A structure-function analysis shows SARS-CoV-2 BA.2.86 balances antibody escape and ACE2 affinity.

BA.2.86, a recently described sublineage of SARS-CoV-2 Omicron, contains many mutations in the spike gene. It appears to have originated from BA.2 and is distinct from the XBB variants responsible for many infections in 2023. The global spread and plethora of mutations in BA.2.86 has caused concern that it may possess greater immune-evasive potential, leading to a new wave of infection. Here, we examine the ability of BA.2.86 to evade the antibody response to infection using a panel of vaccinated or naturally infected sera and find that it shows marginally less immune evasion than XBB.1.5. We locate BA.2.86 in the antigenic landscape of recent variants and look at its ability to escape panels of potent monoclonal antibodies generated against contemporary SARS-CoV-2 infections. We demonstrate, and provide a structural explanation for, increased affinity of BA.2.86 to ACE2, which may increase transmissibility.

Emerging variants develop total escape from potent monoclonal antibodies induced by BA.4/5 infection.

The rapid evolution of SARS-CoV-2 is driven in part by a need to evade the antibody response in the face of high levels of immunity. Here, we isolate spike (S) binding monoclonal antibodies (mAbs) from vaccinees who suffered vaccine break-through infections with Omicron sub lineages BA.4 or BA.5. Twenty eight potent antibodies are isolated and characterised functionally, and in some cases structurally. Since the emergence of BA.4/5, SARS-CoV-2 has continued to accrue mutations in the S protein, to understand this we characterize neutralization of a large panel of variants and demonstrate a steady attrition of neutralization by the panel of BA.4/5 mAbs culminating in total loss of function with recent XBB.1.5.70 variants containing the so-called 'FLip' mutations at positions 455 and 456. Interestingly, activity of some mAbs is regained on the recently reported variant BA.2.86.

A pipeline for identification of causal mutations in barley identifies Xantha-j as the chlorophyll synthase gene.

Thousands of barley (Hordeum vulgare L.) mutants have been isolated over the last century, and many are stored in gene banks across various countries. In the present work, we developed a pipeline to efficiently identify causal mutations in barley. The pipeline is also efficient for mutations located in centromeric regions. Through bulked-segregant analyses using whole genome sequencing of pooled F2 seedlings, we mapped two mutations and identified a limited number of candidate genes. We applied the pipeline on F2-mapping populations made from xan-j.59 (unknown mutation) and xan-l.82 (previously known). The Xantha-j (xan-j) gene was identified as encoding chlorophyll synthase, which catalyzes the last step in the chlorophyll biosynthetic pathway: the addition of a phytol moiety to the propionate side chain of chlorophyllide. Key amino-acid residues in the active site, including the binding sites of the isoprenoid and chlorophyllide substrates, were analyzed in an AlphaFold2-generated structural model of the barley chlorophyll synthase. Three allelic mutants, xan-j.19, xan-j.59, and xan-j.64 were characterized. While xan-j.19 is a one-base pair deletion and xan-j.59 is a nonsense mutation, xan-j.64 causes an S212F substitution in chlorophyll synthase. Our analyses of xan-j.64 and treatment of growing barley with clomazone, an inhibitor of chloroplastic isoprenoid biosynthesis, suggest that binding of the isoprenoid substrate is a prerequisite for the stable maintenance of chlorophyll synthase in the plastid. We further suggest that chlorophyll synthase is a sensor for coordinating chlorophyll and isoprenoid biosynthesis.

The SARS-CoV-2 neutralizing antibody response to SD1 and its evasion by BA.2.86.

Under pressure from neutralising antibodies induced by vaccination or infection the SARS-CoV-2 spike gene has become a hotspot for evolutionary change, leading to the failure of all mAbs developed for clinical use. Most potent antibodies bind to the receptor binding domain which has become heavily mutated. Here we study responses to a conserved epitope in sub-domain-1 (SD1) of spike which have become more prominent because of mutational escape from antibodies directed to the receptor binding domain. Some SD1 reactive mAbs show potent and broad neutralization of SARS-CoV-2 variants. We structurally map the dominant SD1 epitope and provide a mechanism of action by blocking interaction with ACE2. Mutations in SD1 have not been sustained to date, but one, E554K, leads to escape from mAbs. This mutation has now emerged in several sublineages including BA.2.86, reflecting selection pressure on the virus exerted by the increasing prominence of the anti-SD1 response.

