p53 and its related genes, p63 and p73 constitute the p53 gene family. While p53 is the most frequently mutated gene in human tumors, p63 and p73 are rarely mutated or deleted in cancers. Many studies have reported p63/p73 overexpression in human cancers while others showed that a loss of p63/p73 is associated with tumor progression and metastasis. Thus, whether p63 or p73 is a tumor suppressor gene or an oncogene has been a matter of debate. This controversy has been attributed to the existence of multiple splicing isoforms with distinct functions; the full-length TA isoform of p63 has structural and functional similarity to wild-type p53, whereas the ?Np63 acts primarily in dominant-negative fashion against all family members of p53. Differential activities of TA and ?N isoforms have been shown in vivo by creating isform-specific gene knockout mice. All p53, p63, p73 proteins bind to and activate target genes with p53-response elements; p63 also binds to distinct p63-response elements and regulate expression of specific target genes involved in skin, limb, and craniofacial development. Interestingly, several studies have shown that both p63 and p73 are involved in cellular response to cancer therapy and others have indicated that both of these molecules are required for p53-induced apoptosis, suggesting functional interplay among p53 family proteins. Consistent with these findings, aberrant splicing that result in ?Np63 or ?Np73 overexpression are frequently found in human cancers, and is associated with poor clinical outcomes of patients in the latter. Thus immunohistochemical staining of tumor specimen with ?Np73-specific antibody might have diagnostic values in cancer clinics.
Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.
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.
Enterovirus 71 (HEV71) epidemics in children and infants result mainly in mild symptoms; however, especially in the Asia-Pacific region, infection can be fatal. At present, no therapies are available. We have used structural analysis of the complete virus to guide the design of HEV71 inhibitors. Analysis of complexes with four 3-(4-pyridyl)-2-imidazolidinone derivatives with varying anti-HEV71 activities pinpointed key structure-activity correlates. We then identified additional potentially beneficial substitutions, developed methods to reliably triage compounds by quantum mechanics-enhanced ligand docking and synthesized two candidates. Structural analysis and in vitro assays confirmed the predicted binding modes and their ability to block viral infection. One ligand (with IC50 of 25 pM) is an order of magnitude more potent than the best previously reported inhibitor and is also more soluble. Our approach may be useful in the design of effective drugs for enterovirus infections.
The integral membrane protein LIMP-2 has been a paradigm for mannose 6-phosphate receptor (MPR) independent lysosomal targeting, binding to ?-glucocerebrosidase (?-GCase) and directing it to the lysosome, before dissociating in the late-endosomal/lysosomal compartments. Here we report structural results illuminating how LIMP-2 binds and releases ?-GCase according to changes in pH, via a histidine trigger, and suggesting that LIMP-2 localizes the ceramide portion of the substrate adjacent to the ?-GCase catalytic site. Remarkably, we find that LIMP-2 bears P-Man9GlcNAc2 covalently attached to residue N325, and that it binds MPR, via mannose 6-phosphate, with a similar affinity to that observed between LIMP-2 and ?-GCase. The binding sites for ?-GCase and the MPR are functionally separate, so that a stable ternary complex can be formed. By fluorescence lifetime imaging microscopy, we also demonstrate that LIMP-2 interacts with MPR in living cells. These results revise the accepted view of LIMP-2-?-GCase lysosomal targeting.
Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1-cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1-induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1(+/+);MMTV-cyclin D1 and Dmp1(+/+);MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer.
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.
Successful cryogenic X-ray structure determination from a single high-pressure-frozen bovine enterovirus 2 crystal is reported. The presented high-pressure-freezing procedure is based on a commercially available device and allows the cryocooling of macromolecular crystals directly in their mother liquor without the time- and crystal-consuming search for optimal cryoconditions. The method is generally applicable and will allow cryogenic data collection from all types of macromolecular crystals.
