Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.

The SARS-CoV-2 spike (S) protein variant D614G supplanted the ancestral virus worldwide, reaching near fixation in a matter of months. Here we show that D614G was more infectious than the ancestral form on human lung cells, colon cells, and on cells rendered permissive by ectopic expression of human ACE2 or of ACE2 orthologs from various mammals, including Chinese rufous horseshoe bat and Malayan pangolin. D614G did not alter S protein synthesis, processing, or incorporation into SARS-CoV-2 particles, but D614G affinity for ACE2 was reduced due to a faster dissociation rate. Assessment of the S protein trimer by cryo-electron microscopy showed that D614G disrupts an interprotomer contact and that the conformation is shifted toward an ACE2 binding-competent state, which is modeled to be on pathway for virion membrane fusion with target cells. Consistent with this more open conformation, neutralization potency of antibodies targeting the S protein receptor-binding domain was not attenuated.

Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers.

Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane.

The DO-KB Knowledgebase: a 20-year journey developing the disease open science ecosystem.

In 2003, the Human Disease Ontology (DO, https://disease-ontology.org/) was established at Northwestern University. In the intervening 20 years, the DO has expanded to become a highly-utilized disease knowledge resource. Serving as the nomenclature and classification standard for human diseases, the DO provides a stable, etiology-based structure integrating mechanistic drivers of human disease. Over the past two decades the DO has grown from a collection of clinical vocabularies, into an expertly curated semantic resource of over 11300 common and rare diseases linking disease concepts through more than 37000 vocabulary cross mappings (v2023-08-08). Here, we introduce the recently launched DO Knowledgebase (DO-KB), which expands the DO's representation of the diseaseome and enhances the findability, accessibility, interoperability and reusability (FAIR) of disease data through a new SPARQL service and new Faceted Search Interface. The DO-KB is an integrated data system, built upon the DO's semantic disease knowledge backbone, with resources that expose and connect the DO's semantic knowledge with disease-related data across Open Linked Data resources. This update includes descriptions of efforts to assess the DO's global impact and improvements to data quality and content, with emphasis on changes in the last two years.

The combination of three CD4-induced antibodies targeting highly conserved Env regions with a small CD4-mimetic achieves potent ADCC activity.

The majority of naturally elicited antibodies against the HIV-1 envelope glycoproteins (Env) are non-neutralizing (nnAbs) because they are unable to recognize the Env trimer in its native "closed" conformation. Nevertheless, it has been shown that nnAbs have the potential to eliminate HIV-1-infected cells by antibody-dependent cellular cytotoxicity (ADCC) provided that Env is present on the cell surface in its "open" conformation. This is because most nnAbs recognize epitopes that become accessible only after Env interaction with CD4 and the exposure of epitopes that are normally occluded in the closed trimer. HIV-1 limits this vulnerability by downregulating CD4 from the surface of infected cells, thus preventing a premature encounter of Env with CD4. Small CD4-mimetics (CD4mc) sensitize HIV-1-infected cells to ADCC by opening the Env glycoprotein and exposing CD4-induced (CD4i) epitopes. There are two families of CD4i nnAbs, termed anti-cluster A and anti-CoRBS Abs, which are known to mediate ADCC in the presence of CD4mc. Here, we performed Fab competition experiments and found that anti-gp41 cluster I antibodies comprise a major fraction of the plasma ADCC activity in people living with HIV (PLWH). Moreover, addition of gp41 cluster I antibodies to cluster A and CoRBS antibodies greatly enhanced ADCC-mediated cell killing in the presence of a potent indoline CD4mc, CJF-III-288. This cocktail outperformed broadly neutralizing antibodies and even showed activity against HIV-1-infected monocyte-derived macrophages. Thus, combining CD4i antibodies with different specificities achieves maximal ADCC activity, which may be of utility in HIV cure strategies.IMPORTANCEThe elimination of HIV-1-infected cells remains an important medical goal. Although current antiretroviral therapy decreases viral loads below detection levels, it does not eliminate latently infected cells that form the viral reservoir. Here, we developed a cocktail of non-neutralizing antibodies targeting highly conserved Env regions and combined it with a potent indoline CD4mc. This combination exhibited potent ADCC activity against HIV-1-infected primary CD4 + T cells as well as monocyte-derived macrophages, suggesting its potential utility in decreasing the size of the viral reservoir.

Regulation of Ebola GP conformation and membrane binding by the chemical environment of the late endosome.