A guide to barley mutants.

BACKGROUND: Mutants have had a fundamental impact upon scientific and applied genetics. They have paved the way for the molecular and genomic era, and most of today's crop plants are derived from breeding programs involving mutagenic treatments. RESULTS: Barley (Hordeum vulgare L.) is one of the most widely grown cereals in the world and has a long history as a crop plant. Barley breeding started more than 100 years ago and large breeding programs have collected and generated a wide range of natural and induced mutants, which often were deposited in genebanks around the world. In recent years, an increased interest in genetic diversity has brought many historic mutants into focus because the collections are regarded as valuable resources for understanding the genetic control of barley biology and barley breeding. The increased interest has been fueled also by recent advances in genomic research, which provided new tools and possibilities to analyze and reveal the genetic diversity of mutant collections. CONCLUSION: Since detailed knowledge about phenotypic characters of the mutants is the key to success of genetic and genomic studies, we here provide a comprehensive description of mostly morphological barley mutants. The review is closely linked to the International Database for Barley Genes and Barley Genetic Stocks ( bgs.nordgen.org ) where further details and additional images of each mutant described in this review can be found.

Structural comparison of typical and atypical E2 pestivirus glycoproteins.

Pestiviruses, within the family Flaviviridae, are economically important viruses of livestock. In recent years, new pestiviruses have been reported in domestic animals and non-cloven-hoofed animals. Among them, atypical porcine pestivirus (APPV) and Norway rat pestivirus (NRPV) have relatively little sequence conservation in their surface glycoprotein E2. Despite E2 being the main target for neutralizing antibodies and necessary for cell attachment and viral fusion, the mechanism of viral entry remains elusive. To gain further insights into the pestivirus E2 mechanism of action and to assess its diversity within the genus, we report X-ray structures of the pestivirus E2 proteins from APPV and NRPV. Despite the highly divergent structures, both are able to dimerize through their C-terminal domain and contain a solvent-exposed ß-hairpin reported to be involved in host receptor binding. Functional analysis of this ß-hairpin in the context of BVDV revealed its ability to rescue viral infectivity.

An efficient and chemoselective method to generate arynes.

Arynes hold immense potential as reactive intermediates in organic synthesis as they engage in a diverse range of mechanistically distinct chemical reactions. However, the poor functional group compatibility of generating arynes or their precursors has stymied their widespread use. Here, we show that generating arynes by deprotonation of an arene and elimination of an "onium" leaving group is mild, efficient and broad in scope. This is achieved by using aryl(TMP)iodonium salts (TMP = 2,4,6-trimethoxyphenyl) as the aryne precursor and potassium phosphate as the base, and a range of arynophiles are compatible. Additionally, we have performed the first quantitative analysis of functional group compatibility for several methods to generate arynes, including the method developed here and the current state of the art. Finally, we show that a range of "sensitive" functional groups such as Lewis and Brønsted acids and electrophiles are compatible under our conditions.

Atypical Presentation of a Preexisting Pancreatic Pseudocyst with Glucagon-Like Peptide-1 Agonist as a Possible Trigger for Exacerbation: A Case Report.

Pancreatitis encompasses pancreatic tissue inflammation due to enzymatic autodigestion, leading to complications such as pancreatic pseudocysts. This report details a case in which the administration of glucagon-like peptide-1 (GLP-1) agonists rendered a regressing, asymptomatic pseudocyst symptomatic. We posit that, absent other triggers, GLP-1 agonists might exacerbate pseudocysts. This emphasizes timely diagnosis and proper management. Our investigation delves into patient-specific nuances, potential mechanistic insights, the need to study this phenomenon among a broader cohort, alternative pathologies, long-term consequences, and clinical ramifications in response to a shifting pseudocyst behavior.

