VitroJet: new features and case studies.

Single-particle cryo-electron microscopy has become a widely adopted method in structural biology due to many recent technological advances in microscopes, detectors and image processing. Before being able to inspect a biological sample in an electron microscope, it needs to be deposited in a thin layer on a grid and rapidly frozen. The VitroJet was designed with this aim, as well as avoiding the delicate manual handling and transfer steps that occur during the conventional grid-preparation process. Since its creation, numerous technical developments have resulted in a device that is now widely utilized in multiple laboratories worldwide. It features plasma treatment, low-volume sample deposition through pin printing, optical ice-thickness measurement and cryofixation of pre-clipped Autogrids through jet vitrification. This paper presents recent technical improvements to the VitroJet and the benefits that it brings to the cryo-EM workflow. A wide variety of applications are shown: membrane proteins, nucleosomes, fatty-acid synthase, Tobacco mosaic virus, lipid nanoparticles, tick-borne encephalitis viruses and bacteriophages. These case studies illustrate the advancement of the VitroJet into an instrument that enables accurate control and reproducibility, demonstrating its suitability for time-efficient cryo-EM structure determination.

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Antiviral action of a functionalized plastic surface against human coronaviruses.

Viruses may persist on solid surfaces for long periods, which may contribute to indirect transmission. Thus, it is imperative to develop functionalized surfaces that will lower the infectious viral load in everyday life. Here, we have tested a plastic surface functionalized with tall oil rosin against the seasonal human coronavirus OC43 as well as severe acute respiratory syndrome coronavirus 2. All tested non-functionalized plastic surfaces showed virus persistence up to 48 h. In contrast, the functionalized plastic showed good antiviral action already within 15 min of contact and excellent efficacy after 30 min over 90% humidity. Excellent antiviral effects were also observed at lower humidities of 20% and 40%. Despite the hydrophilic nature of the functionalized plastic, viruses did not adhere strongly to it. According to helium ion microscopy, viruses appeared flatter on the rosin-functionalized surface, but after flushing away from the rosin-functionalized surface, they showed no apparent structural changes when imaged by transmission electron microscopy of cryogenic or negatively stained specimens or by atomic force microscopy. Flushed viruses were able to bind to their host cell surface and enter endosomes, suggesting that the fusion with the endosomal membrane was halted. The eluted rosin from the functionalized surface demonstrated its ability to inactivate viruses, indicating that the antiviral efficacy relied on the active leaching of the antiviral substances, which acted on the viruses coming into contact. The rosin-functionalized plastic thus serves as a promising candidate as an antiviral surface for enveloped viruses.IMPORTANCEDuring seasonal and viral outbreaks, the implementation of antiviral plastics can serve as a proactive strategy to limit the spread of viruses from contaminated surfaces, complementing existing hygiene practices. In this study, we show the efficacy of a rosin-functionalized plastic surface that kills the viral infectivity of human coronaviruses within 15 min of contact time, irrespective of the humidity levels. In contrast, non-functionalized plastic surfaces retain viral infectivity for an extended period of up to 48 h. The transient attachment on the surface or the leached active components do not cause major structural changes in the virus or prevent receptor binding; instead, they effectively block viral infection at the endosomal stage.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Structural basis for different membrane-binding properties of E. coli anaerobic and human mitochondrial ß-oxidation trifunctional enzymes.

Facultative anaerobic bacteria such as Escherichia coli have two α2ß2 heterotetrameric trifunctional enzymes (TFE), catalyzing the last three steps of the ß-oxidation cycle: soluble aerobic TFE (EcTFE) and membrane-associated anaerobic TFE (anEcTFE), closely related to the human mitochondrial TFE (HsTFE). The cryo-EM structure of anEcTFE and crystal structures of anEcTFE-α show that the overall assembly of anEcTFE and HsTFE is similar. However, their membrane-binding properties differ considerably. The shorter A5-H7 and H8 regions of anEcTFE-α result in weaker α-ß as well as α-membrane interactions, respectively. The protruding H-H region of anEcTFE-ß is therefore more critical for membrane-association. Mutational studies also show that this region is important for the stability of the anEcTFE-ß dimer and anEcTFE heterotetramer. The fatty acyl tail binding tunnel of the anEcTFE-α hydratase domain, as in HsTFE-α, is wider than in EcTFE-α, accommodating longer fatty acyl tails, in good agreement with their respective substrate specificities.

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.

SARS-CoV-2 Production, Purification Methods and UV Inactivation for Proteomics and Structural Studies.