It remains largely mysterious how the genomes of non-enveloped eukaryotic viruses are transferred across a membrane into the host cell. Picornaviruses are simple models for such viruses, and initiate this uncoating process through particle expansion, which reveals channels through which internal capsid proteins and the viral genome presumably exit the particle, although this has not been clearly seen until now. Here we present the atomic structure of an uncoating intermediate for the major human picornavirus pathogen CAV16, which reveals VP1 partly extruded from the capsid, poised to embed in the host membrane. Together with previous low-resolution results, we are able to propose a detailed hypothesis for the ordered egress of the internal proteins, using two distinct sets of channels through the capsid, and suggest a structural link to the condensed RNA within the particle, which may be involved in triggering RNA release.
Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1? transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21(Cip1), markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1? and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1? prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1? transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1? protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1? expression.
Gain-of-function mutations in oncogenes and loss-of-function mutations in tumor suppressor genes (TSG) lead to cancer. In most human cancers, these mutations occur in somatic tissues. However, hereditary forms of cancer exist for which individuals are heterozygous for a germline mutation in a TSG locus at birth. The second allele is frequently inactivated by gene deletion, point mutation, or promoter methylation in classical TSGs that meet Knudsons two-hit hypothesis. Conversely, the second allele remains as wild-type, even in tumors in which the gene is haplo-insufficient for tumor suppression. This article highlights the importance of PTEN, APC, and other tumor suppressors for counteracting aberrant PI3K, ?-catenin, and other oncogenic signaling pathways. We discuss the use of gene-engineered mouse models (GEMM) of human cancer focusing on Pten and Apc knockout mice that recapitulate key genetic events involved in initiation and progression of human neoplasia. Finally, the therapeutic potential of targeting these tumor suppressor and oncogene signaling networks is discussed.
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.
Cancer is caused by multiple genetic alterations leading to uncontrolled cell proliferation through multiple pathways. Malignant cells arise from a variety of genetic factors, such as mutations in tumor suppressor genes (TSGs) that are involved in regulating the cell cycle, apoptosis, or cell differentiation, or maintenance of genomic integrity. Tumor suppressor mouse models are the most frequently used animal models in cancer research. The anti-tumorigenic functions of TSGs, and their role in development and differentiation, and inhibition of oncogenes are discussed. In this review, we summarize some of the important transgenic and knockout mouse models for TSGs, including Rb, p53, Ink4a/Arf, Brca1/2, and their related genes.
Inhibition of protein kinases has validated therapeutic utility for cancer, with at least seven kinase inhibitor drugs on the market. Protein kinase inhibition also has significant potential for a variety of other diseases, including diabetes, pain, cognition, and chronic inflammatory and immunologic diseases. However, as the vast majority of current approaches to kinase inhibition target the highly conserved ATP-binding site, the use of kinase inhibitors in treating nononcology diseases may require great selectivity for the target kinase. As protein kinases are signal transducers that are involved in binding to a variety of other proteins, targeting alternative, less conserved sites on the protein may provide an avenue for greater selectivity. Here we report an affinity-based, high-throughput screening technique that allows nonbiased interrogation of small molecule libraries for binding to all exposed sites on a protein surface. This approach was used to screen both the c-Jun N-terminal protein kinase Jnk-1 (involved in insulin signaling) and p38? (involved in the formation of TNF? and other cytokines). In addition to canonical ATP-site ligands, compounds were identified that bind to novel allosteric sites. The nature, biological relevance, and mode of binding of these ligands were extensively characterized using two-dimensional (1)H/(13)C NMR spectroscopy, protein X-ray crystallography, surface plasmon resonance, and direct enzymatic activity and activation cascade assays. Jnk-1 and p38? both belong to the MAP kinase family, and the allosteric ligands for both targets bind similarly on a ledge of the protein surface exposed by the MAP insertion present in the CMGC family of protein kinases and distant from the active site. Medicinal chemistry studies resulted in an improved Jnk-1 ligand able to increase adiponectin secretion in human adipocytes and increase insulin-induced protein kinase PKB phosphorylation in human hepatocytes, in similar fashion to Jnk-1 siRNA and to rosiglitazone treatment. Together, the data suggest that these new ligand series bind to a novel, allosteric, and physiologically relevant site and therefore represent a unique approach to identify kinase inhibitors.