COVID-19 therapeutics: stewardship in England and considerations for antimicrobial resistance.

The COVID-19 pandemic saw unprecedented resources and funds driven into research for the development, and subsequent rapid distribution, of vaccines, diagnostics and directly acting antivirals (DAAs). DAAs have undeniably prevented progression and life-threatening conditions in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. However, there are concerns of antimicrobial resistance (AMR), antiviral resistance specifically, for DAAs. To preserve activity of DAAs for COVID-19 therapy, as well as detect possible mutations conferring resistance, antimicrobial stewardship and surveillance were rapidly implemented in England. This paper expands on the ubiquitous ongoing public health activities carried out in England, including epidemiologic, virologic and genomic surveillance, to support the stewardship of DAAs and assess the deployment, safety, effectiveness and resistance potential of these novel and repurposed therapeutics.

Persistence of SARS-CoV-2 antibodies over 18 months following infection: UK Biobank COVID-19 Serology Study.

BACKGROUND: Little is known about the persistence of antibodies after the first year following SARS-CoV-2 infection. We aimed to determine the proportion of individuals that maintain detectable levels of SARS-CoV-2 antibodies over an 18-month period following infection. METHODS: Population-based prospective study of 20 000 UK Biobank participants and their adult relatives recruited in May 2020. The proportion of SARS-CoV-2 cases testing positive for immunoglobulin G (IgG) antibodies against the spike protein (IgG-S), and the nucleocapsid protein (IgG-N), was calculated at varying intervals following infection. RESULTS: Overall, 20 195 participants were recruited. Their median age was 56 years (IQR 39-68), 56% were female and 88% were of white ethnicity. The proportion of SARS-CoV-2 cases with IgG-S antibodies following infection remained high (92%, 95% CI 90%-93%) at 6 months after infection. Levels of IgG-N antibodies following infection gradually decreased from 92% (95% CI 88%-95%) at 3 months to 72% (95% CI 70%-75%) at 18 months. There was no strong evidence of heterogeneity in antibody persistence by age, sex, ethnicity or socioeconomic deprivation. CONCLUSION: This study adds to the limited evidence on the long-term persistence of antibodies following SARS-CoV-2 infection, with likely implications for waning immunity following infection and the use of IgG-N in population surveys.

ChAdOx1 COVID vaccines express RBD open prefusion SARS-CoV-2 spikes on the cell surface.

Vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been proven to be an effective means of decreasing COVID-19 mortality, hospitalization rates, and transmission. One of the vaccines deployed worldwide is ChAdOx1 nCoV-19, which uses an adenovirus vector to drive the expression of the original SARS-CoV-2 spike on the surface of transduced cells. Using cryo-electron tomography and subtomogram averaging, we determined the native structures of the vaccine product expressed on cell surfaces in situ. We show that ChAdOx1-vectored vaccines expressing the Beta SARS-CoV-2 variant produce abundant native prefusion spikes predominantly in one-RBD-up conformation. Furthermore, the ChAdOx1-vectored HexaPro-stabilized spike yields higher cell surface expression, enhanced RBD exposure, and reduced shedding of S1 compared to the wild type. We demonstrate in situ structure determination as a powerful means for studying antigen design options in future vaccine development against emerging novel SARS-CoV-2 variants and broadly against other infectious viruses.

Oxidative Cycloaddition Reactions of Arylboron Reagents via a One-pot Formal Dehydroboration Sequence.

Arylboron compounds are widely available and synthetically useful reagents in which the boron group is typically substituted. Herein, we show that the boron group and ortho-hydrogen atom are substituted in a formal cycloaddition reaction. This transformation is enabled by a one-pot sequence involving diaryliodonium and aryne intermediates. The scope of arylboron reagents and arynophiles is demonstrated, and the method is applied to the formal synthesis of an investigational drug candidate.

Structure of Bovine CD46 Ectodomain.

