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1.  Expression, purification and crystallization of the ectodomain of the envelope glycoprotein E2 from Bovine viral diarrhoea virus  
The cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the ectodomain of BVDV E2 are described.
Bovine viral diarrhoea virus (BVDV) is an economically important animal pathogen which is closely related to Hepatitis C virus. Of the structural proteins, the envelope glycoprotein E2 of BVDV is the major antigen which induces neutralizing antibodies; thus, BVDV E2 is considered as an ideal target for use in subunit vaccines. Here, the expression, purification of wild-type and mutant forms of the ectodomain of BVDV E2 and subsequent crystallization and data collection of two crystal forms grown at low and neutral pH are reported. Native and multiple-wavelength anomalous dispersion (MAD) data sets have been collected and structure determination is in progress.
PMCID: PMC3539699  PMID: 23295482
Pestivirus; BVDV; envelope glycoprotein E2
2.  A national facility for biological cryo-electron microscopy 
This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron.
Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.
PMCID: PMC4304693  PMID: 25615867
three-dimensional electron microscopy
3.  New methods for indexing multi-lattice diffraction data 
A new indexing method is presented which is capable of indexing multiple crystal lattices from narrow wedges of data. The efficacy of this method is demonstrated with both semi-synthetic multi-lattice data and real multi-lattice data recorded from microcrystals of ∼1 µm in size.
A new indexing method is presented which is capable of indexing multiple crystal lattices from narrow wedges of diffraction data. The method takes advantage of a simplification of Fourier transform-based methods that is applicable when the unit-cell dimensions are known a priori. The efficacy of this method is demonstrated with both semi-synthetic multi-lattice data and real multi-lattice data recorded from crystals of ∼1 µm in size, where it is shown that up to six lattices can be successfully indexed and subsequently integrated from a 1° wedge of data. Analysis is presented which shows that improvements in data-quality indicators can be obtained through accurate identification and rejection of overlapping reflections prior to scaling.
PMCID: PMC4188007  PMID: 25286849
indexing; multi-lattice data
4.  Pushing the limits of sulfur SAD phasing: de novo structure solution of the N-terminal domain of the ectodomain of HCV E1 
The sulfur SAD phasing method was successfully used to determine the structure of the N-terminal domain of HCV E1 from low-resolution diffracting crystals by combining data from 32 crystals.
Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Å resolution model building was achievable.
PMCID: PMC4118829  PMID: 25084338
sulfur SAD; HCV; envelope glycoprotein E1
5.  Structure of the regulatory domain of the LysR family regulator NMB2055 (MetR-like protein) from Neisseria meningitidis  
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator, was solved at 2.5 Å resolution.
The crystal structure of the regulatory domain of NMB2055, a putative MetR regulator from Neisseria meningitidis, is reported at 2.5 Å resolution. The structure revealed that there is a disulfide bond inside the predicted effector-binding pocket of the regulatory domain. Mutation of the cysteines (Cys103 and Cys106) that form the disulfide bond to serines resulted in significant changes to the structure of the effector pocket. Taken together with the high degree of conservation of these cysteine residues within MetR-related transcription factors, it is suggested that the Cys103 and Cys106 residues play an important role in the function of MetR regulators.
PMCID: PMC3388910  PMID: 22750853
MetR; Neisseria meningitidis; LysR-type regulator
6.  In cellulo structure determination of a novel cypovirus polyhedrin 
The crystal structure of a previously unsolved type of cypovirus polyhedrin has been determined from data collected directly from frozen live insect cells.
This work demonstrates that with the use of a microfocus synchrotron beam the structure of a novel viral polyhedrin could be successfully determined from microcrystals within cells, removing the preparatory step of sample isolation and maintaining a favourable biological environment. The data obtained are of high quality, comparable to that obtained from isolated crystals, and enabled a facile structure determination. A small but significant difference is observed between the unit-cell parameters and the mosaic spread of in cellulo and isolated crystals, suggesting that even these robust crystals are adversely affected by removal from the cell.
PMCID: PMC4014125  PMID: 24816111
microcrystals; viral protein; data collection; in cellulo
7.  Exploiting fast detectors to enter a new dimension in room-temperature crystallography 
A departure from a linear or an exponential decay in the diffracting power of macromolecular crystals is observed and accounted for through consideration of a multi-state sequential model.