A surprisingly strong spin rate dependence of (15)N and (13)C NMR T(1) times in magic angle spinning experiments on solid peptides is demonstrated. Using a variety of isotopomers, the phenomenon is shown to be the result of chemical shift anisotropy-mediated spin diffusion. This effect has the potential to be used to detect long-range distance constraints in macromolecular systems.
Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence--by controlling for phylogenetic structure--for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease.
Equine rhinitis A virus (ERAV) shares many features with foot-and-mouth disease virus (FMDV) and both are classified within the genus Aphthovirus of the family Picornaviridae. ERAV is used as a surrogate for FMDV research as it does not require high-level biosecurity. In contrast to FMDV, which uses integrins as cellular receptors, the receptor for ERAV has been reported to involve the sugar moiety sialic acid. This study confirmed the importance of sialic acid for cell entry by ERAV and reports the crystal structure of ERAV particles complexed with the receptor analogue 3-sialyllactose. The receptor is attached to the rim of a capsid pit adjacent to the major immunogenic site and distinct from the sialic acid binding site used by a related picornavirus, the cardiovirus Theilers murine encephalitis virus. The structure of the major antigenic determinant of the virus, previously identified from antibody escape mutations, is also described as the EF loop of VP1, which forms a hairpin stretching across the capsid surface close to the icosahedral fivefold axis, neighbouring the receptor-binding site, and spanning two protomeric units.
The use of biomarkers ensures breast cancer patients receive optimal treatment. Established biomarkers such as estrogen receptor (ER) and progesterone receptor (PR) have been playing significant roles in the selection and management of patients for endocrine therapy. HER2 is a strong predictor of response to trastuzumab. Recently, the roles of ER as a negative and HER2 as a positive indicator for chemotherapy have been established. Ki67 has traditionally been recognized as a poor prognostic factor, but recent studies suggest that measurement of Ki67-positive cells during treatment will more effectively predict treatment efficacy for both anti-hormonal and chemotherapy. p53 mutations are found in 20-35% of human breast cancers and are associated with aggressive disease with poor clinical outcome when the DNA-binding domain is mutated. The utility of cyclin D1 as a predictor of breast cancer prognosis is controversial, but cyclin D1b overexpression is associated with poor prognosis. Likewise, overexpression of the low molecular weight form of cyclin E1 protein predicts poor prognosis. Breast cancers from BRCA1/2 carriers often show high nuclear grades, negativity to ER/PR/HER2, and p53 mutations, and thus, are associated with poor prognosis. The prognostic values of other molecular markers, such as p14(ARF), TBX2/3, VEGF in breast cancer are also discussed. Careful evaluation of these biomarkers with current treatment modality is required to determine whether their measurement or monitoring offer significant clinical benefits.
We have developed a series of phenylpyrrolidine- and phenylpiperidine-substituted benzimidazole carboxamide poly(ADP-ribose) polymerase (PARP) inhibitors with excellent PARP enzyme potency as well as single-digit nanomolar cellular potency. These efforts led to the identification of (S)-2-(2-fluoro-4-(pyrrolidin-2-yl)phenyl)-1H-benzimidazole-4-carboxamide (22b, A-966492). Compound 22b displayed excellent potency against the PARP-1 enzyme with a K(i) of 1 nM and an EC(50) of 1 nM in a whole cell assay. In addition, 22b is orally bioavailable across multiple species, crosses the blood-brain barrier, and appears to distribute into tumor tissue. It also demonstrated good in vivo efficacy in a B16F10 subcutaneous murine melanoma model in combination with temozolomide and in an MX-1 breast cancer xenograft model both as a single agent and in combination with carboplatin.