Interaction between the Ebola virus envelope glycoprotein (GP) and the endosomal membrane is an essential step during virus entry into the cell. Acidic pH and Ca2+ have been implicated in mediating the GP-membrane interaction. However, the molecular mechanism by which these environmental factors regulate the conformational changes that enable engagement of GP with the target membrane is unknown. Here, we apply fluorescence correlation spectroscopy (FCS) and single-molecule Förster resonance energy transfer (smFRET) imaging to elucidate how the acidic pH, Ca2+ and anionic phospholipids in the late endosome promote GP-membrane interaction, thereby facilitating virus entry. We find that bis(monoacylglycero)phosphate (BMP), which is specific to the late endosome, is especially critical in determining the Ca2+-dependence of the GP-membrane interaction. Molecular dynamics (MD) simulations suggested residues in GP that sense pH and induce conformational changes that make the fusion loop available for insertion into the membrane. We similarly confirm residues in the fusion loop that mediate GP's interaction with Ca2+, which likely promotes local conformational changes in the fusion loop and mediates electrostatic interactions with the anionic phospholipids. Collectively, our results provide a mechanistic understanding of how the environment of the late endosome regulates the timing and efficiency of virus entry.

Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

The Human Disease Ontology 2022 update.

The Human Disease Ontology (DO) (www.disease-ontology.org) database, has significantly expanded the disease content and enhanced our userbase and website since the DO's 2018 Nucleic Acids Research DATABASE issue paper. Conservatively, based on available resource statistics, terms from the DO have been annotated to over 1.5 million biomedical data elements and citations, a 10× increase in the past 5 years. The DO, funded as a NHGRI Genomic Resource, plays a key role in disease knowledge organization, representation, and standardization, serving as a reference framework for multiscale biomedical data integration and analysis across thousands of clinical, biomedical and computational research projects and genomic resources around the world. This update reports on the addition of 1,793 new disease terms, a 14% increase of textual definitions and the integration of 22 137 new SubClassOf axioms defining disease to disease connections representing the DO's complex disease classification. The DO's updated website provides multifaceted etiology searching, enhanced documentation and educational resources.

ECO: the Evidence and Conclusion Ontology, an update for 2022.

The Evidence and Conclusion Ontology (ECO) is a community resource that provides an ontology of terms used to capture the type of evidence that supports biomedical annotations and assertions. Consistent capture of evidence information with ECO allows tracking of annotation provenance, establishment of quality control measures, and evidence-based data mining. ECO is in use by dozens of data repositories and resources with both specific and general areas of focus. ECO is continually being expanded and enhanced in response to user requests as well as our aim to adhere to community best-practices for ontology development. The ECO support team engages in multiple collaborations with other ontologies and annotating groups. Here we report on recent updates to the ECO ontology itself as well as associated resources that are available through this project. ECO project products are freely available for download from the project website (https://evidenceontology.org/) and GitHub (https://github.com/evidenceontology/evidenceontology). ECO is released into the public domain under a CC0 1.0 Universal license.

The Hypervariable Tpr Multigene Family of Theileria Parasites, Defined by a Conserved, Membrane-Associated, C-Terminal Domain, Includes Several Copies with Defined Orthology Between Species.

Multigene families often play an important role in host-parasite interactions. One of the largest multigene families in Theileria parva, the causative agent of East Coast fever, is the T. parva repeat (Tpr) gene family. The function of the putative Tpr proteins remains unknown. The initial publication of the T. parva reference genome identified 39 Tpr family open reading frames (ORFs) sharing a conserved C-terminal domain. Twenty-eight of these are clustered in a central region of chromosome 3, termed the "Tpr locus", while others are dispersed throughout all four nuclear chromosomes. The Tpr locus contains three of the four assembly gaps remaining in the genome, suggesting the presence of additional, as yet uncharacterized, Tpr gene copies. Here, we describe the use of long-read sequencing to attempt to close the gaps in the reference assembly of T. parva (located among multigene families clusters), characterize the full complement of Tpr family ORFs in the T. parva reference genome, and evaluate their evolutionary relationship with Tpr homologs in other Theileria species. We identify three new Tpr family genes in the T. parva reference genome and show that sequence similarity among paralogs in the Tpr locus is significantly higher than between genes outside the Tpr locus. We also identify sequences homologous to the conserved C-terminal domain in five additional Theileria species. Using these sequences, we show that the evolution of this gene family involves conservation of a few orthologs across species, combined with gene gains/losses, and species-specific expansions.