CD46, or membrane cofactor protein, is a type-one transmembrane protein from the complement regulatory protein family. Alongside its role in complement activation, CD46 is involved in many other processes, from T-cell activation to reproduction. It is also referred to as a pathogen magnet, because it is used as a receptor by multiple bacteria and viruses. Bovine CD46 (bovCD46) in particular is involved in bovine viral diarrhoea virus entry, an economically important disease in cattle industries. This study presents the X-ray crystallographic structure of the extracellular region of bovCD46, revealing a four-short-consensus-repeat (SCR) structure similar to that in human CD46. SCR1-3 are arranged linearly, while SCR 4 has a reduced interface angle, resulting in a hockey stick-like appearance. The structure also reveals the bovine viral diarrhoea virus interaction site in SCR1, which is likely to confer pestivirus specificity for their target host, CD46. Insights gained from the structural information on pestivirus receptors, such as CD46, could offer valuable guidance for future control strategies.

Broadly neutralizing antibodies against COVID-19.

The COVID-19 pandemic caused by SARS-CoV-2 has led to hundreds of millions of infections and millions of deaths, however, human monoclonal antibodies (mAbs) can be an effective treatment. Since SARS-CoV-2 emerged, a variety of strains have acquired increasing numbers of mutations to gain increased transmissibility and escape from the immune response. Most reported neutralizing human mAbs, including all approved therapeutic ones, have been knocked down or out by these mutations. Broadly neutralizing mAbs are therefore of great value, to treat current and possible future variants. Here, we review four types of neutralizing mAbs against the spike protein with broad potency against previously and currently circulating variants. These mAbs target the receptor-binding domain, the subdomain 1, the stem helix, or the fusion peptide. Understanding how these mAbs retain potency in the face of mutational change could guide future development of therapeutic antibodies and vaccines.

Saccharomyces cerevisiae Δ9-desaturase Ole1 forms a supercomplex with Slc1 and Dga1.

Biosynthesis of the various lipid species that compose cellular membranes and lipid droplets depends on the activity of multiple enzymes functioning in coordinated pathways. The flux of intermediates through lipid biosynthetic pathways is regulated to respond to nutritional and environmental demands placed on the cell necessitating that there be flexibility in pathway activity and organization. This flexibility can in part be achieved through the organization of enzymes into metabolon supercomplexes. However, the composition and organization of such supercomplexes remain unclear. Here, we identified protein-protein interactions between acyltransferases Sct1, Gpt2, Slc1, Dga1, and the Δ9 acyl-CoA desaturase Ole1 in Saccharomyces cerevisiae. We further determined that a subset of these acyltransferases interact with each other independent of Ole1. We show that truncated versions of Dga1 lacking the carboxyl-terminal 20 amino acid residues are nonfunctional and unable to bind Ole1. Furthermore, charged-to-alanine scanning mutagenesis revealed that a cluster of charged residues near the carboxyl terminus was required for the interaction with Ole1. Mutation of these charged residues disrupted the interaction between Dga1 and Ole1 but allowed Dga1 to retain catalytic activity and to induce lipid droplet formation. These data support the formation of a complex of acyltransferases involved in lipid biosynthesis that interacts with Ole1, the sole acyl-CoA desaturase in S. cerevisiae, that can channel unsaturated acyl chains toward phospholipid or triacylglycerol synthesis. This desaturasome complex may provide the architecture that allows for the necessary flux of de novo-synthesized unsaturated acyl-CoA to phospholipid or triacylglycerol synthesis as demanded by cellular requirements.

Generation of SARS-CoV-2 escape mutations by monoclonal antibody therapy.

COVID-19 patients at risk of severe disease may be treated with neutralising monoclonal antibodies (mAbs). To minimise virus escape from neutralisation these are administered as combinations e.g. casirivimab+imdevimab or, for antibodies targeting relatively conserved regions, individually e.g. sotrovimab. Unprecedented genomic surveillance of SARS-CoV-2 in the UK has enabled a genome-first approach to detect emerging drug resistance in Delta and Omicron cases treated with casirivimab+imdevimab and sotrovimab respectively. Mutations occur within the antibody epitopes and for casirivimab+imdevimab multiple mutations are present on contiguous raw reads, simultaneously affecting both components. Using surface plasmon resonance and pseudoviral neutralisation assays we demonstrate these mutations reduce or completely abrogate antibody affinity and neutralising activity, suggesting they are driven by immune evasion. In addition, we show that some mutations also reduce the neutralising activity of vaccine-induced serum.

