Heterologous RNA Encapsidated in Pariacoto Virus-Like Particles Forms a Dodecahedral Cage Similar to Genomic RNA in Wild-Type Virions (original) (raw)
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The structure of pariacoto virus reveals a dodecahedral cage of duplex RNA
Nature structural biology, 2001
The 3.0 A resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for approximately 35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7-54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394-401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.
Journal of Virology, 2004
The nodavirus Flock house virus (FHV) has a bipartite, positive-sense RNA genome that is packaged into an icosahedral particle displaying T=3 symmetry. The high-resolution X-ray structure of FHV has shown that 10 bp of well-ordered, double-stranded RNA are located at each of the 30 twofold axes of the virion, but it is not known which portions of the genome form these duplex regions. The regular distribution of double-stranded RNA in the interior of the virus particle indicates that large regions of the encapsidated genome are engaged in secondary structure interactions. Moreover, the RNA is restricted to a topology that is unlikely to exist during translation or replication. We used electron cryomicroscopy and image reconstruction to determine the structure of four types of FHV particles that differed in RNA and protein content. RNA-capsid interactions were primarily mediated via the N and C termini, which are essential for RNA recognition and particle assembly. A substantial fract...
Structural and electrostatic characterization of Pariacoto virus: Implications for viral assembly
Biopolymers, 2009
We present the first all-atom model for the structure of a T=3 virus, pariacoto virus (PaV), which is a non-enveloped, icosahedral RNA virus and a member of the Nodaviridae family. The model is an extension of the crystal structure, which reveals about 88% of the protein structure but only about 35% of the RNA structure. Evaluation of alternative models confirms our earlier observation that the polycationic protein tails must penetrate deeply into the core of the virus, where they stabilize the structure by neutralizing a substantial fraction of the RNA charge. This leads us to propose a model for the assembly of small icosahedral RNA viruses: nonspecific binding of the protein tails to the RNA leads to a collapse of the complex, in a fashion reminiscent of DNA condensation. The globular protein domains are excluded from the condensed phase but are tethered to it, so they accumulate in a shell around the condensed phase, where their concentration is high enough to trigger oligomerization and formation of the mature virus. Pariacoto virus (PaV), a T=3, non-enveloped, icosahedral virus, is a member of the Nodaviridae family. It was originally isolated in Peru from the Southern Armyworm, Spodoptera eridania (1). Its genome consists of two positive-sense ssRNAs (2). RNA1 (3011 nucleotides) codes for protein A, the catalytic subunit for the host RNA replicase, which enables the RNAdependent RNA replicase to start replicating the viral RNA. RNA2 (1311 nucleotides) codes for capsid precursor protein α. 180 of these α proteins and the genome assemble together to make up the virus. Ever since it was isolated, PaV has been extensively studied using various techniques (3-6). The relatively small size (20nm diameter) and the ease with which it can be produced in various cell lines (7) make PaV and other members of the Nodaviridae family easy to characterize at the molecular level (8-10). Structural studies of viruses are very important to understand protein-protein and protein-RNA interactions as well as to understand assembly pathways in RNA viruses (11-14). In the last few years, many studies have been done on RNA viruses using molecular modeling as supplementary method when other methods such as x-ray crystallography and cryo-electron microscopy (cryo-EM) do not give sufficient structural information. An all-atom model was derived for Satellite Tobacco Mosaic Virus (STMV), a T=1 virus, using molecular modeling (15). Those authors also carried out molecular dynamics simulations on the model to study the stability of the protein capsid and the RNA genome (15). Electrostatic interactions between
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
Architecture of small RNA viruses
Progress in Crystal Growth and Characterization of Materials, 1997
Symmetric organization of biological macromolecules is necessary fi)r certain structural and functional requirements of living cells. The mechanisms by which biomoleeules assemble unambiguously into unique structures has been a central theme of investigation in molecular biology. Simple viruses consist of a nucleic acid core which (:()des for the genetic information surrounded and protected by a protein coat or capsid. In a large majority of the eases, the protein coats possess exact icosahedral symmetry. Developments in experimental X-ray crystallography and computer technology has led recently m the elucidation of the architecture of several viruses. Systematic studies on the structure of the protein subunits, their location and orientation on the ieosahedral eapsid, and the details of interaction between subunits has provided some insights into the mechanisms of error free virus assembly. However, the structures of even the simplest viruses are sufficiently complex and do not lead to eomplete understanding of the pathway of assembly by an examination of the final structure. The current state of research in this fast advancing area is briefly reviewed.
