The EMBL-EBI Job Dispatcher sequence analysis tools framework in 2024.

The EMBL-EBI Job Dispatcher sequence analysis tools framework (https://www.ebi.ac.uk/jdispatcher) enables the scientific community to perform a diverse range of sequence analyses using popular bioinformatics applications. Free access to the tools and required sequence datasets is provided through user-friendly web applications, as well as via RESTful and SOAP-based APIs. These are integrated into popular EMBL-EBI resources such as UniProt, InterPro, ENA and Ensembl Genomes. This paper overviews recent improvements to Job Dispatcher, including its brand new website and documentation, enhanced visualisations, improved job management, and a rising trend of user reliance on the service from low- and middle-income regions.

Simultaneous membrane and RNA binding by tick-borne encephalitis virus capsid protein.

Tick-borne encephalitis virus is an enveloped, pathogenic, RNA virus in the family Flaviviridae, genus Flavivirus. Viral particles are formed when the nucleocapsid, consisting of an RNA genome and multiple copies of the capsid protein, buds through the endoplasmic reticulum membrane and acquires the viral envelope and the associated proteins. The coordination of the nucleocapsid components to the sites of assembly and budding are poorly understood. Here, we investigate the interactions of the wild-type and truncated capsid proteins with membranes with biophysical methods and model membrane systems. We show that capsid protein initially binds membranes via electrostatic interactions with negatively-charged lipids, which is followed by membrane insertion. Additionally, we show that membrane-bound capsid protein can recruit viral genomic RNA. We confirm the biological relevance of the biophysical findings by using mass spectrometry to show that purified virions contain negatively-charged lipids. Our results suggest that nucleocapsid assembly is coordinated by negatively-charged membrane patches on the endoplasmic reticulum and that the capsid protein mediates direct contacts between the nucleocapsid and the membrane.

Safe Access Zone Legislation and Its Compliance with the Human Rights of Anti-Abortion Protesters in Australia.

Terminating a pregnancy is now lawful in all Australian jurisdictions, although on diverse bases. While abortions have not been subject to the same degree of heated debate in Australia as elsewhere, protests aimed at persuading women not to have a termination of their pregnancy have occurred outside abortion service providers in the past. Over the last decade, this has led to the introduction of laws setting out so-called safe access zones around provider premises. Anti-abortion protests are prohibited within a specific distance from abortion services and infringements attract criminal liability. As safe access zone laws prevent protesters from expressing their views in certain spaces, the question arises as to the laws' compliance with protesters' human rights. This article analyses this by considering the human rights compliance of the Queensland ban in light of Queensland human rights legislation. It concludes that the imposed prohibition of anti-abortion protests near abortion clinics is compatible with human rights.

Structural Studies Reveal that Endosomal Cations Promote Formation of Infectious Coxsackievirus A9 A-Particles, Facilitating RNA and VP4 Release.

Coxsackievirus A9 (CVA9), an enterovirus, is a common cause of pediatric aseptic meningitis and neonatal sepsis. During cell entry, enterovirus capsids undergo conformational changes leading to expansion, formation of large pores, externalization of VP1 N termini, and loss of the lipid factor from VP1. Factors such as receptor binding, heat, and acidic pH can trigger capsid expansion in some enteroviruses. Here, we show that fatty acid-free bovine serum albumin or neutral endosomal ionic conditions can independently prime CVA9 for expansion and genome release. Our results showed that CVA9 treatment with albumin or endosomal ions generated a heterogeneous population of virions, which could be physically separated by asymmetric flow field flow fractionation and computationally by cryo-electron microscopy (cryo-EM) and image processing. We report cryo-EM structures of CVA9 A-particles obtained by albumin or endosomal ion treatment and a control nonexpanded virion to 3.5, 3.3, and 2.9 Å resolution, respectively. Whereas albumin promoted stable expanded virions, the endosomal ionic concentrations induced unstable CVA9 virions which easily disintegrated, losing their genome. Loss of most of the VP4 molecules and exposure of negatively charged amino acid residues in the capsid's interior after expansion created a repulsive viral RNA-capsid interface, aiding genome release. IMPORTANCE Coxsackievirus A9 (CVA9) is a common cause of meningitis and neonatal sepsis. The triggers and mode of action of RNA release into the cell unusually do not require receptor interaction. Rather, a slow process in the endosome, independent of low pH, is required. Here, we show by biophysical separation, cryogenic electron microscopy, and image reconstruction that albumin and buffers mimicking the endosomal ion composition can separately and together expand and prime CVA9 for uncoating. Furthermore, we show in these expanded particles that VP4 is present at only ~10% of the occupancy found in the virion, VP1 is externalized, and the genome is repelled by the negatively charged, repulsive inner surface of the capsid that occurs due to the expansion. Thus, we can now link observations from cell biology of infection with the physical processes that occur in the capsid to promote genome uncoating.