Severe acute respiratory syndrome coronavirus-2 is the causative agent of COVID-19. During the pandemic of 2019-2022, at least 500 million have been infected and over 6.3 million people have died from COVID-19. The virus is pleomorphic, and due to its pathogenicity is often handled in very restrictive biosafety containments laboratories. We developed two effective and rapid purification methods followed by UV inactivation that allow easy downstream handling of the virus. We monitored the purification through titering, sequencing, mass spectrometry and electron cryogenic microscopy. Although pelleting through a sucrose cushion, followed by gentle resuspension overnight gave the best particle recovery, infectivity decreased, and the purity was significantly worse than if using the size exclusion resin Capto Core. Capto Core can be used in batch mode, and was seven times faster than the pelleting method, obviating the need for ultracentrifugation in the containment laboratory, but resulting in a dilute virus. UV inactivation was readily optimized to allow handling of the inactivated samples under standard operating conditions. When containment laboratory space is limited, we recommend the use of Capto Core for purification and UV for inactivation as a simple, rapid workflow prior, for instance, to electron cryogenic microscopy or cell activation experiments.

Molecular Organisation of Tick-Borne Encephalitis Virus.

Tick-borne encephalitis virus (TBEV) is a pathogenic, enveloped, positive-stranded RNA virus in the family Flaviviridae. Structural studies of flavivirus virions have primarily focused on mosquito-borne species, with only one cryo-electron microscopy (cryo-EM) structure of a tick-borne species published. Here, we present a 3.3 Å cryo-EM structure of the TBEV virion of the Kuutsalo-14 isolate, confirming the overall organisation of the virus. We observe conformational switching of the peripheral and transmembrane helices of M protein, which can explain the quasi-equivalent packing of the viral proteins and highlights their importance in stabilising membrane protein arrangement in the virion. The residues responsible for M protein interactions are highly conserved in TBEV but not in the structurally studied Hypr strain, nor in mosquito-borne flaviviruses. These interactions may compensate for the lower number of hydrogen bonds between E proteins in TBEV compared to the mosquito-borne flaviviruses. The structure reveals two lipids bound in the E protein which are important for virus assembly. The lipid pockets are comparable to those recently described in mosquito-borne Zika, Spondweni, Dengue, and Usutu viruses. Our results thus advance the understanding of tick-borne flavivirus architecture and virion-stabilising interactions.

Virus structure and structure-based antivirals.

Structure-based antiviral developments in the past two years have been dominated by the structure determination and inhibition of SARS-CoV-2 proteins and new lead molecules for picornaviruses. The SARS-CoV-2 spike protein has been targeted successfully with antibodies, nanobodies, and receptor protein mimics effectively blocking receptor binding or fusion. The two most promising non-structural proteins sharing strong structural and functional conservation across virus families are the main protease and the RNA-dependent RNA polymerase, for which design and reuse of broad range inhibitors already approved for use has been an attractive avenue. For picornaviruses, the increasing recognition of the transient expansion of the capsid as a critical transition towards RNA release has been targeted through a newly identified, apparently widely conserved, druggable, interprotomer pocket preventing viral entry. We summarize some of the key papers in these areas and ponder the practical uses and contributions of molecular modeling alongside empirical structure determination.

Host-Pathogen Adhesion as the Basis of Innovative Diagnostics for Emerging Pathogens.

Infectious diseases are an existential health threat, potentiated by emerging and re-emerging viruses and increasing bacterial antibiotic resistance. Targeted treatment of infectious diseases requires precision diagnostics, especially in cases where broad-range therapeutics such as antibiotics fail. There is thus an increasing need for new approaches to develop sensitive and specific in vitro diagnostic (IVD) tests. Basic science and translational research are needed to identify key microbial molecules as diagnostic targets, to identify relevant host counterparts, and to use this knowledge in developing or improving IVD. In this regard, an overlooked feature is the capacity of pathogens to adhere specifically to host cells and tissues. The molecular entities relevant for pathogen-surface interaction are the so-called adhesins. Adhesins vary from protein compounds to (poly-)saccharides or lipid structures that interact with eukaryotic host cell matrix molecules and receptors. Such interactions co-define the specificity and sensitivity of a diagnostic test. Currently, adhesin-receptor binding is typically used in the pre-analytical phase of IVD tests, focusing on pathogen enrichment. Further exploration of adhesin-ligand interaction, supported by present high-throughput "omics" technologies, might stimulate a new generation of broadly applicable pathogen detection and characterization tools. This review describes recent results of novel structure-defining technologies allowing for detailed molecular analysis of adhesins, their receptors and complexes. Since the host ligands evolve slowly, the corresponding adhesin interaction is under selective pressure to maintain a constant receptor binding domain. IVD should exploit such conserved binding sites and, in particular, use the human ligand to enrich the pathogen. We provide an inventory of methods based on adhesion factors and pathogen attachment mechanisms, which can also be of relevance to currently emerging pathogens, including SARS-CoV-2, the causative agent of COVID-19.

