Geometric mismatches within the concentric layers of rotavirus particles: A potential regulatory switch of viral particle transcription activity (original) (raw)
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Journal of …, 2008
Rotaviruses are prototypical double-stranded RNA viruses whose triple-layered icosahedral capsid constitutes transcriptional machinery activated by the release of the external layer. To understand the molecular basis of this activation, we studied the structural interplay between the three capsid layers by electron cryo-microscopy and digital image processing. Two viral particles and four virus-like particles containing various combinations of inner (VP2)-, middle (VP6)-, and outer (VP7)-layer proteins were studied. We observed that the absence of the VP2 layer increases the particle diameter and changes the type of quasi-equivalent icosahedral symmetry, as described by the shift in triangulation number (T) of the VP6 layer (from T ؍ 13 to T ؍ 19 or more). By fitting X-ray models of VP6 into each reconstruction, we determined the quasi-atomic structures of the middle layers. These models showed that the VP6 lattices, i.e., curvature and trimer contacts, are characteristic of the particle composition. The different functional states of VP6 thus appear as being characterized by trimers having similar conformations but establishing different intertrimeric contacts. Remarkably, the external protein VP7 reorients the VP6 trimers located around the fivefold axes of the icosahedral capsid, thereby shrinking the channel through which mRNA exits the transcribing rotavirus particle. We conclude that the constraints arising from the different geometries imposed by the external and internal layers of the rotavirus capsid constitute a potential switch regulating the transcription activity of the viral particles.
Biological research, 1994
The structural relationship between VP6 (inner capsid polypeptide) and the viral core was studied using chemical cross-linking with dithiobis(succinimidyl propionate). Crosslinked single shelled and reconstituted rotavirus particles, suggest the existence of a complex organization of VP6 molecules in the inner capsid and a direct interaction with the core polypeptide VP3. The inhibition of the recovery of RNA polymerase activity associated with the reconstitution of the single shelled particle in the presence of antiVP6 monoclonal antibodies indicates that a VP6 domain between amino acids 56 and 58 seems to be important in viral transcription. A VP6 gene temperature-sensitive mutant (ts G) carrying a mutation affecting assembly of single shelled particles was used in reconstitution experiments. The mutant was able to recover RNA polymerase activity at restrictive temperature. Wild type cores or VP6 were able to reconstitute the particle with both the mutant cores and VP6. These resu...
Structure, 2005
Rotaviruses are important human pathogens with a triple-layered icosahedral capsid. The major capsid protein VP6 is shown here to self-assemble into spherical or helical particles mainly depending upon pH. Assembly is inhibited either by low pH (<3.0) or by a high concentration (>100 mM) of divalent cations (Ca 2+ and Zn 2+). The structures of two types of helical tubes were determined by electron cryomicroscopy and image analysis to a resolution of 2.0 and 2.5 nm. In both reconstructions, the molecular envelope of VP6 ®ts the atomic model determined by X-ray crystallography remarkably well. The 3-fold symmetry of the VP6 trimer, being incompatible with the helical symmetry, is broken at the level of the trimer contacts. One type of contact is maintained within all VP6 particles (tubes and virus), strongly suggesting that VP6 assemblies arise from different packings of a unique dimer of trimers. Our data show that the protonation state and thus the charge distribution are important switches governing the assembly of macromolecular assemblies.
Proceedings of the National Academy of Sciences, 1999
During genome transcription in rotavirus, as with many segmented double-stranded RNA viruses, mRNA is transcribed within the intact subviral particle and translocated through specific channels in the capsid. To understand how the conformation of the capsid affects the efficiency of transcriptional events in the viral core, we carried out a series of comparative structural and biochemical studies to characterize four different structural forms of the virus exhibiting differing transcriptional behavior. Two of these were virus-antibody complexes having contrasting transcriptional capabilities, and two were variant structural forms of the virus that exist during the life cycle and also exhibit contrasting transcriptional behavior. Three-dimensional structural studies using electron cryomicroscopy showed that the binding of one Fab (8H2͞G5) does not affect the conformation of the capsid, and the efficiency of mRNA production is similar to that of the native subviral particle. The other Fab (2A11͞E9) introduces conformational changes in the capsid similar to those seen in the transcriptionally incompetent mature particle. In both of the transcriptionally incompetent particle types, mRNA synthesis was arrested after limited elongation with the resulting oligonucleotide transcripts remaining trapped inside the particles. Our results indicate that the continuous translocation of nascent mRNA through the capsid is critical for efficient transcript elongation and that the blockage of translocation causes premature termination of transcription.
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
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
Journal of General Virology, 1986
The inner capsid structure of the OSU strain of porcine rotavirus was studied by electron microscopy of freeze-dried preparations and of negatively stained chemically disrupted virus particles. The analysis of the particles by the freeze-drying technique revealed a T : 13 1 (laevo) symmetry for the organization of the inner capsid. Treatment of single-capsid rotavirus particles with 30~ formamide or 5 M-urea resulted in their degradation, giving rise to very similar products, corresponding to isolated vertices, edges and faces of the virus icosahedron. An analysis of such structures confirmed the triangulation number and handedness of the rotavirus inner capsid, and provided evidence for the open-mesh model, in which the five-and six-coordinated axes are represented by 'holes" formed by smaller trimeric morphological subunits.
Location of the dsRNA-Dependent Polymerase, VP1, in Rotavirus Particles
Journal of Molecular Biology, 2013
Double-stranded RNA (dsRNA) viruses transcribe and replicate RNA within an assembled, inner capsid particle; only plus-sense mRNA emerges into the intracellular milieu. During infectious entry of a rotavirus particle, the outer layer of its three-layer structure dissociates, delivering the inner double-layered particle (DLP) into the cytosol. DLP structures determined by X-ray crystallography and electron cryomicroscopy (cryoEM) show that the RNA coils uniformly into the particle interior, avoiding a "fivefold hub" of more structured density projecting inward from the VP2 shell of the DLP along each of the twelve 5-fold axes. Analysis of the X-ray crystallographic electron density map suggested that principal contributors to the hub are the N-terminal arms of VP2, but reexamination of the cryoEM map has shown that many features come from a molecule of VP1, randomly occupying five equivalent and partly overlapping positions. We confirm here that the electron density in the X-ray map leads to the same conclusion, and we describe the functional implications of the orientation and position of the polymerase. The exit channel for the nascent transcript directs the nascent transcript toward an opening along the 5-fold axis. The template strand enters from within the particle, and the dsRNA product of the initial replication step exits in a direction tangential to the inner surface of the VP2 shell, allowing it to coil optimally within the DLP. The polymerases of reoviruses appear to have similar positions and functional orientations.
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.