A novel druggable interprotomer pocket in the capsid of rhino- and enteroviruses.

Rhino- and enteroviruses are important human pathogens, against which no antivirals are available. The best-studied inhibitors are "capsid binders" that fit in a hydrophobic pocket of the viral capsid. Employing a new class of entero-/rhinovirus inhibitors and by means of cryo-electron microscopy (EM), followed by resistance selection and reverse genetics, we discovered a hitherto unknown druggable pocket that is formed by viral proteins VP1 and VP3 and that is conserved across entero-/rhinovirus species. We propose that these inhibitors stabilize a key region of the virion, thereby preventing the conformational expansion needed for viral RNA release. A medicinal chemistry effort resulted in the identification of analogues targeting this pocket with broad-spectrum activity against Coxsackieviruses B (CVBs) and compounds with activity against enteroviruses (EV) of groups C and D, and even rhinoviruses (RV). Our findings provide novel insights in the biology of the entry of entero-/rhinoviruses and open new avenues for the design of broad-spectrum antivirals against these pathogens.

A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody.

Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCE Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

Extracellular Albumin and Endosomal Ions Prime Enterovirus Particles for Uncoating That Can Be Prevented by Fatty Acid Saturation.

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release.IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses.

Advances in high-throughput methods for the identification of virus receptors.

Viruses have evolved many mechanisms to invade host cells and establish successful infections. The interaction between viral attachment proteins and host cell receptors is the first and decisive step in establishing such infections, initiating virus entry into the host cells. Therefore, the identification of host receptors is fundamental in understanding pathogenesis and tissue tropism. Furthermore, receptor identification can inform the development of antivirals, vaccines, and diagnostic technologies, which have a substantial impact on human health. Nevertheless, due to the complex nature of virus entry, the redundancy in receptor usage, and the limitations in current identification methods, many host receptors remain elusive. Recent advances in targeted gene perturbation, high-throughput screening, and mass spectrometry have facilitated the discovery of virus receptors in recent years. In this review, we compare the current methods used within the field to identify virus receptors, focussing on genomic- and interactome-based approaches.

Complementary substrate specificity and distinct quaternary assembly of the Escherichia coli aerobic and anaerobic ß-oxidation trifunctional enzyme complexes.

The trifunctional enzyme (TFE) catalyzes the last three steps of the fatty acid ß-oxidation cycle. Two TFEs are present in Escherichia coli, EcTFE and anEcTFE. EcTFE is expressed only under aerobic conditions, whereas anEcTFE is expressed also under anaerobic conditions, with nitrate or fumarate as the ultimate electron acceptor. The anEcTFE subunits have higher sequence identity with the human mitochondrial TFE (HsTFE) than with the soluble EcTFE. Like HsTFE, here it is found that anEcTFE is a membrane-bound complex. Systematic enzyme kinetic studies show that anEcTFE has a preference for medium- and long-chain enoyl-CoAs, similar to HsTFE, whereas EcTFE prefers short chain enoyl-CoA substrates. The biophysical characterization of anEcTFE and EcTFE shows that EcTFE is heterotetrameric, whereas anEcTFE is purified as a complex of two heterotetrameric units, like HsTFE. The tetrameric assembly of anEcTFE resembles the HsTFE tetramer, although the arrangement of the two anEcTFE tetramers in the octamer is different from the HsTFE octamer. These studies demonstrate that EcTFE and anEcTFE have complementary substrate specificities, allowing for complete degradation of long-chain enoyl-CoAs under aerobic conditions. The new data agree with the notion that anEcTFE and HsTFE are evolutionary closely related, whereas EcTFE belongs to a separate subfamily.

k-SLAM: accurate and ultra-fast taxonomic classification and gene identification for large metagenomic data sets.

k-SLAM is a highly efficient algorithm for the characterization of metagenomic data. Unlike other ultra-fast metagenomic classifiers, full sequence alignment is performed allowing for gene identification and variant calling in addition to accurate taxonomic classification. A k-mer based method provides greater taxonomic accuracy than other classifiers and a three orders of magnitude speed increase over alignment based approaches. The use of alignments to find variants and genes along with their taxonomic origins enables novel strains to be characterized. k-SLAM's speed allows a full taxonomic classification and gene identification to be tractable on modern large data sets. A pseudo-assembly method is used to increase classification accuracy by up to 40% for species which have high sequence homology within their genus.