A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.
PMCID: PMC4014120  PMID: 24816094
radiation damage; room temperature; macromolecular crystallography; dose rate
8.  Antigenic Switching of Hepatitis B Virus by Alternative Dimerization of the Capsid Protein 
Chronic Hepatitis B virus (HBV) infection afflicts millions worldwide with cirrhosis and liver cancer. HBV e-antigen (HBeAg), a clinical marker for disease severity, is a non-particulate variant of the protein (core antigen, HBcAg) that forms the building-blocks of capsids. HBeAg is not required for virion production, but is implicated in establishing immune tolerance and chronic infection. Here, we report the crystal structure of HBeAg, which clarifies how the short N-terminal propeptide of HBeAg induces a radically altered mode of dimerization relative to HBcAg (~140° rotation), locked into place through f ormation of intramolecular disulfide bridges. This structural switch precludes capsid assembly and engenders a distinct antigenic repertoire, explaining why the two antigens are cross-reactive at the T-cell level (through sequence identity) but not at the B-cell level (through conformation). The structure offers insight into how HBeAg may establish immune tolerance for HBcAg while evading its robust immunogenicity.
PMCID: PMC3544974  PMID: 23219881
9.  From lows to highs: using low-resolution models to phase X-ray data 
An unusual example of how virus structure determination pushes the limits of the molecular replacement method is presented.
The study of virus structures has contributed to methodo­logical advances in structural biology that are generally applicable (molecular replacement and noncrystallographic symmetry are just two of the best known examples). Moreover, structural virology has been instrumental in forging the more general concept of exploiting phase information derived from multiple structural techniques. This hybridization of structural methods, primarily electron microscopy (EM) and X-ray crystallography, but also small-angle X-ray scattering (SAXS) and nuclear magnetic resonance (NMR) spectroscopy, is central to integrative structural biology. Here, the interplay of X-ray crystallography and EM is illustrated through the example of the structural determination of the marine lipid-containing bacteriophage PM2. Molecular replacement starting from an ∼13 Å cryo-EM reconstruction, followed by cycling density averaging, phase extension and solvent flattening, gave the X-ray structure of the intact virus at 7 Å resolution This in turn served as a bridge to phase, to 2.5 Å resolution, data from twinned crystals of the major coat protein (P2), ultimately yielding a quasi-atomic model of the particle, which provided significant insights into virus evolution and viral membrane biogenesis.
PMCID: PMC3817700  PMID: 24189238
virus structure; phasing methods; data collection; noncrystallographic symmetry
10.  Structure of a VP1-VP3 Complex Suggests How Birnaviruses Package the VP1 Polymerase 
Journal of Virology  2013;87(6):3229-3236.
Infectious pancreatic necrosis virus (IPNV), a member of the family Birnaviridae, infects young salmon, with a severe impact on the commercial sea farming industry. Of the five mature proteins encoded by the IPNV genome, the multifunctional VP3 has an essential role in morphogenesis; interacting with the capsid protein VP2, the viral double-stranded RNA (dsRNA) genome and the RNA-dependent RNA polymerase VP1. Here we investigate one of these VP3 functions and present the crystal structure of the C-terminal 12 residues of VP3 bound to the VP1 polymerase. This interaction, visualized for the first time, reveals the precise molecular determinants used by VP3 to bind the polymerase. Competition binding studies confirm that this region of VP3 is necessary and sufficient for VP1 binding, while biochemical experiments show that VP3 attachment has no effect on polymerase activity. These results indicate how VP3 recruits the polymerase into birnavirus capsids during morphogenesis.
PMCID: PMC3592137  PMID: 23283942
11.  Tracking in atomic detail the functional specializations in viral RecA helicases that occur during evolution 
Nucleic Acids Research  2013;41(20):9396-9410.
