Biology subjects

Zhu, X.

Publications and source records attributed to Zhu, X..

At least 307 records · Page 17Linked to original sources

bioRxiv2021

On the robustness of inference of association with the gut microbiota in stool, swab and mucosal tissue samples

The gut microbiota plays an important role in human health and disease. Stool, swab and mucosal tissue samples have been used in individual studies to survey the microbial community but the consequences of using these different sample types are not completely understood. We previously reported differences in microbial community composition with 16S rRNA amplicon sequencing between stool, swab and mucosal tissue samples. Here, we extended the previous study to a larger cohort and performed shotgun metagenome sequencing of 1,397 stool, swab and mucosal tissue samples from 240 participants. Consistent with previous results, taxonomic composition of stool and swab samples was distinct, but still more similar to each other than mucosal tissue samples, which had a substantially different community composition, characterized by a high relative abundance of the mucus metabolizers Bacteroides and Subdoligranulum, as well as bacteria with higher tolerance for oxidative stress such as Escherichia. As has been previously reported, functional profiles were more uniform across sample types than taxonomic profiles with differences between stool and swab samples smaller, but mucosal tissue samples remained distinct from the other two types. When the taxonomic and functional profiles of different sample types were used for inference in association with host phenotypes of age, sex, body mass index (BMI), antibiotics or non-steroidal anti-inflammatory drugs (NSAIDs) use, hypothesis testing using either stool or swab gave broadly similar results, but inference performed on mucosal tissue samples gave results that were generally less consistent with either stool or swab. Our study represents an important resource for the experimental design of studies aimed to understand microbiota perturbations specific to defined micro niches within the human intestinal tract.

microbiology↗

bioRxiv2021

Sequence signatures of two IGHV3-53/3-66 public clonotypes to SARS-CoV-2 receptor binding domain

Since the COVID-19 pandemic onset, the antibody response to SARS-CoV-2 has been extensively characterized. Antibodies to the receptor binding domain (RBD) on the spike protein are frequently encoded by IGHV3-53/3-66 with a short CDR H3. Germline-encoded sequence motifs in CDRs H1 and H2 play a major role, but whether any common motifs are present in CDR H3, which is often critical for binding specificity, have not been elucidated. Here, we identify two public clonotypes of IGHV3-53/3-66 RBD antibodies with a 9-residue CDR H3 that pair with different light chains. Distinct sequence motifs on CDR H3 are present in the two public clonotypes that appear to be related to differential light chain pairing. Additionally, we show that Y58F is a common somatic hypermutation that results in increased binding affinity of IGHV3-53/3-66 RBD antibodies with a short CDR H3. Overall, our results advance fundamental understanding of the antibody response to SARS-CoV-2.

microbiology↗

bioRxiv2021

Human colorectal pre-cancer atlas identifies distinct molecular programs underlying two major subclasses of pre-malignant tumors

Most colorectal cancers (CRCs) develop from either adenomas (ADs) or sessile serrated lesions (SSLs). The origins and molecular landscapes of these histologically distinct pre-cancerous polyps remain incompletely understood. Here, we present an atlas at single-cell resolution of sporadic conventional tubular/tubulovillous ADs, SSLs, hyperplastic polyps (HPs), microsatellite stable (MSS) and unstable (MSI-H) CRC, and normal colonic mucosa. Using single-cell transcriptomics and multiplex imaging, we studied 69 datasets from 33 participants. We also examined separate sets of 66 and 274 polyps for RNA and targeted gene sequencing, respectively. We performed multiplex imaging on a tissue microarray of 14 ADs and 15 CRCs, and we integrated pre-cancer polyp data with published single-cell and The Cancer Genome Atlas (TCGA) bulk CRC data to establish potential polyp-cancer relationships. Striking differences were observed between ADs and SSLs that extended to MSS and MSI-H CRCs, respectively, reflecting their distinct origins and trajectories. ADs arose from WNT pathway dysregulation in stem cells, which aberrantly expanded and expressed a Hippo and ASCL2 regenerative program. In marked contrast, SSLs were depleted of stem cell-like populations and instead exhibited a program of gastric metaplasia in the setting of elevated cytotoxic inflammation. Using subtype-specific gene regulatory networks and shared genetic variant analysis, we implicated serrated polyps, including some HPs conventionally considered benign, as arising from a metaplastic program in committed absorptive cells. ADs and SSLs displayed distinct patterns of immune cell infiltration that may influence their natural history. Our multi-omic atlas provides novel insights into the malignant potential of colorectal polyps and serves as a framework for precision surveillance and prevention of sporadic CRC.

