Conformational changes accompany activation of reovirus RNA-dependent RNA transcription (original) (raw)
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Virology, 2002
The 144-kDa 2 protein, a component of the transcriptionally active reovirus core particle, catalyzes the last three enzymatic activities for formation of the 5Ј cap 1 structure on the viral plus-strand transcripts. Limited evidence suggests it may also play a role in transcription per se. Particle-associated 2 forms pentameric turrets ("spikes") around the fivefold axes of the icosahedral core. To address the requirements for 2 in core functions other than the known functions in RNA capping, particles depleted of 2 were generated from cores in vitro by a series of treatments involving heat, protease, and ionic detergent. The resulting particles contained less than 5% of pretreatment levels of 2 but showed negligible loss of the other four core proteins or the 10 double-stranded RNA genome segments. Transmission cryo-electron microscopy (cryo-TEM) and scanning cryo-electron microscopy demonstrated loss of the 2 spikes from these otherwise intact particles. In functional analyses, the "spikeless cores" showed greatly reduced activities not only for RNA capping but also for transcription and nucleoside triphosphate hydrolysis, suggesting enzymatic or structural roles for 2 in all these activities. Comparison of the core and spikeless core structures obtained by cryo-TEM and three-dimensional image reconstruction revealed changes in the 1 core shell that accompany 2 loss, most notably the elimination of small pores that span the shell near the icosahedral fivefold axes. Changes in the shell may explain the reductions in transcriptase-related activities by spikeless cores.
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.
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.
RNA Synthesis in a Cage—Structural Studies of Reovirus Polymerase λ3
Cell, 2002
Studies of Reovirus Polymerase 3 redoxin molecule (Doublie et al., 1998)-that provides a barrier to loss of template:primer. DNA replicases associate with a so-called sliding-clamp assembly (Stukenberg et al., 1991), which encircles a DNA strand when Department of Molecular and Cellular Biology loaded by an ATP-dependent loading enzyme and which Harvard University produces an extremely processive association. mRNA Cambridge, Massachusetts 02138 synthases, such as RNA polymerase II (Gnatt et al., 2001) 2 Cellular and Molecular Biology Program and E. coli DNA-dependent RNA polymerase (Murakami Institute for Molecular Virology et al., 2002), contain a central polymerase site sur-University of Wisconsin-Madison rounded by elaborations to guide template and product Madison, Wisconsin 53706 in and out of the catalytic cleft. 3 Department of Microbiology and Molecular The 3 polymerase of reoviruses (Starnes and Joklik, Genetics 1993) and its homologs in other double-strand RNA vi-Harvard Medical School ruses function within the confined interior of a viral core Boston, Massachusetts 02115 particle (Gillies et al., 1971; Shatkin and Sipe, 1968; Skehel and Joklik, 1969). For members of the Reoviridae family of dsRNA viruses, core assembly requires recruit-Summary ment of one capped, plus-sense strand for each of the 10-12 genomic segments, along with a roughly equal The reovirus polymerase and those of other dsRNA number of RNA-dependent RNA polymerase complexes viruses function within the confines of a protein capsid (Nibert and Schiff, 2001)
Journal of Cell Biology, 1993
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 capsid proteins. Comparisons of the virion, ISVP and core structures and examination of difference maps reveal dramatic changes in supra-molecular structure and protein conformation that are related to the early steps of reovirus infection. The intact virion (approximately 850-A diam) is designed for environmental stability in which the dsRNA genome is protected not only by tight sigma 3-mu 1, lambda 2-sigma 3, and lambda 2-mu 1 interactions in the outer capsid but also by a densely packed core shell formed primarily by lambda 1 and sigma 2. The segmented genome appe...