A comparative analysis of parechovirus protein structures with other picornaviruses.

Parechoviruses belong to the genus Parechovirus within the family Picornaviridae and are non-enveloped icosahedral viruses with a single-stranded RNA genome. Parechoviruses include human and animal pathogens classified into six species. Those that infect humans belong to the Parechovirus A species and can cause infections ranging from mild gastrointestinal or respiratory illness to severe neonatal sepsis. There are no approved antivirals available to treat parechovirus (nor any other picornavirus) infections. In this parechovirus review, we focus on the cleaved protein products resulting from the polyprotein processing after translation comparing and contrasting their known or predicted structures and functions to those of other picornaviruses. The review also includes our original analysis from sequence and structure prediction. This review highlights significant structural differences between parechoviral and other picornaviral proteins, suggesting that parechovirus drug development should specifically be directed to parechoviral targets.

Identification of a conserved virion-stabilizing network inside the interprotomer pocket of enteroviruses.

Enteroviruses pose a persistent and widespread threat to human physical health, with no specific treatments available. Small molecule capsid binders have the potential to be developed as antivirals that prevent virus attachment and entry into host cells. To aid with broad-range drug development, we report here structures of coxsackieviruses B3 and B4 bound to different interprotomer-targeting capsid binders using single-particle cryo-EM. The EM density maps are beyond 3 Å resolution, providing detailed information about interactions in the ligand-binding pocket. Comparative analysis revealed the residues that form a conserved virion-stabilizing network at the interprotomer site, and showed the small molecule properties that allow anchoring in the pocket to inhibit virus disassembly.

Structure of Nora virus at 2.7 Å resolution and implications for receptor binding, capsid stability and taxonomy.

Nora virus, a virus of Drosophila, encapsidates one of the largest single-stranded RNA virus genomes known. Its taxonomic affinity is uncertain as it has a picornavirus-like cassette of enzymes for virus replication, but the capsid structure was at the time for genome publication unknown. By solving the structure of the virus, and through sequence comparison, we clear up this taxonomic ambiguity in the invertebrate RNA virosphere. Despite the lack of detectable similarity in the amino acid sequences, the 2.7 Å resolution cryoEM map showed Nora virus to have T = 1 symmetry with the characteristic capsid protein ß-barrels found in all the viruses in the Picornavirales order. Strikingly, α-helical bundles formed from the extended C-termini of capsid protein VP4B and VP4C protrude from the capsid surface. They are similar to signalling molecule folds and implicated in virus entry. Unlike other viruses of Picornavirales, no intra-pentamer stabilizing annulus was seen, instead the intra-pentamer stability comes from the interaction of VP4C and VP4B N-termini. Finally, intertwining of the N-termini of two-fold symmetry-related VP4A capsid proteins and RNA, provides inter-pentamer stability. Based on its distinct structural elements and the genetic distance to other picorna-like viruses we propose that Nora virus, and a small group of related viruses, should have its own family within the order Picornavirales.

Neuropilin-1 facilitates SARS-CoV-2 cell entry and infectivity.

The causative agent of coronavirus disease 2019 (COVID-19) is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For many viruses, tissue tropism is determined by the availability of virus receptors and entry cofactors on the surface of host cells. In this study, we found that neuropilin-1 (NRP1), known to bind furin-cleaved substrates, significantly potentiates SARS-CoV-2 infectivity, an effect blocked by a monoclonal blocking antibody against NRP1. A SARS-CoV-2 mutant with an altered furin cleavage site did not depend on NRP1 for infectivity. Pathological analysis of olfactory epithelium obtained from human COVID-19 autopsies revealed that SARS-CoV-2 infected NRP1-positive cells facing the nasal cavity. Our data provide insight into SARS-CoV-2 cell infectivity and define a potential target for antiviral intervention.

Extracellular vesicles provide a capsid-free vector for oncolytic adenoviral DNA delivery.

Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering therapeutic cargo, including oncolytic viruses for cancer treatment. Delivery of oncolytic viruses in EVs could provide considerable advantages, hiding the viruses from the immune system and providing alternative entry pathways into cancer cells. Here we describe the formation and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected cell-derived EVs) as a function of time after infection. IEVs were secreted already before the lytic release of virions and their structure resembled normally secreted EVs, suggesting that they were not just apoptotic fragments of infected cells. IEVs were able to carry the viral genome and induce infection in other cancer cells. As such, the role of EVs in the life cycle of adenoviruses may be an important part of a successful infection and may also be harnessed for cancer- and gene therapy.

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.

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.

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.