cancer biology↗

bioRxiv2021

Cryo-EM Structure of the N501Y SARS-CoV-2 Spike Protein in Complex with a Potent Neutralizing Antibody

The recently reported "UK variant" of SARS-CoV-2 is thought to be more infectious than previously circulating strains as a result of several changes, including the N501Y mutation. We present a 2.9-[A] resolution cryo-EM structure of the complex between the ACE2 receptor and N501Y spike protein ectodomains that shows Y501 inserted into a cavity at the binding interface near Y41 of ACE2. The additional interactions result in increased affinity of ACE2 for the N501Y mutant, accounting for its increased infectivity. However, this mutation does not result in large structural changes, enabling important neutralization epitopes to be retained in the spike receptor binding domain. We confirmed this through biophysical assays and by determining cryo-EM structures of spike protein ectodomains bound to two representative potent neutralizing antibody fragments. Short summaryThe N501Y mutation found in the coronavirus UK variant increases infectivity but some neutralizing antibodies can still bind.

biophysics↗

bioRxiv2021

Synaptojanin1 deficiency upregulates basal level autophagosome formation in astrocytes

Macroautophagy (hereafter, autophagy) dysregulation is implicated in multiple neurological disorders. While the autophagy pathways are heavily investigated in heterologous cells and neurons, how autophagy is regulated in the astrocyte, the most abundant cell type in the mammalian brain, is less understood. Here we report that Synaptojanin1 (Synj1), a neuron enriched lipid phosphatase, is expressed in low levels in astrocytes and represses autophagy at the basal level. Synj1 is encoded by the Synj1 gene, whose missense mutations are linked to Parkinsonism with seizure. While the best-known role of Synj1 is to facilitate synaptic vesicle recycling, recent studies suggest that Synj1 also regulates autophagy. Our previous study using the Synj1 haploinsufficient (Synj1+/-) mouse demonstrated that Synj1 deficiency was associated with an age-dependent autophagy impairment in multiple brain regions. We now use cultured astrocytes from Synj1 deficient mice to investigate its role in astrocyte autophagy. We demonstrate that Synj1 deficient astrocytes exhibit increased LC3 puncta, which is more pronounced when lysosomal acidification is blocked. The increased autophagosome formation is accompanied by reduced autophagy substrate, p62, but an insensitivity to starvation induced autophagy clearance. Moreover, we show, for the first time, that the Parkinsonism associated R839C mutation impacts astrocyte autophagy. The profound impact of this mutation on Synj1s phosphatase functions results in elevated basal level autophagosome formation and clearance that mimics Synj1 deletion. We find that energy sensing molecules, including mTOR and AMPK, are altered in Synj1 deficient astrocytes, which may contribute to the enhanced basal level autophagy.

cell biology↗

bioRxiv2020

N-glycolylneuraminic acid binding of avian H7 influenza A viruses

Influenza A viruses initiate infection by binding to glycans with terminal sialic acids present on the cell surface. Hosts of influenza A viruses variably express two major forms of sialic acid, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). NeuGc is produced in the majority of mammals including horses, pigs, and mice, but is absent in humans, ferrets, and birds. Intriguingly, the only known naturally occurring influenza A viruses that exclusively bind NeuGc are the extinct highly pathogenic equine H7N7 viruses. We determined the crystal structure of a representative equine H7 hemagglutinin (HA) in complex with its NeuGc ligand and observed a high similarity in the receptor-binding domain with an avian H7 HA. To determine the molecular basis for NeuAc and NeuGc specificity, we performed systematic mutational analyses, based on the structural insights, on two distant avian H7 HAs. We found that mutation A135E is key for binding 2,3-linked NeuGc but does not abolish NeuAc binding. Interestingly, additional mutations S128T, I130V, or a combination of T189A and K193R, converted from NeuAc to NeuGc specificity as determined by glycan microarrays. However, specific binding to NeuGc-terminal glycans on our glycan array did not always correspond with full NeuGc specificity on chicken and equine erythrocytes and tracheal epithelium sections. Phylogenetic analysis of avian and equine H7 HAs that investigated the amino acids at positions 128, 130, 135, 189, and 193 reveals a clear distinction between equine and avian residues. The highest variability in amino acids (four different residues) is observed at key position 135, of which only the equine glutamic acid leads to binding of NeuGc. The results demonstrate that avian H7 viruses, although genetically distinct from equine H7 viruses, can bind NeuGc after the introduction of two to three mutations, providing insights into the adaptation of H7 viruses to NeuGc receptors. Author summaryInfluenza A viruses cause millions of cases of severe illness and deaths annually. To initiate infection and replicate, the virus first needs to bind to a structure on the cell surface, like a key fitting in a lock. For influenza A virus, these keys (receptors) on the cell surface are chains of sugar molecules (glycans). The terminal sugar on these glycans is often either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc). Most influenza A viruses bind NeuAc, but a small minority binds NeuGc. NeuGc is present in species like horses, pigs, and mice, but not in humans, ferrets, and birds. Therefore, NeuGc binding could be a determinant of an Influenza A virus species barrier. Here, we investigated the molecular determinants of NeuGc specificity and the origin of viruses that bind NeuGc.