The morphogenesis of oligodendrocytes is essential for central nervous system myelin formation and the rapid propagation of axon potentials through saltatory conduction. However, the discrete cellular events involved in the three-dimensional maturation of oligodendrocytes remain to be fully described. To address this, we followed the developmental stages of oligodendrocytes in mouse organotypic hippocampal slice cultures for 7-60 days using viral-mediated gene delivery of membrane-targeted fluorescent proteins. Using static and time-lapse confocal imaging, we find that postmigratory NG2-expressing cells exhibit slow anatomical reorganization over the course of hours. This is in direct contrast to oligodendrocytes that take on a promyelinating and transitional phenotype, which display a more complex morphology and undergo dramatic actin-dependent structural remodeling over just minutes. More mature myelinating oligodendrocytes, which have pruned most of their processes, still retain some local remodeling behavior at developing internodes, but in general, revert to a relatively stable state. Our findings provide a detailed characterization of cellular events that help shape oligodendrocyte morphology and likely participate in neuron-glial cell interactions and the process of myelination.
Equine rhinitis A virus (ERAV) is closely related to foot-and-mouth disease virus (FMDV), belonging to the genus Aphthovirus of the Picornaviridae. How picornaviruses introduce their RNA genome into the cytoplasm of the host cell to initiate replication is unclear since they have no lipid envelope to facilitate fusion with cellular membranes. It has been thought that the dissociation of the FMDV particle into pentameric subunits at acidic pH is the mechanism for genome release during cell entry, but this raises the problem of how transfer across the endosome membrane of the genome might be facilitated. In contrast, most other picornaviruses form altered particle intermediates (not reported for aphthoviruses) thought to induce membrane pores through which the genome can be transferred. Here we show that ERAV, like FMDV, dissociates into pentamers at mildly acidic pH but demonstrate that dissociation is preceded by the transient formation of empty 80S particles which have released their genome and may represent novel biologically relevant intermediates in the aphthovirus cell entry process. The crystal structures of the native ERAV virus and a low pH form have been determined via highly efficient crystallization and data collection strategies, required due to low virus yields. ERAV is closely similar to FMDV for VP2, VP3 and part of VP4 but VP1 diverges, to give a particle with a pitted surface, as seen in cardioviruses. The low pH particle has internal structure consistent with it representing a pre-dissociation cell entry intermediate. These results suggest a unified mechanism of picornavirus cell entry.
Based on screening hit 1, a series of tricyclic quinoxalinones have been designed and evaluated for inhibition of PARP-1. Substitutions at the 7- and 8-positions of the quinoxalinone ring led to a number of compounds with good enzymatic and cellular potency. The tricyclic quinoxalinone class is sensitive to modifications of both the amine substituent and the tricyclic core. The synthesis and structure-activity relationship studies are presented.
B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention in human physiology. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using the high-affinity monoclonal IgG1 antibody 106.3, which binds an epitope spanning residues 5-13 of the mature bioactive peptide. To understand the structural basis of this molecular recognition, we crystallized the Fab fragment complexed with the peptide epitope and determined the three-dimensional structure by X-ray diffraction to 2.1 A resolution. The structure reveals the detailed interactions that five of the complementarity-determining regions make with the partially folded peptide. Thermodynamic measurements using fluorescence spectroscopy suggest that the interaction is enthalpy driven, with an overall change in free energy of binding, DeltaG = -54 kJ/mol, at room temperature. The parameters are interpreted on the basis of the structural information. The kinetics of binding suggest a diffusion-limited mechanism, whereby the peptide easily adopts a bound conformation upon interaction with the antibody. Moreover, comparative analysis with alanine-scanning results of the epitope explains the basis of selectivity for BNP over other related natriuretic peptides.