Accelerating drug target inhibitor discovery with a deep generative foundation model.

Inhibitor discovery for emerging drug-target proteins is challenging, especially when target structure or active molecules are unknown. Here, we experimentally validate the broad utility of a deep generative framework trained at-scale on protein sequences, small molecules, and their mutual interactions-unbiased toward any specific target. We performed a protein sequence-conditioned sampling on the generative foundation model to design small-molecule inhibitors for two dissimilar targets: the spike protein receptor-binding domain (RBD) and the main protease from SARS-CoV-2. Despite using only the target sequence information during the model inference, micromolar-level inhibition was observed in vitro for two candidates out of four synthesized for each target. The most potent spike RBD inhibitor exhibited activity against several variants in live virus neutralization assays. These results establish that a single, broadly deployable generative foundation model for accelerated inhibitor discovery is effective and efficient, even in the absence of target structure or binder information.

Protection against SARS-CoV-2 Omicron BA.4/5 variant following booster vaccination or breakthrough infection in the UK.

Following primary SARS-CoV-2 vaccination, whether boosters or breakthrough infections provide greater protection against SARS-CoV-2 infection is incompletely understood. Here we investigated SARS-CoV-2 antibody correlates of protection against new Omicron BA.4/5 (re-)infections and anti-spike IgG antibody trajectories after a third/booster vaccination or breakthrough infection following second vaccination in 154,149 adults ≥18 y from the United Kingdom general population. Higher antibody levels were associated with increased protection against Omicron BA.4/5 infection and breakthrough infections were associated with higher levels of protection at any given antibody level than boosters. Breakthrough infections generated similar antibody levels to boosters, and the subsequent antibody declines were slightly slower than after boosters. Together our findings show breakthrough infection provides longer-lasting protection against further infections than booster vaccinations. Our findings, considered alongside the risks of severe infection and long-term consequences of infection, have important implications for vaccine policy.

Cryogenic electron ptychographic single particle analysis with wide bandwidth information transfer.

Advances in cryogenic transmission electron microscopy have revolutionised the determination of many macromolecular structures at atomic or near-atomic resolution. This method is based on conventional defocused phase contrast imaging. However, it has limitations of weaker contrast for small biological molecules embedded in vitreous ice, in comparison with cryo-ptychography, which shows increased contrast. Here we report a single-particle analysis based on the use of ptychographic reconstruction data, demonstrating that three dimensional reconstructions with a wide information transfer bandwidth can be recovered by Fourier domain synthesis. Our work suggests future applications in otherwise challenging single particle analyses, including small macromolecules and heterogeneous or flexible particles. In addition structure determination in situ within cells without the requirement for protein purification and expression may be possible.

Characterization of the rotavirus assembly pathway in situ using cryoelectron tomography.

Rotavirus assembly is a complex process that involves the stepwise acquisition of protein layers in distinct intracellular locations to form the fully assembled particle. Understanding and visualization of the assembly process has been hampered by the inaccessibility of unstable intermediates. We characterize the assembly pathway of group A rotaviruses observed in situ within cryo-preserved infected cells through the use of cryoelectron tomography of cellular lamellae. Our findings demonstrate that the viral polymerase VP1 recruits viral genomes during particle assembly, as revealed by infecting with a conditionally lethal mutant. Additionally, pharmacological inhibition to arrest the transiently enveloped stage uncovered a unique conformation of the VP4 spike. Subtomogram averaging provided atomic models of four intermediate states, including a pre-packaging single-layered intermediate, the double-layered particle, the transiently enveloped double-layered particle, and the fully assembled triple-layered virus particle. In summary, these complementary approaches enable us to elucidate the discrete steps involved in forming an intracellular rotavirus particle.