molecular biology↗

bioRxiv2020

Patient-Derived Orthotopic Xenografts and Cell Lines from Pediatric High-Grade Glioma Recapitulate the Heterogeneity of Histopathology, Molecular Signatures, and Drug Response

Pediatric high-grade glioma (pHGG) is a major contributor to cancer-related death in children. In vitro and in vivo disease models reflecting the intimate connection between developmental context and pathogenesis of pHGG are essential to advance understanding and identify therapeutic vulnerabilities. We established 21 patient-derived pHGG orthotopic xenograft (PDOX) models and eight matched cell lines from diverse groups of pHGG. These models recapitulated histopathology, DNA methylation signatures, mutations and gene expression patterns of the patient tumors from which they were derived, and included rare subgroups not well-represented by existing models. We deployed 16 new and existing cell lines for high-throughput screening (HTS). In vitro HTS results predicted variable in vivo response to inhibitors of PI3K/mTOR and MEK signaling pathways. These unique new models and an online interactive data portal to enable exploration of associated detailed molecular characterization and HTS chemical sensitivity data provide a rich resource for pediatric brain tumor research.

cancer biology↗

bioRxiv2020

Loss of phosphatidylserine flippase β-subunit Tmem30a in podocytes leads to albuminuria and glomerulosclerosis

Phosphatidylserine (PS) is asymmetrically concentrated in the cytoplasmic leaflet of eukaryotic cell plasma membranes. This asymmetry is regulated by a group of P4 ATPases (named PS flippases) and its {beta}-subunit TMEM30A. The disruption of PS flippase leads to severe human diseases. Tmem30a is essential in the mouse retina, cerebellum and liver. However, the role of Tmem30a in the kidney, where it is highly expressed, remains unclear. Podocytes in the glomerulus form a branched interdigitating filtration barrier that can prevent the traversing of large cellular elements and macromolecules from the blood into the urinary space. Damage to podocytes can disrupt the filtration barrier and lead to proteinuria and podocytopathy, including focal segmental glomerulosclerosis, minimal change disease, membranous nephropathy, and diabetic nephropathy. To investigate the role of Tmem30a in the kidney, we generated a podocyte-specific Tmem30a knockout (cKO) mouse model using the NPHS2-Cre line. Tmem30a KO mice displayed albuminuria, podocyte degeneration, mesangial cell proliferation with prominent extracellular matrix accumulation and eventual progression to focal segmental glomerulosclerosis (FSGS). Reduced TMEM30A expression was observed in patients with minimal change disease and membranous nephropathy, highlighting the clinical importance of TMEM30A in podocytopathy. Our data demonstrate a critical role of Tmem30a in maintaining podocyte survival and glomerular filtration barrier integrity. Understanding the dynamic regulation of the PS distribution in the glomerulus provides a unique perspective to pinpoint the mechanism of podocyte damage and potential therapeutic targets.

pathology↗

bioRxiv2020

Deficiency in DNA mismatch repair of methylation damage is a major mutational process in cancer

