We have examined the roles of RNA-coat protein (CP) interactions in the assembly of satellite tobacco necrosis virus (STNV). The viral genomic RNA encodes only the CP, which comprises aβ-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem-loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of T=1 capsids, which is indifferent to sequences within the stem. The B3-containing virus-like particle has been crystallised and its structure was determined to 2.3Å. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8-11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal-CP interactions.
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Understanding the fundamental principles of virus architecture is one of the most important challenges in biology and medicine. Crick and Watson were the first to propose that viruses exhibit symmetry in the organization of their protein containers for reasons of genetic economy. Based on this, Caspar and Klug introduced quasi-equivalence theory to predict the relative locations of the coat proteins within these containers and classified virus structure in terms of T-numbers. Here it is shown that quasi-equivalence is part of a wider set of structural constraints on virus structure. These constraints can be formulated using an extension of the underlying symmetry group and this is demonstrated with a number of case studies. This new concept in virus biology provides for the first time predictive information on the structural constraints on coat protein and genome topography, and reveals a previously unrecognized structural interdependence of the shapes and sizes of different viral components. It opens up the possibility of distinguishing the structures of different viruses with the same T-number, suggesting a refined viral structure classification scheme. It can moreover be used as a basis for models of virus function, e.g. to characterize the start and end configurations of a structural transition important for infection.© 2013 International Union of Crystallography Printed in Singapore-all rights reserved.
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We have examined the roles of RNA-coat protein (CP) interactions in the assembly of satellite tobacco necrosis virus (STNV). The viral genomic RNA encodes only the CP, which comprises aβ-barrel domain connected to a positively charged N-terminal extension. In the previous crystal structures of this system, the first 11 residues of the protein are disordered. Using variants of an RNA aptamer sequence isolated against the CP, B3, we have studied the sequence specificity of RNA-induced assembly. B3 consists of a stem-loop presenting the tetra-loop sequence ACAA. There is a clear preference for RNAs encompassing this loop sequence, as measured by the yield of T = 1 capsids, which is indifferent to sequences within the stem. The B3-containing virus-like particle has been crystallised and its structure was determined to 2.3 Å. A lower-resolution map encompassing density for the RNA has also been calculated. The presence of B3 results in increased ordering of the N-terminal helices located at the particle 3-fold axes, which extend by roughly one and a half turns to encompass residues 8-11, including R8 and K9. Under assembly conditions, STNV CP in the absence of RNA is monomeric and does not self-assemble. These facts suggest that a plausible model for assembly initiation is the specific RNA-induced stabilisation of a trimeric capsomere. The basic nature of the helical extension suggests that electrostatic repulsion between CPs prevents assembly in the absence of RNA and that this barrier is overcome by correct placement of appropriately orientated helical RNA stems. Such a mechanism would be consistent with the data shown here for assembly with longer RNA fragments, including an STNV genome. The results are discussed in light of a first stage of assembly involving compaction of the genomic RNA driven by multiple RNA packaging signal-CP interactions.© 2013 Elsevier Ltd. All rights reserved.
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The formation of a protective protein container is an essential step in the life-cycle of most viruses. In the case of single-stranded (ss)RNA viruses, this step occurs in parallel with genome packaging in a co-assembly process. Previously, it had been thought that this process can be explained entirely by electrostatics. Inspired by recent single-molecule fluorescence experiments that recapitulate the RNA packaging specificity seen in vivo for two model viruses, we present an alternative theory, which recognizes the important cooperative roles played by RNA-coat protein interactions, at sites we have termed packaging signals. The hypothesis is that multiple copies of packaging signals, repeated according to capsid symmetry, aid formation of the required capsid protein conformers at defined positions, resulting in significantly enhanced assembly efficiency. The precise mechanistic roles of packaging signal interactions may vary between viruses, as we have demonstrated for MS2 and STNV. We quantify the impact of packaging signals on capsid assembly efficiency using a dodecahedral model system, showing that heterogeneous affinity distributions of packaging signals for capsid protein out-compete those of homogeneous affinities. These insights pave the way to a new anti-viral therapy, reducing capsid assembly efficiency by targeting of the vital roles of the packaging signals, and opens up new avenues for the efficient construction of protein nanocontainers in bionanotechnology.© 2013 The Author(s).
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All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N-RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into whichRNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.
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We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.