Influenza virus NS1 protein binds cellular DNA to block transcription of antiviral genes.

Influenza NS1 protein is an important virulence factor that is capable of binding double-stranded (ds) RNA and inhibiting dsRNA-mediated host innate immune responses. Here we show that NS1 can also bind cellular dsDNA. This interaction prevents loading of transcriptional machinery to the DNA, thereby attenuating IAV-mediated expression of antiviral genes. Thus, we identified a previously undescribed strategy, by which RNA virus inhibits cellular transcription to escape antiviral response and secure its replication.

Human picornaviruses associated with neurological diseases and their neutralization by antibodies.

Picornaviruses are the most commonly encountered infectious agents in mankind. They typically cause mild infections of the gastrointestinal or respiratory tract, but sometimes also invade the central nervous system. There, they can cause severe diseases with long-term sequelae and even be lethal. The most infamous picornavirus is poliovirus, for which significant epidemics of poliomyelitis were reported from the end of the nineteenth century. A successful vaccination campaign has brought poliovirus close to eradication, but neurological diseases caused by other picornaviruses have increasingly been reported since the late 1990s. In this review we focus on enterovirus 71, coxsackievirus A16, enterovirus 68 and human parechovirus 3, which have recently drawn attention because of their links to severe neurological diseases. We discuss the clinical relevance of these viruses and the primary role of humoral immunity in controlling them, and summarize current knowledge on the neutralization of such viruses by antibodies.

Crystal Structure of the Measles Virus Nucleoprotein Core in Complex with an N-Terminal Region of Phosphoprotein.

UNLABELLED: The enveloped negative-stranded RNA virus measles virus (MeV) is an important human pathogen. The nucleoprotein (N(0)) assembles with the viral RNA into helical ribonucleocapsids (NC) which are, in turn, coated by a helical layer of the matrix protein. The viral polymerase complex uses the NC as its template. The N(0) assembly onto the NC and the activity of the polymerase are regulated by the viral phosphoprotein (P). In this study, we pulled down an N(0)1₋408 fragment lacking most of its C-terminal tail domain by several affinity-tagged, N-terminal P fragments to map the N(0)-binding region of P to the first 48 amino acids. We showed biochemically and using P mutants the importance of the hydrophobic interactions for the binding. We fused an N(0) binding peptide, P1₋48, to the C terminus of an N(0)21₋408 fragment lacking both the N-terminal peptide and the C-terminal tail of N protein to reconstitute and crystallize the N(0)-P complex. We solved the X-ray structure of the resulting N(0)-P chimeric protein at a resolution of 2.7 Å. The structure reveals the molecular details of the conserved N(0)-P interface and explains how P chaperones N(0), preventing both self-assembly of N(0) and its binding to RNA. Finally, we propose a model for a preinitiation complex for RNA polymerization. IMPORTANCE: Measles virus is an important, highly contagious human pathogen. The nucleoprotein N binds only to viral genomic RNA and forms the helical ribonucleocapsid that serves as a template for viral replication. We address how N is regulated by another protein, the phosphoprotein (P), to prevent newly synthesized N from binding to cellular RNA. We describe the atomic model of an N-P complex and compare it to helical ribonucleocapsid. We thus provide insight into how P chaperones N and helps to start viral RNA synthesis. Our results provide a new insight into mechanisms of paramyxovirus replication. New data on the mechanisms of phosphoprotein chaperone action allows better understanding of virus genome replication and nucleocapsid assembly. We describe a conserved structural interface for the N-P interaction which could be a target for drug development to treat not only measles but also potentially other paramyxovirus diseases.

Structural Basis of Human Parechovirus Neutralization by Human Monoclonal Antibodies.