DNA mismatch repair (MMR) is essential for maintaining genome integrity with its deficiency predisposing to cancer1. MMR is well known for its role in the post-replicative repair of mismatched base pairs that escape proofreading by DNA polymerases following cell division2. Yet, cancer genome sequencing has revealed that MMR deficient cancers not only have high mutation burden but also harbour multiple mutational signatures3, suggesting that MMR has pleotropic effects on DNA repair. The mechanisms underlying these mutational signatures have remained unclear despite studies using a range of in vitro4,5 and in vivo6 models of MMR deficiency. Here, using mutation data from cancer genomes, we identify a previously unknown function of MMR, showing that the loss of non-canonical replication-independent MMR activity is a major mutational process in human cancers. MMR is comprised of the MutS (MSH2/MSH6) and MutL (MLH1/PMS2) complexes7. Cancers with deficiency of MutS exhibit mutational signature contributions distinct from those deficient of MutL. This disparity is attributed to mutations arising from the unrepaired deamination of 5-methylcytosine (5mC), i.e. methylation damage, as opposed to replicative errors by DNA polymerases induced mismatches. Repair of methylation damage is strongly associated with H3K36me3 chromatin but independent of binding of MBD4, a DNA glycosylase that recognise 5mC and can repair methylation damage. As H3K36me3 recruits MutS, our results suggest that MutS is the essential factor in mediating the repair of methylation damage. Cell line models of MMR deficiency display little evidence of 5mC deamination-induced mutations as their rapid rate of proliferation limits for the opportunity for methylation damage. We thus uncover a non-canonical role of MMR in the protection against methylation damage in non-dividing cells.

genomics↗

bioRxiv2020

STOMAGEN-like Regulates Stomatal Generation Causing Increased Water Use Efficiency during C4 Evolution

C4 photosynthesis optimizes plants carbon and water relations, allowing high photosynthetic rate with low stomatal conductance. Stomata have long been believed as a part of C4 syndrome. However, it remains unclear how stomata traits evolved along the path from C3 to C4. Stomatal patterning was examined in Flaveria genus, a model for studying C4 evolution. Comparative, transgenic and semi-in-vitro experiments were used to study molecular basis that underlies stomatal traits along C4 evolution. Novel results: the evolution from C3 to C4 species through intermediate species is accompanied by a stepwise rather than an abrupt change in the stomatal traits. The initial change occurs near Type II and dramatic change occurs at the C4-like species. On the road to C4, stomata become less in number but bigger in size and changes in stomatal density dominates changes in maximum stomatal conductance (gsmax). The reduction of FSTOMAGEN expression underlies altered gsmax between Flaveria species with different photosynthetic pathways and likely occurs in other C4 lineages. Our study provides insight into the pattern, mechanism and role of stomatal evolution along the road towards C4. This work highlights the stomatal traits in the current C4 evolutionary model and the co-evolution of photosynthetic pathway and stomata.

plant biology↗

bioRxiv2020

Potent SARS-CoV-2 neutralizing antibodies selected from a human antibody library constructed decades ago

Combinatorial antibody libraries not only effectively reduce antibody discovery to a numbers game, but enable documentation of the history of antibody responses in an individual. The SARS-CoV-2 pandemic has prompted a wider application of this technology to meet the public health challenge of pandemic threats in the modern era. Herein, we used a combinatorial human antibody library constructed 20 years before the COVID-19 pandemic to discover three highly potent antibodies that selectively bind SARS-CoV-2 spike protein and neutralize authentic SARS-CoV-2 virus. Compared to neutralizing antibodies from COVID-19 patients with generally low somatic hypermutation (SHM), these antibodies contain over 13-22 SHMs, many of which are involved in specific interactions in crystal structures with SARS-CoV-2 spike RBD. The identification of these somatically mutated antibodies in a pre-pandemic library raises intriguing questions about the origin and evolution of human immune responses to SARS-CoV-2.

microbiology↗

bioRxiv2020

Mitochondrial copper and phosphate transporter specificity was defined early in the evolution of eukaryotes