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We have determined the three-dimensional structures of both native and expanded forms of turnip crinkle virus (TCV), using cryo-electron microscopy, which allows direct visualization of the encapsidated single-stranded RNA and coat protein (CP) N-terminal regions not seen in the high-resolution X-ray structure of the virion. The expanded form, which is a putative disassembly intermediate during infection, arises from a separation of the capsid-forming domains of the CP subunits. Capsid expansion leads to the formation of pores that could allow exit of the viral RNA. A subset of the CP N-terminal regions becomes proteolytically accessible in the expanded form, although the RNA remains inaccessible to nuclease. Sedimentation velocity assays suggest that the expanded state is metastable and that expansion is not fully reversible. Proteolytically cleaved CP subunits dissociate from the capsid, presumably leading to increased electrostatic repulsion within the viral RNA. Consistent with this idea, electron microscopy images show that proteolysis introduces asymmetry into the TCV capsid and allows initial extrusion of the genome from a defined site. The apparent formation of polysomes in wheat germ extracts suggests that subsequent uncoating is linked to translation. The implication is that the viral RNA and its capsid play multiple roles during primary infections, consistent with ribosome-mediated genome uncoating to avoid host antiviral activity.© 2012 Elsevier Ltd.
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Single-stranded RNA viruses package their genomes into capsids enclosing fixed volumes. We assayed the ability of bacteriophage MS2 coat protein to package large, defined fragments of its genomic, single-stranded RNA. We show that the efficiency of packaging into a T=3 capsid in vitro is inversely proportional to RNA length, implying that there is a free-energy barrier to be overcome during assembly. All the RNAs examined have greater solution persistence lengths than the internal diameter of the capsid into which they become packaged, suggesting that protein-mediated RNA compaction must occur during assembly. Binding ethidium bromide to one of these RNA fragments, which would be expected to reduce its flexibility, severely inhibited packaging, consistent with this idea. Cryo-EM structures of the capsids assembled in these experiments with the sub-genomic RNAs show a layer of RNA density beneath the coat protein shell but lack density for the inner RNA shell seen in the wild-type virion. The inner layer is restored when full-length virion RNA is used in the assembly reaction, implying that it becomes ordered only when the capsid is filled, presumably because of the effects of steric and/or electrostatic repulsions. The cryo-EM results explain the length dependence of packaging. In addition, they show that for the sub-genomic fragments the strongest ordered RNA density occurs below the coat protein dimers forming the icosahedral 5-fold axes of the capsid. There is little such density beneath the proteins at the 2-fold axes, consistent with our model in which coat protein dimers binding to RNA stem-loops located at sites throughout the genome leads to switching of their preferred conformations, thus regulating the placement of the quasi-conformers needed to build the T=3 capsid. The data are consistent with mutual chaperoning of both RNA and coat protein conformations, partially explaining the ability of such viruses to assemble so rapidly and accurately.
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The protein Ure2 from baker's yeast is associated with a heritable and transmissible phenotypic change in the yeast Saccharomyces cerevisiae. Such prion properties are thought to arise from the fact that Ure2p is able to self-assemble into insoluble fibrils. Assemblies of Ure2p are composed of full-length proteins in which the structure of the globular, functional, C-terminal domain is retained. We have carried out structural studies on full-length, wild-type Ure2p fibrils with a regularly twisted morphology. Using electron microscopy and cryo-electron microscopy with image analysis we show high-resolution images of the twisted filaments revealing details within the fibrillar structure. We examine these details in light of recent proposed models and discuss how this new information contributes to an understanding of the architecture of Ure2p yeast prion fibrils.
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We have developed an angular refinement procedure incorporating correction for the microscope contrast transfer function, to determine cryoelectron microscopy (cryo-EM) structures of the Escherichia coli chaperonin GroEL in its apo and ATP-bound forms. This image reconstruction procedure is verified to 13-Angstrom resolution by comparison of the cryo-EM structure of unliganded GroEL with the crystal structure. Binding, encapsulation, and release of nonnative proteins by GroEL and its cochaperone GroES are controlled by the binding and hydrolysis of ATP. Seven ATP molecules bind cooperatively to one heptameric ring of GroEL. This binding causes long-range conformational changes that determine the orientations of remote substrate-binding sites, and it also determines the conformation of subunits in the opposite ring of GroEL, in a negatively cooperative mechanism. The conformation of GroEL-ATP was determined at similar to 15-Angstrom resolution. In one ring of GroEL-ATP, the apical (substrate-binding) domains are extremely disordered, consistent with the high mobility needed for them to achieve the 60 degrees elevation and 90 degrees twist of the GroES-bound state. Unexpectedly, ATP binding also increases the separation between the two rings, although the interring contacts are present in the density map. (C) 2001 Academic Press.
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