UNLABELLED: Since it was first recognized in 2004 that human parechoviruses (HPeV) are a significant cause of central nervous system and neonatal sepsis, their clinical importance, primarily in children, has started to emerge. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases and has given moderate success. Direct inhibition of parechovirus infection using monoclonal antibodies is a potential treatment. We have developed two neutralizing monoclonal antibodies against HPeV1 and HPeV2, namely, AM18 and AM28, which also cross-neutralize other viruses. Here, we present the mapping of their epitopes using peptide scanning, surface plasmon resonance, fluorescence-based thermal shift assays, electron cryomicroscopy, and image reconstruction. We determined by peptide scanning and surface plasmon resonance that AM18 recognizes a linear epitope motif including the arginine-glycine-aspartic acid on the C terminus of capsid protein VP1. This epitope is normally used by the virus to attach to host cell surface integrins during entry and is found in 3 other viruses that AM18 neutralizes. Therefore, AM18 is likely to cause virus neutralization by aggregation and by blocking integrin binding to the capsid. Further, we show by electron cryomicroscopy, three-dimensional reconstruction, and pseudoatomic model fitting that ordered RNA interacts with HPeV1 VP1 and VP3. AM28 recognizes quaternary epitopes on the capsid composed of VP0 and VP3 loops from neighboring pentamers, thereby increasing the RNA accessibility temperature for the virus-AM28 complex compared to the virus alone. Thus, inhibition of RNA uncoating probably contributes to neutralization by AM28. IMPORTANCE: Human parechoviruses can cause mild infections to severe diseases in young children, such as neonatal sepsis, encephalitis, and cardiomyopathy. Intravenous immunoglobulin treatment is the only treatment available in such life-threatening cases. In order to develop more targeted treatment, we have searched for human monoclonal antibodies that would neutralize human parechoviruses 1 and 2, associated with mild infections such as gastroenteritis and severe infections of the central nervous system, and thus allow safe treatment. In the current study, we show how two such promising antibodies interact with the virus, modeling the atomic interactions between the virus and the antibody to propose how neutralization occurs. Both antibodies can cause aggregation; in addition, one antibody interferes with the virus recognizing its target cell, while the other, recognizing only the whole virus, inhibits the genome uncoating and replication in the cell.

Insights into virus evolution and membrane biogenesis from the structure of the marine lipid-containing bacteriophage PM2.

Recent, primarily structural observations indicate that related viruses, harboring no sequence similarity, infect hosts of different domains of life. One such clade of viruses, defined by common capsid architecture and coat protein fold, is the so-called PRD1-adenovirus lineage. Here we report the structure of the marine lipid-containing bacteriophage PM2 determined by crystallographic analyses of the entire approximately 45 MDa virion and of the outer coat proteins P1 and P2, revealing PM2 to be a primeval member of the PRD1-adenovirus lineage with an icosahedral shell and canonical double beta barrel major coat protein. The view of the lipid bilayer, richly decorated with membrane proteins, constitutes a rare visualization of an in vivo membrane. The viral membrane proteins P3 and P6 are organized into a lattice, suggesting a possible assembly pathway to produce the mature virus.

Architecture of respiratory syncytial virus revealed by electron cryotomography.

Human respiratory syncytial virus is a human pathogen that causes severe infection of the respiratory tract. Current information about the structure of the virus and its interaction with host cells is limited. We carried out an electron cryotomographic characterization of cell culture-grown human respiratory syncytial virus to determine the architecture of the virion. The particles ranged from 100 nm to 1,000 nm in diameter and were spherical, filamentous, or a combination of the two. The filamentous morphology correlated with the presence of a cylindrical matrix protein layer linked to the inner leaflet of the viral envelope and with local ordering of the glycoprotein spikes. Recombinant viruses with only the fusion protein in their envelope showed that these glycoproteins were predominantly in the postfusion conformation, but some were also in the prefusion form. The ribonucleocapsids were left-handed, randomly oriented, and curved inside the virions. In filamentous particles, they were often adjacent to an intermediate layer of protein assigned to M2-1 (an envelope-associated protein known to mediate association of ribonucleocapsids with the matrix protein). Our results indicate important differences in structure between the Paramyxovirinae and Pneumovirinae subfamilies within the Paramyxoviridae, and provide fresh insights into host cell exit of a serious pathogen.

Structure of the archaeal head-tailed virus HSTV-1 completes the HK97 fold story.