Many complex viruses package their genomes into empty protein shells and bacteriophages of the Cystoviridae family provide some of the simplest models for this. The cystoviral hexameric NTPase, P4, uses chemical energy to translocate single-stranded RNA genomic precursors into the procapsid. We previously dissected the mechanism of RNA translocation for one such phage, ɸ12, and have now investigated three further highly divergent, cystoviral P4 NTPases (from ɸ6, ɸ8 and ɸ13). High-resolution crystal structures of the set of P4s allow a structure-based phylogenetic analysis, which reveals that these proteins form a distinct subfamily of the RecA-type ATPases. Although the proteins share a common catalytic core, they have different specificities and control mechanisms, which we map onto divergent N- and C-terminal domains. Thus, the RNA loading and tight coupling of NTPase activity with RNA translocation in ɸ8 P4 is due to a remarkable C-terminal structure, which wraps right around the outside of the molecule to insert into the central hole where RNA binds to coupled L1 and L2 loops, whereas in ɸ12 P4, a C-terminal residue, serine 282, forms a specific hydrogen bond to the N7 of purines ring to confer purine specificity for the ɸ12 enzyme.
PMCID: PMC3814363  PMID: 23939620
12.  Plate Tectonics of Virus Shell Assembly and Reorganization in Phage Φ8, a Distant Relative of Mammalian Reoviruses 
Structure(London, England:1993)  2013;21(8):1384-1395.
The hallmark of a virus is its capsid, which harbors the viral genome and is formed from protein subunits, which assemble following precise geometric rules. dsRNA viruses use an unusual protein multiplicity (120 copies) to form their closed capsids. We have determined the atomic structure of the capsid protein (P1) from the dsRNA cystovirus Φ8. In the crystal P1 forms pentamers, very similar in shape to facets of empty procapsids, suggesting an unexpected assembly pathway that proceeds via a pentameric intermediate. Unlike the elongated proteins used by dsRNA mammalian reoviruses, P1 has a compact trapezoid-like shape and a distinct arrangement in the shell, with two near-identical conformers in nonequivalent structural environments. Nevertheless, structural similarity with the analogous protein from the mammalian viruses suggests a common ancestor. The unusual shape of the molecule may facilitate dramatic capsid expansion during phage maturation, allowing P1 to switch interaction interfaces to provide capsid plasticity.
Graphical Abstract
•Crystal structure of the major capsid protein P1 of the Pseudomonas phage Φ8•Φ8 P1 shares a common ancestor with mammalian reoviruses•Φ8 P1’s trapezoidal shape may facilitate capsid expansion during maturation•The pentameric organization of Φ8 P1 suggests a revised assembly pathway
El Omari et al. report a structure of the dsRNA bacteriophage ϕ8 capsid protein P1. P1 crystallizes as a pentamer, suggesting a new pathway for procapsid assembly. P1 displays a novel fold and a trapezoidal shape, distinct from that of other dsRNA virus, which may facilitate capsid expansion during maturation.
PMCID: PMC3737474  PMID: 23891291
13.  Crystal structure of signal regulatory protein gamma (SIRPγ) in complex with an antibody Fab fragment 
Signal Regulatory Protein γ (SIRPγ) is a member of a closely related family of three cell surface receptors implicated in modulating immune/inflammatory responses. SIRPγ is expressed on T lymphocytes where it appears to be involved in the integrin-independent adhesion of lymphocytes to antigen-presenting cells. Here we describe the first full length structure of the extracellular region of human SIRPγ.
We obtained crystals of SIRPγ by making a complex of the protein with the Fab fragment of the anti-SIRP antibody, OX117, which also binds to SIRPα and SIRPβ. We show that the epitope for FabOX117 is formed at the interface of the first and second domains of SIRPγ and comprises residues which are conserved between all three SIRPs. The FabOX117 binding site is distinct from the region in domain 1 which interacts with CD47, the physiological ligand for both SIRPγ and SIRPα but not SIRPβ. Comparison of the three domain structures of SIRPγ and SIRPα showed that these receptors can adopt different overall conformations due to the flexibility of the linker between the first two domains. SIRPγ in complex with FabOX117 forms a dimer in the crystal. Binding to the Fab fixes the position of domain 1 relative to domains 2/3 exposing a surface which favours formation of a homotypic dimer. However, the interaction appears to be relatively weak since only monomers of SIRPγ were observed in sedimentation velocity analytical ultracentrifugation of the protein alone. Studies of complex formation by equilibrium ultracentrifugation showed that only a 1:1 complex of SIRPγ: FabOX117 was formed with a dissociation constant in the low micromolar range (Kd = 1.2 +/− 0.3 μM).