Conformational changes in the Ebola virus membrane fusion machine induced by pH, Ca2+, and receptor binding.

The Ebola virus (EBOV) envelope glycoprotein (GP) is a membrane fusion machine required for virus entry into cells. Following endocytosis of EBOV, the GP1 domain is cleaved by cellular cathepsins in acidic endosomes, removing the glycan cap and exposing a binding site for the Niemann-Pick C1 (NPC1) receptor. NPC1 binding to cleaved GP1 is required for entry. How this interaction translates to GP2 domain-mediated fusion of viral and endosomal membranes is not known. Here, using a bulk fluorescence dequenching assay and single-molecule Förster resonance energy transfer (smFRET)-imaging, we found that acidic pH, Ca2+, and NPC1 binding synergistically induce conformational changes in GP2 and permit virus-liposome lipid mixing. Acidic pH and Ca2+ shifted the GP2 conformational equilibrium in favor of an intermediate state primed for NPC1 binding. Glycan cap cleavage on GP1 enabled GP2 to transition from a reversible intermediate to an irreversible conformation, suggestive of the postfusion 6-helix bundle; NPC1 binding further promoted transition to the irreversible conformation. Thus, the glycan cap of GP1 may allosterically protect against inactivation of EBOV by premature triggering of GP2.

Developing a research community within an online healthcare feedback platform.

INTRODUCTION: Care Opinion is an online feedback platform supporting patients to author stories about their care. It is not known whether authors would be willing to be involved in improving care through research. The aims of this study were to explore the views and preferences of Care Opinion authors about joining an online research community and to pilot new research community functionality. METHODS: Five hundred and nine Care Opinion authors were invited to take part in an online survey in June 2019. Survey items included questions about participants' willingness to take part in research and their preferences for supporting processes. Data were analysed descriptively. Authors were invited to consent to join a research community and were asked to participate in three pilot studies. RESULTS: One hundred and sixty-three people consented to take part in the survey (32%). Participants indicated they would like to know the time commitment to the project (146, 90%), details about the organization carrying out the research (124, 76%) and safeguarding information (124, 76%). Over half indicated that they did not know how to get involved in healthcare research (87, 53%). Subsequently, 667 authors were invited to join the research community, 183 (27%) accepted, and three studies were matched to their expressed preferences for project attributes or organization type. CONCLUSION: Many people who leave online feedback about their experiences of healthcare are also willing to join a research community via that platform. They have strong preferences for supporting University and NHS research. Eligibility and acceptance rates to join pilot research studies varied. Further work is needed to grow the research community, increase its diversity, and create relevant and varied opportunities to support research. PATIENT OR PUBLIC CONTRIBUTION: Four members of the Safety In Numbers patient and public involvement and engagement (PPIE) group advised about survey development.

STRIDE: a command-line HMM-based identifier and sub-classifier of Plasmodium falciparum RIFIN and STEVOR variant surface antigen families.

BACKGROUND: RIFINs and STEVORs are variant surface antigens expressed by P. falciparum that play roles in severe malaria pathogenesis and immune evasion. These two highly diverse multigene families feature multiple paralogs, making their classification challenging using traditional bioinformatic methods. RESULTS: STRIDE (STevor and RIfin iDEntifier) is an HMM-based, command-line program that automates the identification and classification of RIFIN and STEVOR protein sequences in the malaria parasite Plasmodium falciparum. STRIDE is more sensitive in detecting RIFINs and STEVORs than available PFAM and TIGRFAM tools and reports RIFIN subtypes and the number of sequences with a FHEYDER amino acid motif, which has been associated with severe malaria pathogenesis. CONCLUSIONS: STRIDE will be beneficial to malaria research groups analyzing genome sequences and transcripts of clinical field isolates, providing insight into parasite biology and virulence.

Gp41-targeted antibodies restore infectivity of a fusion-deficient HIV-1 envelope glycoprotein.