It has been proposed that viruses can be divided into a small number of structure-based viral lineages. One of these lineages is exemplified by bacterial virus Hong Kong 97 (HK97), which represents the head-tailed dsDNA bacteriophages. Seemingly similar viruses also infect archaea. Here we demonstrate using genomic analysis, electron cryomicroscopy, and image reconstruction that the major coat protein fold of newly isolated archaeal Haloarcula sinaiiensis tailed virus 1 has the canonical coat protein fold of HK97. Although it has been anticipated previously, this is physical evidence that bacterial and archaeal head-tailed viruses share a common architectural principle. The HK97-like fold has previously been recognized also in herpesviruses, and this study expands the HK97-like lineage to viruses from all three domains of life. This is only the second established lineage to include archaeal, bacterial, and eukaryotic viruses. Thus, our findings support the hypothesis that the last common universal ancestor of cellular organisms was infected by a number of different viruses.

Hydrophobin film structure for HFBI and HFBII and mechanism for accelerated film formation.

Hydrophobins represent an important group of proteins from both a biological and nanotechnological standpoint. They are the means through which filamentous fungi affect their environment to promote growth, and their properties at interfaces have resulted in numerous applications. In our study we have combined protein docking, molecular dynamics simulation, and electron cryo-microscopy to gain atomistic level insight into the surface structure of films composed of two class II hydrophobins: HFBI and HFBII produced by Trichoderma reesei. Together our results suggest a unit cell composed of six proteins; however, our computational results suggest P6 symmetry, while our experimental results show P3 symmetry with a unit cell size of 56 Å. Our computational results indicate the possibility of an alternate ordering with a three protein unit cell with P3 symmetry and a smaller unit cell size, and we have used a Monte Carlo simulation of a spin model representing the hydrophobin film to show how this alternate metastable structure may play a role in increasing the rate of surface coverage by hydrophobin films, possibly indicating a mechanism of more general significance to both biology and nanotechnology.

Combined approaches to flexible fitting and assessment in virus capsids undergoing conformational change.

Fitting of atomic components into electron cryo-microscopy (cryoEM) density maps is routinely used to understand the structure and function of macromolecular machines. Many fitting methods have been developed, but a standard protocol for successful fitting and assessment of fitted models has yet to be agreed upon among the experts in the field. Here, we created and tested a protocol that highlights important issues related to homology modelling, density map segmentation, rigid and flexible fitting, as well as the assessment of fits. As part of it, we use two different flexible fitting methods (Flex-EM and iMODfit) and demonstrate how combining the analysis of multiple fits and model assessment could result in an improved model. The protocol is applied to the case of the mature and empty capsids of Coxsackievirus A7 (CAV7) by flexibly fitting homology models into the corresponding cryoEM density maps at 8.2 and 6.1Å resolution. As a result, and due to the improved homology models (derived from recently solved crystal structures of a close homolog - EV71 capsid - in mature and empty forms), the final models present an improvement over previously published models. In close agreement with the capsid expansion observed in the EV71 structures, the new CAV7 models reveal that the expansion is accompanied by ∼5° counterclockwise rotation of the asymmetric unit, predominantly contributed by the capsid protein VP1. The protocol could be applied not only to viral capsids but also to many other complexes characterised by a combination of atomic structure modelling and cryoEM density fitting.

Using a periclinal chimera to unravel layer-specific gene expression in plants.

Plant organs are made from multiple cell types, and defining the expression level of a gene in any one cell or group of cells from a complex mixture is difficult. Dicotyledonous plants normally have three distinct layers of cells, L1, L2 and L3. Layer L1 is the single layer of cells making up the epidermis, layer L2 the single cell sub-epidermal layer and layer L3 constitutes the rest of the internal cells. Here we show how it is possible to harvest an organ and characterise the level of layer-specific expression by using a periclinal chimera that has its L1 layer from Solanum pennellii and its L2 and L3 layers from Solanum lycopersicum. This is possible by measuring the level of the frequency of species-specific transcripts. RNA-seq analysis enabled the genome-wide assessment of whether a gene is expressed in the L1 or L2/L3 layers. From 13 277 genes that are expressed in both the chimera and the parental lines and with at least one polymorphism between the parental alleles, we identified 382 genes that are preferentially expressed in L1 in contrast to 1159 genes in L2/L3. Gene ontology analysis shows that many genes preferentially expressed in L1 are involved in cutin and wax biosynthesis, whereas numerous genes that are preferentially expressed in L2/L3 tissue are associated with chloroplastic processes. These data indicate the use of such chimeras and provide detailed information on the level of layer-specific expression of genes.