Foot-and-mouth disease virus (FMDV) VP1 G-H loop contains the major antigenic site. By replacing the sequence upstream of the RGD motif with a FLAG epitope, a marker virus for pathogenesis studies was generated. In cell culture, the recombinant virus containing FLAG (A24-FLAG) exhibited similar plaque phenotypes and growth kinetics to parental virus. A24-FLAG was distinguished, neutralized, and immunoprecipitated by FLAG anti-sera. A24-FLAG infected cattle exhibited FMD and an antibody response similar to parental virus. FLAG epitope stability was confirmed both in vitro and in vivo. Interestingly, no anti-FLAG antibodies were detectable in cattle up to 21 days post-inoculation. A24-FLAG G-H loop modeling suggested FLAG was rendered a cryptic site, inaccessible to the host immune system. These studies demonstrate the FMDV VP1 G-H loop tolerance to substitutions without detriment to pathogenesis and antigenicity. Finally, A24-FLAG manifested virulence in cattle as parental virus, and could be distinguished and tracked by tag-specific anti-sera.
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.
Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease of cloven-hoofed animals with an almost-worldwide distribution. Conventional FMD vaccines consisting of chemically inactivated viruses have aided in the eradication of FMD from Europe and remain the main tool for control in endemic countries. Although significant steps have been made to improve the quality of vaccines, such as improved methods of antigen concentration and purification, manufacturing processes are technically demanding and expensive. Consequently, there is large variation in the quality of vaccines distributed in FMD-endemic countries compared with those manufactured for emergency use in FMD-free countries. Here, we have used reverse genetics to introduce haemagglutinin (HA) and FLAG tags into the foot-and-mouth disease virus (FMDV) capsid. HA- and FLAG-tagged FMDVs were infectious, with a plaque morphology similar to the non-tagged parental infectious copy virus and the field virus. The tagged viruses utilized integrin-mediated cell entry and retained the tag epitopes over serial passages. In addition, infectious HA- and FLAG-tagged FMDVs were readily purified from small-scale cultures using commercial antibodies. Tagged FMDV offers a feasible alternative to the current methods of vaccine concentration and purification, a potential to develop FMD vaccine conjugates and a unique tool for FMDV research.
A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100?K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin ? Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A(2A) adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macromolecular crystallography.
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.
Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.
Enterovirus 71 (EV71) is a major agent of hand, foot and mouth disease in children that can cause severe central nervous system disease and death. No vaccine or antiviral therapy is available. High-resolution structural analysis of the mature virus and natural empty particles shows that the mature virus is structurally similar to other enteroviruses. In contrast, the empty particles are markedly expanded and resemble elusive enterovirus-uncoating intermediates not previously characterized in atomic detail. Hydrophobic pockets in the EV71 capsid are collapsed in this expanded particle, providing a detailed explanation of the mechanism for receptor-binding triggered virus uncoating. These structures provide a model for enterovirus uncoating in which the VP1 GH loop acts as an adaptor-sensor for cellular receptor attachment, converting heterologous inputs to a generic uncoating mechanism, highlighting new opportunities for therapeutic intervention.
The transcription factor Dmp1 is a Ras/HER2-activated haplo-insufficient tumor suppressor that activates the Arf/p53 pathway of cell-cycle arrest. Recent evidence suggests that Dmp1 may activate p53 independently of Arf in certain cell types. Here, we report findings supporting this concept with the definition of an Arf-independent function for Dmp1 in tumor suppression. We found that Dmp1 and p53 can interact directly in mammalian cells via the carboxyl-terminus of p53 and the DNA-binding domain of Dmp1. Expression of Dmp1 antagonized ubiquitination of p53 by Mdm2 and promoted nuclear localization of p53. Dmp1-p53 binding significantly increased the level of p53, independent of the DNA-binding activity of Dmp1. Mechanistically, p53 target genes were activated synergistically by the coexpression of Dmp1 and p53 in p53(-/-);Arf(-/-) cells, and genotoxic responses of these genes were hampered more dramatically in Dmp1(-/-) and p53(-/-) cells than in Arf(-/-) cells. Together, our findings identify a robust new mechanism of p53 activation mediated by direct physical interaction between Dmp1 and p53.
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