomic resolution, the details of how some multi-protein transcriptase complexes actively produce mRNA, as well as conformational changes associated with activation of such complexes, remain poorly understood. The mammalian reovirus innermost capsid (core) manifests all enzymatic activities necessary to produce mRNA from each of the 10 encased double-stranded RNA genes. We used rapid freezing and electron cryo-microscopy to trap and visualize transcriptionally active reovirus core particles and compared them to inactive core images. Rod-like density centered within actively transcribing core spike channels was attributed to exiting nascent mRNA. Comparative radial density plots of active and inactive core particles identified several structural changes in both internal and external regions of the icosahedral core capsid. Inactive and transcriptionally active cores were partially digested with trypsin and identities of initial tryptic peptides determined by mass spectrometry. Differentially-digested peptides, which also suggest transcription-associated conformational changes, were placed within the known 3-dimensional structures of major core proteins.
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.
Subunit Folds and Maturation Pathway of a dsRNA Virus Capsid
Structure, 2013
The cystovirus f6 shares several distinct features with other double-stranded RNA (dsRNA) viruses, including the human pathogen, rotavirus: segmented genomes, nonequivalent packing of 120 subunits in its icosahedral capsid, and capsids as compartments for transcription and replication. f6 assembles as a dodecahedral procapsid that undergoes major conformational changes as it matures into the spherical capsid. We determined the crystal structure of the capsid protein, P1, revealing a flattened trapezoid subunit with an a-helical fold. We also solved the procapsid with cryo-electron microscopy to comparable resolution. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation that is coupled to sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine.
Journal of Molecular Biology, 2003
Although double-stranded (ds) RNA viruses are a rather diverse group, they share general architectural principles and numerous functional features. All dsRNA viruses, from the mammalian reoviruses to the bacteriophage f6, including fungal viruses, share a specialized capsid involved in transcription and replication of the dsRNA genome, and release of the viral plus strand RNA. This ubiquitous capsid consists of 120 protein subunits in a so-called T ¼ 2 organization. The stringent requirements of dsRNA metabolism may explain the similarities observed in capsid architecture among a broad spectrum of dsRNA viruses. We have used cryoelectron microscopy combined with three-dimensional reconstruction techniques and complementary biophysical techniques, to determine the structure at 26 Å resolution of the Penicillium chrysogenum virus (PcV) capsid. In contrast to all previous studies of dsRNA viruses, PcV capsid is an authentic T ¼ 1 capsid with 60 equivalent protein subunits. This T ¼ 1 capsid is built with the largest structural protein (110 kDa). Structural comparison between viral particles and capsids devoid of RNA show changes along the inner surface of the capsid, mostly located around the icosahedral 5 and 3-fold axis. Considering that there may be numerous interactions between the inner surface of the protein shell and the underlying RNA, the genome could have an important role in the conformation of the structural subunits. The empty capsid structure suggests a mechanism for transcript release from actively transcribing particles. Furthermore, sequence analysis of the PcV coat protein revealed that both halves of the protein share numerous regions of similar amino acid residues. These results open new perspectives when considering the structural organization of dsRNA virus capsids.
Crystal Structure of the Avian Reovirus Inner Capsid Protein A
Journal of Virology, 2008
Avian reovirus, an important avian pathogen, expresses eight structural and four nonstructural proteins. The structural A protein is a major component of the inner capsid, clamping together A building blocks. A has also been implicated in the resistance of avian reovirus to the antiviral action of interferon by strongly binding double-stranded RNA in the host cell cytoplasm and thus inhibiting activation of the double-stranded RNA-dependent protein kinase. We have solved the structure of bacterially expressed A by molecular replacement and refined it using data to 2.3-Å resolution. Twelve A molecules are present in the P1 unit cell, arranged as two short double helical hexamers. A positively charged patch is apparent on the surface of A on the inside of this helix and mutation of either of two key arginine residues (Arg155 and Arg273) within this patch abolishes double-stranded RNA binding. The structural data, together with gel shift assay, electron microscopy, and sedimentation velocity centrifugation results, provide evidence for cooperative binding of A to double-stranded RNA. The minimal length of double-stranded RNA required for A binding was observed to be 14 to 18 bp.
The Journal of Cell Biology
Three structural forms of type 1 Lang reovirus (virions, intermediate subviral particles [ISVPs], and cores) have been examined by cryoelectron microscopy (cryoEM) and image reconstruction at 27 to 32-A resolution. Analysis of the three-dimensional maps and known biochemical composition allows determination of capsid protein location, globular shape, stoichiometry, quaternary organization, and interactions with adjacent eapsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supramolecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (,,o850-/~ diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight o3-#1, h2-or3, and h2-#l interactions in the outer capsid but also by a densely packed core shell formed primarily by M and 02. The segmented genome appears to be packed in a liquid crystalline fashion at radii < 240 A. Depending on viral growth conditions, virions undergo cleavage by enteric or endosomal/lysosomal proteases, to generate the activated ISVP (,x,800-/~ diana). This transition involves the release of an outer capsid layer spanning radii from 360 to 427 .~ that is formed by 60 tetra-w_eric and 60 hexameric clusters of ellipsoidal subunits Address reprint requests to T'tmothy S. Baker,