The three-domain extracellular regions of SIRPs are structurally conserved but show conformational flexibility in the disposition of the amino terminal ligand-binding Ig domain relative to the two membrane proximal Ig domains. Binding of a cross-reactive anti-SIRP Fab fragment to SIRPγ stabilises a conformation that favours SIRP dimer formation in the crystal structure, though this interaction does not appear sufficiently stable to be observed in solution.
PMCID: PMC3716694  PMID: 23826770
Antigen-binding complex; Signal regulatory protein; Receptor structure
14.  Evaluation and Use of In-Silico Structure-Based Epitope Prediction with Foot-and-Mouth Disease Virus 
PLoS ONE  2013;8(5):e61122.
Understanding virus antigenicity is of fundamental importance for the development of better, more cross-reactive vaccines. However, as far as we are aware, no systematic work has yet been conducted using the 3D structure of a virus to identify novel epitopes. Therefore we have extended several existing structural prediction algorithms to build a method for identifying epitopes on the appropriate outer surface of intact virus capsids (which are structurally different from globular proteins in both shape and arrangement of multiple repeated elements) and applied it here as a proof of principle concept to the capsid of foot-and-mouth disease virus (FMDV). We have analysed how reliably several freely available structure-based B cell epitope prediction programs can identify already known viral epitopes of FMDV in the context of the viral capsid. To do this we constructed a simple objective metric to measure the sensitivity and discrimination of such algorithms. After optimising the parameters for five methods using an independent training set we used this measure to evaluate the methods. Individually any one algorithm performed rather poorly (three performing better than the other two) suggesting that there may be value in developing virus-specific software. Taking a very conservative approach requiring a consensus between all three top methods predicts a number of previously described antigenic residues as potential epitopes on more than one serotype of FMDV, consistent with experimental results. The consensus results identified novel residues as potential epitopes on more than one serotype. These include residues 190–192 of VP2 (not previously determined to be antigenic), residues 69–71 and 193–197 of VP3 spanning the pentamer-pentamer interface, and another region incorporating residues 83, 84 and 169–174 of VP1 (all only previously experimentally defined on serotype A). The computer programs needed to create a semi-automated procedure for carrying out this epitope prediction method are presented.
PMCID: PMC3646828  PMID: 23667434
15.  Bacteriophage P23-77 Capsid Protein Structures Reveal the Archetype of an Ancient Branch from a Major Virus Lineage 
Structure(London, England:1993)  2013;21(5):718-726.
It has proved difficult to classify viruses unless they are closely related since their rapid evolution hinders detection of remote evolutionary relationships in their genetic sequences. However, structure varies more slowly than sequence, allowing deeper evolutionary relationships to be detected. Bacteriophage P23-77 is an example of a newly identified viral lineage, with members inhabiting extreme environments. We have solved multiple crystal structures of the major capsid proteins VP16 and VP17 of bacteriophage P23-77. They fit the 14 Å resolution cryo-electron microscopy reconstruction of the entire virus exquisitely well, allowing us to propose a model for both the capsid architecture and viral assembly, quite different from previously published models. The structures of the capsid proteins and their mode of association to form the viral capsid suggest that the P23-77-like and adeno-PRD1 lineages of viruses share an extremely ancient common ancestor.
•High-resolution structures of the two major capsid proteins of bacteriophage P23-77•P23-77 capsid proteins exhibit a conserved single β-barrel core fold•P23-77 is an ancient relative of the double β-barrel lineage of viruses•Capsid model illustrates that P23-77 uses a novel method of organization
Rissanen et al. propose a model for the architecture and assembly of bacteriophage P23-77 quite different from those previously published. The capsid proteins and their mode of association to form the virus particle suggest that P23-77 share a common evolutionary origin with the PRD1/Adenovirus lineage.
PMCID: PMC3919167  PMID: 23623731
16.  Rational Engineering of Recombinant Picornavirus Capsids to Produce Safe, Protective Vaccine Antigen 
PLoS Pathogens  2013;9(3):e1003255.