The HIV-1 envelope glycoprotein (Env) mediates viral entry via conformational changes associated with binding the cell surface receptor (CD4) and coreceptor (CCR5/CXCR4), resulting in subsequent fusion of the viral and cellular membranes. While the gp120 Env surface subunit has been extensively studied for its role in viral entry and evasion of the host immune response, the gp41 transmembrane glycoprotein and its role in natural infection are less well characterized. Here, we identified a primary HIV-1 Env variant that consistently supports >300% increased viral infectivity in the presence of autologous or heterologous HIV-positive plasma. However, in the absence of HIV-positive plasma, viruses with this Env exhibited reduced infectivity that was not due to decreased CD4 binding. Using Env chimeras and sequence analysis, we mapped this phenotype to a change Q563R, in the gp41 heptad repeat 1 (HR1) region. We demonstrate that Q563R reduces viral infection by disrupting formation of the gp41 six-helix bundle required for virus-cell membrane fusion. Intriguingly, antibodies that bind cluster I epitopes on gp41 overcome this inhibitory effect, restoring infectivity to wild-type levels. We further demonstrate that the Q563R change increases HIV-1 sensitivity to broadly neutralizing antibodies (bNAbs) targeting the gp41 membrane-proximal external region (MPER). In summary, we identify an HIV-1 Env variant with impaired infectivity whose Env functionality is restored through the binding of host antibodies. These data contribute to our understanding of gp41 residues involved in membrane fusion and identify a mechanism by which host factors can alleviate a viral defect.

Intrinsic conformational dynamics of the HIV-1 genomic RNA 5'UTR.

The highly conserved 5' untranslated region (5'UTR) of the HIV-1 RNA genome is central to the regulation of virus replication. NMR and biochemical experiments support a model in which the 5'UTR can transition between at least two conformational states. In one state the genome remains a monomer, as the palindromic dimerization initiation site (DIS) is sequestered via base pairing to upstream sequences. In the second state, the DIS is exposed, and the genome is competent for kissing loop dimerization and packaging into assembling virions where an extended dimer is formed. According to this model the conformation of the 5'UTR determines the fate of the genome. In this work, the dynamics of this proposed conformational switch and the factors that regulate it were probed using multiple single-molecule and in-gel ensemble FRET assays. Our results show that the HIV-1 5'UTR intrinsically samples conformations that are stabilized by both viral and host factor binding. Annealing of tRNALys3, the primer for initiation of reverse transcription, can promote the kissing dimer but not the extended dimer. In contrast, HIV-1 nucleocapsid (NC) promotes formation of the extended dimer in both the absence and presence of tRNALys3 Our data are consistent with an ordered series of events that involves primer annealing, genome dimerization, and virion assembly.

Proven and not proven: A potential alternative to the current Scottish verdict system.

The current Scottish verdict system includes three verdicts: 'guilty', 'not guilty' and 'not proven'. The Scottish Government are currently reviewing the utility of the not proven verdict. Proponents of the not proven verdict suggest that it directs jurors to their true role of determining whether the prosecution's case has, or has not, been 'proven'. Reformists suggest a move to a system similar to England and Wales, with only guilty and not guilty verdicts. However, legal professionals have indicated a preference for an alternative system of proven and not proven. The aim of the current study was to test the effects of a proven and not proven system on verdicts given, when compared to alternative verdict systems (specifically, the current Scottish and Anglo-American verdict systems). 227 mock jurors watched a staged murder trial, filmed in a real-life courtroom, with legal professionals questioning witnesses and a judge giving legal direction. Jurors were significantly more likely to convict in a guilty and not guilty verdict system than either a proven and not proven or a guilty, not guilty and not proven verdict system. Future research should replicate this study with a focus on the impact of the not proven verdict in sexual offences.

Verdict spotting: investigating the effects of juror bias, evidence anchors and verdict system in jurors.

The Scottish verdict of not proven represents a second acquittal verdict which is not legally defined. Existing research into the influence of the not proven verdict on jury decision making is modest. The main aim of the current study was therefore to investigate the influence of verdict systems (two vs three) on juror decision making. The effect of pre-trial bias and evidence anchors on juror judgements were also examined. One-hundred and twenty-eight mock jurors listened to two homicide vignettes and were asked to rate their belief of guilt of the accused and to give a verdict in both trials. The results suggest that pre-trial bias was a significant predictor of both verdict choice and belief of guilt, whereas evidence anchors were not a significant predictor of either. Finally, both guilty and not guilty verdicts were given with increased frequency in the two-verdict system when compared to the three-verdict system.

Stability and conformation of the dimeric HIV-1 genomic RNA 5'UTR.