Journal of Biological Chemistry, 1983
Polypeptides of M, = 50,000 and 80,000 in rabbit reticulocyte initiation factor preparations can be specifically cross-linked to the oxidized 5' cap structure of native reovirus mRNA in an ATP-M%'-dependent manner (Sonenberg, N., Guertin, D., Cleveland, D., and Trachsel, H. (1981) Cell 27,563-572). However, specific cross-linking of these polypeptides can occur in the absence of ATP"&+ when m'I-capped inosine substituted mRNA, which contains less secondary structure than native reovirus mRNA, is used. We also found, using wheat germ extract, that inhibition of initiation complex formation by high salt concentrations is directly related to the degree of secondary structure of the mRNA. Binding of ribosomes to bromouridine-substituted reovirus mRNA is severely inhibited at high K+ concentrations, while binding to inosine-substituted mRNA is only slightly inhibited and binding of native reovirus mRNA is inhibited to an intermediate degree. These results are consistent with the hypothesis that cap recognition factors mediate an ATP-dependent melting of secondary structures involving 5' proximal sequences to the initiation codon in order to facilitate binding of ribosomes during translation initiation.
Intrinsically-disordered N-termini in human parechovirus 1 capsid proteins bind encapsidated RNA
Scientific Reports, 2018
Human parechoviruses (HPeV) are picornaviruses with a highly-ordered RNA genome contained within icosahedrally-symmetric capsids. Ordered RNA structures have recently been shown to interact with capsid proteins VP1 and VP3 and facilitate virus assembly in HPeV1. Using an assay that combines reversible cross-linking, RNA affinity purification and peptide mass fingerprinting (RCAP), we mapped the RNA-interacting regions of the capsid proteins from the whole HPeV1 virion in solution. The intrinsically-disordered N-termini of capsid proteins VP1 and VP3, and unexpectedly, VP0, were identified to interact with RNA. Comparing these results to those obtained using recombinantlyexpressed VP0 and VP1 confirmed the virion binding regions, and revealed unique RNA binding regions in the isolated VP0 not previously observed in the crystal structure of HPeV1. We used RNA fluorescence anisotropy to confirm the RNA-binding competency of each of the capsid proteins' N-termini. These findings suggests that dynamic interactions between the viral RNA and the capsid proteins modulate virus assembly, and suggest a novel role for VP0. Human parechoviruses (HPeV) are important human pathogens for which we lack antivirals or vaccines. They have a positive-sense, single-stranded RNA genome and belong to the Picornaviridae family. The mature virion is icosahedrally-symmetric with a triangulation number of T = 1 (pseudo T = 3) and is composed of capsid proteins VP0, VP1 and VP3 1-4. Unlike in other picornaviruses, the parechovirus VP0 is not proteolytically cleaved in the final maturation of the virions 5. There is also an extensive network of VP0 N-termini on the inner capsid surface that enhance inter-pentamer stability, along with an annulus of VP3 termini under the vertex 3. Regions of structured RNA were recently identified as packaging signals (PSs) that interact with VP1 and VP3 in the HPeV virion 4. Upon interaction with viral pentameric assembly intermediates, these PSs drive capsid assembly. Multiple VP1 and VP3 residues were found to contact the viral RNA in the atomic models of HPeV1 (PDB: 4Z92 & 5MJV) 3,4. When these residues were mutated to alanine, virus assembly was prevented 4. The atomic models do not cover the complete sequences of the capsid proteins or the full genome. The N-terminal regions of all three capsid proteins were apparently disordered 3,4. The virion population may contain multiple states of both the RNA and the capsid, as was recently observed for bacteriophage MS2 6-8. Hence, we expect that there are more RNA-protein interactions to be discovered in the virion. More direct methods could be utilized to identify the regions of the HPeV1 capsid that interact with the encapsidated RNA. One such method is reversible cross-linking, affinity purification, and peptide-mass fingerprinting (RCAP) which has previously been used to map protein-nucleic acid interaction sites. RCAP has been successfully used to map regions of the capsid protein that interact with the virion RNA in brome mosaic virus, adenovirus, and bacteriophage MS2 9-12. The MS2 protein-RNA interactions identified by the RCAP assay have since been confirmed in asymmetric cryoEM reconstructions of MS2 6,13. Here we mapped regions within the HPeV1 capsid proteins that interact with the encapsidated RNA using RCAP. Several regions within VP1 and VP3 were found to interact with the RNA. Surprisingly, VP0 was also identified to contact the genomic RNA within the HPeV virion. The N-terminal regions of all capsid proteins not visualized in the HPeV1 atomic model apparently contact viral RNA. Recombinantly-expressed VP0 and
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.