Foot-and-mouth disease remains a major plague of livestock and outbreaks are often economically catastrophic. Current inactivated virus vaccines require expensive high containment facilities for their production and maintenance of a cold-chain for their activity. We have addressed both of these major drawbacks. Firstly we have developed methods to efficiently express recombinant empty capsids. Expression constructs aimed at lowering the levels and activity of the viral protease required for the cleavage of the capsid protein precursor were used; this enabled the synthesis of empty A-serotype capsids in eukaryotic cells at levels potentially attractive to industry using both vaccinia virus and baculovirus driven expression. Secondly we have enhanced capsid stability by incorporating a rationally designed mutation, and shown by X-ray crystallography that stabilised and wild-type empty capsids have essentially the same structure as intact virus. Cattle vaccinated with recombinant capsids showed sustained virus neutralisation titres and protection from challenge 34 weeks after immunization. This approach to vaccine antigen production has several potential advantages over current technologies by reducing production costs, eliminating the risk of infectivity and enhancing the temperature stability of the product. Similar strategies that will optimize host cell viability during expression of a foreign toxic gene and/or improve capsid stability could allow the production of safe vaccines for other pathogenic picornaviruses of humans and animals.
Author Summary
Picornaviruses are small RNA viruses, responsible for important human and animal diseases for example polio, some forms of the common cold and foot-and-mouth disease. Safe and effective picornavirus vaccines could in principle be produced from recombinant virus-like particles, which lack the viral genome and so cannot propagate. However the synthesis of stable forms of such particles at scale has proved very difficult. Two key problems have been that a protease required for the proper processing of the polyprotein precursor is toxic for host cells and the empty recombinant particles tend to be physically unstable in comparison to virus particles containing nucleic acid. This is particularly true in the case of Foot-and-Mouth Disease Virus (FMDV). Here we report the production and evaluation of a novel vaccine against FMDV that addresses both of these shortcomings. Importantly, the strategies we have devised to produce improved FMDV vaccines can be directly applied to viruses pathogenic for humans.
PMCID: PMC3609824  PMID: 23544011
17.  Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity 
Journal of Virological Methods  2013;187(2):406-412.
► Efficient expression of FMDV empty capsids in insect cells after moderation of 3C protease action. ► Expression cassette productive in multiple insect cell lines. ► Empty capsids visualised by transmission electron microscopy. ► Empty capsids react with wide range of positive sera as well as authentic virus. ► Efficient empty capsid synthesis may allow development as a vaccine.
Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or incomplete inactivation. Non-infectious empty capsids are structural mimics of authentic particles with no associated risk and constitute an alternate vaccine candidate. Capsids self-assemble from the processed virus structural proteins, VP0, VP3 and VP1, which are released from the structural protein precursor P1-2A by the action of the virus-encoded 3C protease. To date recombinant empty capsid assembly has been limited by poor expression levels, restricting the development of empty capsids as a viable vaccine. Here expression of the FMDV structural protein precursor P1-2A in insect cells is shown to be efficient but linkage of the cognate 3C protease to the C-terminus reduces expression significantly. Inactivation of the 3C enzyme in a P1-2A-3C cassette allows expression and intermediate levels of 3C activity resulted in efficient processing of the P1-2A precursor into the structural proteins which assembled into empty capsids. Expression was independent of the insect host cell background and leads to capsids that are recognised as authentic by a range of anti-FMDV bovine sera suggesting their feasibility as an alternate vaccine.
PMCID: PMC3558679  PMID: 23174161
Foot-and-mouth disease virus; Recombinant baculovirus; Empty capsids; Protein processing; Frameshift; 3C protease; Vaccine
18.  Structure of a Pestivirus Envelope Glycoprotein E2 Clarifies Its Role in Cell Entry 
Cell Reports  2013;3(1):30-35.
Enveloped viruses have developed various adroit mechanisms to invade their host cells. This process requires one or more viral envelope glycoprotein to achieve cell attachment and membrane fusion. Members of the Flaviviridae such as flaviviruses possess only one envelope glycoprotein, E, whereas pestiviruses and hepacivirus encode two glycoproteins, E1 and E2. Although E2 is involved in cell attachment, it has been unclear which protein is responsible for membrane fusion. We report the crystal structures of the homodimeric glycoprotein E2 from the pestivirus bovine viral diarrhea virus 1 (BVDV1) at both neutral and low pH. Unexpectedly, BVDV1 E2 does not have a class II fusion protein fold, and at low pH the N-terminal domain is disordered, similarly to the intermediate postfusion state of E2 from sindbis virus, an alphavirus. Our results suggest that the pestivirus and possibly the hepacivirus fusion machinery are unlike any previously observed.