Journal of General Virology, 2005
Birnaviruses possess a capsid with a single protein layer in contrast to most double-stranded RNA viruses infecting multicellular eukaryotes. Using freeze-drying and heavy metal shadowing, the capsids of two birnaviruses, infectious bursal disease virus (IBDV) and infectious pancreatic necrosis virus, as well as of an IBDV virus-like particle (VLP) are shown to follow the same T=13 laevo icosahedral geometry. The structure of the VLP was determined at a resolution of approximately 15 Å (1?5 nm) by a combination of electron cryomicroscopy and a recently developed three-dimensional reconstruction method, where the scattering density is expressed in terms of symmetry-adapted functions. This reconstruction methodology is well adapted to the icosahedral symmetry of viruses and only requires a small number of images to analyse. The atomic model of the external capsid protein, VP2, recently determined by X-ray crystallography, fits well into the VLP reconstruction and occupies all the electron densities present in the map. Thus, similarly to the IBDV virion, only VP2 forms the icosahedral layer of the VLP. The other components of both VLP and IBDV particles that play a crucial role in the capsid assembly, VP1, VP3 and the peptides arising from the processing of pVP2, do not follow the icosahedral symmetry, allowing them to be involved in other processes such as RNA packaging.
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
Structure, 1994
Background: Studies of simple RNA animal viruses show that cell attachment, particle destabilization and cell entry are complex processes requiring a level of capsid sophistication that is difficult to achieve with a shell containing only a single gene product. Nodaviruses [such as Flock House virus (FHV)] are an exception. We have previously determined the structure of FHV at 3 A resolution, and now combine this information with data from cryo-electron microscopy in an attempt to clarify the process by which nodaviruses infect animal cells. Results: A difference map was computed in which electron density at 22A resolution, derived from the 3.0A resolution X-ray model of the FHV capsid protein, was subtracted from the electron density derived from the cryo-electron microscopy reconstruction of FHV at 22 A resolution. Comparisons of this density with the X-ray model showed that quasi-equivalent regions of identical polypeptide sequences have markedly different interactions with the bulk RNA density. Previously reported biphasic kinetics of particle maturation and the requirement of subunit cleavage for particle infectivity are consistent with these results. Conclusions: On the basis of this study we propose a model for nodavirus infection that is conceptually similar to that proposed for poliovirus but differs from it in detail. The constraints of a single protein type in the capsid lead to a noteworthy use of quasi-symmetry not only to control the binding of a 'pocket factor' but also to modulate maturation cleavage and to release a pentameric helical bundle (with genomic RNA attached) that may further interact with the cell membrane. April 1994, 2:271-282
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.
The Journal of general virology, 2003
Virions of the alphanodavirus Pariacoto virus (PaV) have T=3 icosahedral symmetry and are assembled from multiple copies of a precursor protein that is cleaved into two mature capsid proteins after assembly. The crystal structure of PaV shows that the N-terminal approximately 30 amino acid residues of the subunits surrounding the 5-fold axes interact extensively with icosahedrally ordered regions of the encapsidated positive-sense genomic RNAs. We found that wild-type PaV particles also contain a minor capsid protein that is truncated by 24 residues at its N terminus. Reverse genetic experiments showed that translation of this protein initiated at the second AUG of the capsid protein open reading frame. When either the longer or shorter version of the capsid protein was expressed independently of the other, it assembled into virus particles and underwent maturational cleavage. Virions that lacked the shorter capsid protein retained infectivity for cultured insect cells and Galleria ...