During HIV-1 assembly, the viral Gag polyprotein specifically selects the dimeric RNA genome for packaging into new virions. The 5' untranslated region (5'UTR) of the dimeric genome may adopt a conformation that is optimal for recognition by Gag. Further conformational rearrangement of the 5'UTR, promoted by the nucleocapsid (NC) domain of Gag, is predicted during virus maturation. Two 5'UTR dimer conformations, the kissing dimer (KD) and the extended dimer (ED), have been identified in vitro, which differ in the extent of intermolecular basepairing. Whether 5'UTRs from different HIV-1 strains with distinct sequences have access to the same dimer conformations has not been determined. Here, we applied fluorescence cross-correlation spectroscopy and single-molecule Förster resonance energy transfer imaging to demonstrate that 5'UTRs from two different HIV-1 subtypes form (KDs) with divergent stabilities. We further show that both 5'UTRs convert to a stable dimer in the presence of the viral NC protein, adopting a conformation consistent with extensive intermolecular contacts. These results support a unified model in which the genomes of diverse HIV-1 strains adopt an ED conformation.

Antibody affinity as a driver of signal generation in a paper-based immunoassay for Ebola virus surveillance.

During epidemics, such as the frequent and devastating Ebola virus outbreaks that have historically plagued regions of Africa, serological surveillance efforts are critical for viral containment and the development of effective antiviral therapeutics. Antibody serology can also be used retrospectively for population-level surveillance to provide a more complete estimate of total infections. Ebola surveillance efforts rely on enzyme-linked immunosorbent assays (ELISAs), which restrict testing to laboratories and are not adaptable for use in resource-limited settings. In this manuscript, we describe a paper-based immunoassay capable of detecting anti-Ebola IgG using Ebola virus envelope glycoprotein ectodomain (GP) as the affinity reagent. We evaluated seven monoclonal antibodies (mAbs) against GP-KZ52, 13C6, 4G7, 2G4, c6D8, 13F6, and 4F3-to elucidate the impact of binding affinity and binding epitope on assay performance and, ultimately, result interpretation. We used biolayer interferometry to characterize the binding of each antibody to GP before assessing their performance in our paper-based device. Binding affinity (KD) and on rate (kon) were major factors influencing the sensitivity of the paper-based immunoassay. mAbs with the best KD (3-25 nM) exhibited the lowest limits of detection (ca. µg mL-1), while mAbs with KD > 25 nM were undetectable in our device. Additionally, and most surprisingly, we determined that observed signals in paper devices were directly proportional to kon. These results highlight the importance of ensuring that the quality of recognition reagents is sufficient to support desired assay performance and suggest that the strength of an individual's immune response can impact the interpretation of assay results.

Human Disease Ontology 2018 update: classification, content and workflow expansion.

The Human Disease Ontology (DO) (http://www.disease-ontology.org), database has undergone significant expansion in the past three years. The DO disease classification includes specific formal semantic rules to express meaningful disease models and has expanded from a single asserted classification to include multiple-inferred mechanistic disease classifications, thus providing novel perspectives on related diseases. Expansion of disease terms, alternative anatomy, cell type and genetic disease classifications and workflow automation highlight the updates for the DO since 2015. The enhanced breadth and depth of the DO's knowledgebase has expanded the DO's utility for exploring the multi-etiology of human disease, thus improving the capture and communication of health-related data across biomedical databases, bioinformatics tools, genomic and cancer resources and demonstrated by a 6.6× growth in DO's user community since 2015. The DO's continual integration of human disease knowledge, evidenced by the more than 200 SVN/GitHub releases/revisions, since previously reported in our DO 2015 NAR paper, includes the addition of 2650 new disease terms, a 30% increase of textual definitions, and an expanding suite of disease classification hierarchies constructed through defined logical axioms.

ECO, the Evidence & Conclusion Ontology: community standard for evidence information.

The Evidence and Conclusion Ontology (ECO) contains terms (classes) that describe types of evidence and assertion methods. ECO terms are used in the process of biocuration to capture the evidence that supports biological assertions (e.g. gene product X has function Y as supported by evidence Z). Capture of this information allows tracking of annotation provenance, establishment of quality control measures and query of evidence. ECO contains over 1500 terms and is in use by many leading biological resources including the Gene Ontology, UniProt and several model organism databases. ECO is continually being expanded and revised based on the needs of the biocuration community. The ontology is freely available for download from GitHub (https://github.com/evidenceontology/) or the project's website (http://evidenceontology.org/). Users can request new terms or changes to existing terms through the project's GitHub site. ECO is released into the public domain under CC0 1.0 Universal.