Graphical Abstract
► Structure of the major antigenically dominant protein of BVDV ► The overall fold of BVDV E2 shows no similarity to the class II fusion proteins ► At low pH, BVDV E2 N-terminal domain is disordered ► Entry mechanism of BVDV is probably applicable to hepatitis C virus
Stuart and colleagues have determined the atomic structure of the ectodomain of bovine viral diarrhea virus E2 glycoprotein, the major, antigenically dominant protein on the virus surface. The structure was expected to resemble the fusion molecules found on the surface of viruses such as dengue virus, but it is unlike anything previously seen. E2 itself is not, in fact, the fusion protein but binds the cell receptor and directs fusion via a pH-dependent conformational switch.
PMCID: PMC3607223  PMID: 23273918
19.  Dame Louise Napier Johnson (1940–2012) 
Obituary for Dame Louise Napier Johnson.
PMCID: PMC3515394
20.  A sensor-adaptor mechanism for enterovirus uncoating from structures of EV71 
Enterovirus 71 (EV71), a major agent of hand-foot-and-mouth disease in children, can cause severe central nervous system disease and mortality. At present no vaccine or antiviral therapy is available. We have determined high-resolution structures for the mature virus and natural empty particles. The structure of the mature virus is similar to that of other enteroviruses, whilst the empty particles are dramatically expanded, with notable fissures, resembling elusive enterovirus uncoating intermediates not previously characterized in atomic detail. Hydrophobic capsid pockets within the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. The results provide a paradigm for enterovirus uncoating, in which the VP1 GH loop acts as an adaptor-sensor for the attachment of cellular receptors, converting heterologous inputs to a generic uncoating mechanism, spotlighting novel points for therapeutic intervention.
PMCID: PMC3378640  PMID: 22388738
21.  A plate-based high-throughput assay for virus stability and vaccine formulation 
Journal of Virological Methods  2012;185(1):166-170.
► A plate-based assay for virus measuring virus stability. ► Two fluorescent dyes measure independently but simultaneously capsid stability and capsid protein stability. ► A fast and efficient high-throughput method to optimise vaccine formulation. ► Facilitates the dissection of virus uncoating.
Standard methods for assessing the thermal stability of viruses can be time consuming and rather qualitative yet such data is a necessary requisite for vaccine formulation. In this study a novel plate-based thermal scanning assay for virus particle stability has been developed (PaSTRy: Particle Stability Thermal Release Assay). Two environment-sensitive fluorescent dyes, with non-overlapping emission spectra and different affinities, are used to accrue simultaneously independent data for the overall stability of the virus capsid, as judged by the exposure of the genome, and for capsid protein stability according to the exposure of hydrophobic side chains which are normally buried. This offers a fast and efficient high-throughput method to optimise vaccine formulation and to investigate the processes of virus uncoating.
PMCID: PMC3470038  PMID: 22744000
High-throughput; Virus stability; Vaccine formulation
22.  Noncatalytic Ions Direct the RNA-Dependent RNA Polymerase of Bacterial Double-Stranded RNA Virus ϕ6 from De Novo Initiation to Elongation 
Journal of Virology  2012;86(5):2837-2849.
RNA-dependent RNA polymerases (RdRps) are key to the replication of RNA viruses. A common divalent cation binding site, distinct from the positions of catalytic ions, has been identified in many viral RdRps. We have applied biochemical, biophysical, and structural approaches to show how the RdRp from bacteriophage ϕ6 uses the bound noncatalytic Mn2+ to facilitate the displacement of the C-terminal domain during the transition from initiation to elongation. We find that this displacement releases the noncatalytic Mn2+, which must be replaced for elongation to occur. By inserting a dysfunctional Mg2+ at this site, we captured two nucleoside triphosphates within the active site in the absence of Watson-Crick base pairing with template and mapped movements of divalent cations during preinitiation. These structures refine the pathway from preinitiation through initiation to elongation for the RNA-dependent RNA polymerization reaction, explain the role of the noncatalytic divalent cation in ϕ6 RdRp, and pinpoint the previously unresolved Mn2+-dependent step in replication.
PMCID: PMC3302264  PMID: 22205747
23.  Crystallization and preliminary crystallographic analysis of the major capsid proteins VP16 and VP17 of bacteriophage P23-77 
The major capsid proteins VP16 and VP17 of bacteriophage P23-77 have been crystallized using both recombinant and purified virus and preliminary diffraction analyses have been performed.
Members of the diverse double-β-barrel lineage of viruses are identified by the conserved structure of their major coat protein. New members of this lineage have been discovered based on structural analysis and we are interested in identifying relatives that utilize unusual versions of the double-β-barrel fold. One candidate for such studies is P23-77, an icosahedral dsDNA bacteriophage that infects the extremophile Thermus thermophilus. P23-77 has two major coat proteins, namely VP16 and VP17, of a size consistent with a single-β-barrel core fold. These previously unstudied proteins have now been successfully expressed as recombinant proteins, purified and crystallized using hanging-drop and sitting-drop vapour-diffusion methods. Crystals of coat proteins VP16 and VP17 have been obtained as well as of a putative complex. In addition, virus-derived material has been crystallized. Diffraction data have been collected to beyond 3 Å resolution for five crystal types and structure determinations are in progress.
PMCID: PMC3374517  PMID: 22691792
bacteriophages; capsid proteins
24.  Inhibition of Apoptosis and NF-κB Activation by Vaccinia Protein N1 Occur via Distinct Binding Surfaces and Make Different Contributions to Virulence 
PLoS Pathogens  2011;7(12):e1002430.
Vaccinia virus (VACV) protein N1 is an intracellular virulence factor and belongs to a family of VACV B-cell lymphoma (Bcl)-2-like proteins whose members inhibit apoptosis or activation of pro-inflammatory transcription factors, such as interferon (IFN) regulatory factor-3 (IRF-3) and nuclear factor-κB (NF-κB). Unusually, N1 inhibits both apoptosis and NF-κB activation. To understand how N1 exerts these different functions, we have mutated residues in the Bcl-2-like surface groove and at the interface used to form N1 homodimers. Mutagenesis of the surface groove abolished only the N1 anti-apoptotic activity and protein crystallography showed these mutants differed from wild-type N1 only at the site of mutation. Conversely, mutagenesis of the dimer interface converted N1 to a monomer and affected only inhibition of NF-κB activation. Collectively, these data show that N1 inhibits pro-inflammatory and pro-apoptotic signalling using independent surfaces of the protein. To determine the relative contribution of each activity to virus virulence, mutant N1 alleles were introduced into a VACV strain lacking N1 and the virulence of these viruses was analysed after intradermal and intranasal inoculation in mice. In both models, VACV containing a mutant N1 unable to inhibit apoptosis had similar virulence to wild-type virus, whereas VACV containing a mutant N1 impaired for NF-κB inhibition induced an attenuated infection similar to that of the N1-deleted virus. This indicates that anti-apoptotic activity of N1 does not drive virulence in these in vivo models, and highlights the importance of pro-inflammatory signalling in the immune response against viral infections.
Author Summary
Viruses have multiple strategies to escape the host immune system. These include proteins to inhibit cellular signalling pathways promoting an inflammatory response, and others that prevent programmed cell death (apoptosis), allowing completion of the virus replication cycle. This paper concerns the vaccinia virus (VACV) protein N1, which forms homodimers and blocks activation of both apoptosis and the pro-inflammatory NF-κB transcription factor. By introducing mutations in N1, we demonstrate that these functions are mediated by different surfaces of the protein. Biochemical and structural analysis of these mutants demonstrates that the anti-apoptotic activity of N1 relies on a hydrophobic groove on the surface of the protein and that the anti-NF-κB activity requires an intact dimer interface. Recombinant VACVs expressing the mutant N1 proteins were made to investigate the contributions of the different properties of N1 to virulence. The results showed that the anti-NF-κB activity of N1, rather than the N1-mediated inhibition of apoptosis, is the major contributor to virulence. This underlines the central role of pro-inflammatory signalling in the host immune response against viral infections.
PMCID: PMC3240604  PMID: 22194685
25.  Automation of large scale transient protein expression in mammalian cells 
Journal of Structural Biology  2011;175(2-2):209-215.
Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI− cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.
PMCID: PMC3477309  PMID: 21571074
Automated tissue culture; Eukaryotic expression system; HEK 293 cells; Transient transfection; HYPERFlask

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