Mitochondrial carrier family (MCF/SLC25) proteins are selective transporters that maintain the mitochondrial metabolome. Here we combine computational, biochemical and phenotypic approaches to understand substrate selectivity of SLC25A3. In mammals, SLC25A3 transports both copper and phosphate, yet in Saccharomyces cerevisiae the transport of these substrates is partitioned across two paralogs: PIC2, which transports copper, and MIR1, which transports phosphate. To understand whether the ancestral state of this transporter was a single promiscuous transporter that duplicated and gained selectivity, we explored the evolutionary relationships of PIC2 and MIR1 orthologs across the eukaryotic tree of life. Phylogenetic analyses reveal that PIC2-like and MIR1-like orthologs are present in all major eukaryotic supergroups, indicating that the gene duplication that created these paralogs occurred early in eukaryotic evolution. Frequent lineage-specific gene duplications and losses suggest that substrate specificity may be evolutionarily labile. To link this phylogenetic signal to protein function and resolve the residues involved in substrate selection, we used structural modelling and site-directed mutagenesis to identify PIC2 residues involved in copper and phosphate transport activities. Based on these analyses, we generated a Leu175Ala variant of mouse SLC25A3 that retains the ability to transport copper, but not phosphate, and rescues the cytochrome c oxidase defect in SLC25A3 knockout cells. Taken together, this work uses an evolutionary framework to uncover amino acids involved in substrate recognition by MCF proteins responsible for copper and phosphate transport.

evolutionary biology↗

bioRxiv2020

Microtubules and Gαo-signaling independently regulate the preferential secretion of newly synthesized insulin granules in pancreatic islet β cells

For sustainable function, each pancreatic islet {beta} cell maintains thousands of insulin granules (IGs) at all times. Glucose stimulation induces the secretion of a small portion of these IGs and simultaneously triggers IG biosynthesis to sustain this stock. The failure of these processes, often induced by sustained high-insulin output, results in type 2 diabetes. Intriguingly, newly synthesized IGs are more likely secreted during glucose-stimulated insulin secretion. The older IGs tend to lose releasability and be degraded, which represents a futile metabolic load that can sensitize {beta} cells to workload-induced dysfunction and even death. Here, we examine the factor(s) that allows the preferential secretion of younger IGs. We show that {beta} cells without either microtubules (MTs) or Go signaling secrete a bigger portion of older IGs, which is associated with increased IG docking on plasma membrane. Yet Go inactivation does not alter the {beta}-cell MT network. These findings suggest that Go and MT regulate the preferential release of newer IGs via parallel pathways and provide two potential models to further explore the underlying mechanisms and physiological significance of this regulation in functional {beta} cells.

cell biology↗

bioRxiv2020

Cooperation-based sperm clusters mediate sperm oviduct entry and fertilization

Sperm cooperation has been observed in multiple species, yet its existence and benefit for reproductive success in mammals remains underexplored. Here, combining tissue-clearing with deep three-dimensional imaging, we demonstrate that postcopulatory mouse sperm congregate into unidirectional sperm cooperative clusters at the utero-tubal junction (UTJ), a key physical barrier for passage into the oviduct. Reducing sperm number in male mice by unilateral vasoligation or busulfan-treatment impairs sperm cluster formation and oviduct entry. Interestingly, sperm derived from Tex101-/- mouse has normal number, motility and morphology, yet they cannot form sperm cluster and fail to pass through the UTJ, which is at least in part due to the altered tail beating pattern of the Tex101-/- sperm. Moreover, Tex101-/- sperms defect in oviduct entry cannot be rescued by the presence of wild-type (WT) sperm in the same uteri by sequential mating, suggesting sperm cooperative cluster as an essential behavior contributing to male fertility, which could be related to human infertility or subfertility.

developmental biology↗

bioRxiv2020

idCOV: a pipeline for quick clade identification of SARS-CoV-2 isolates

idCOV is a phylogenetic pipeline for quickly identifying the clades of SARS-CoV-2 virus isolates from raw sequencing data based on a selected clade-defining marker list. Using a public dataset, we show that idCOV can make equivalent calls as annotated by Nextstrain.org on all three common clade systems using user uploaded FastQ files directly. Web and equivalent command-line interfaces are available. It can be deployed on any Linux environment, including personal computer, HPC and the cloud. The source code is available at https://github.com/xz-stjude/idcov. A documentation for installation can be found at https://github.com/xz-stjude/idcov/blob/master/README.md.

bioinformatics↗

bioRxiv2020

Transferrin receptor (Tfr1) ablation in satellite cells impacts skeletal muscle regeneration through the activation of ferroptosis

Satellite cells (SCs) are critical to the postnatal development and skeletal muscle regeneration. Inactivation of SCs is linked with the skeletal muscle loss. Leveraging on the RNAseq screening, transferrin receptor (Tfr1) is identified to be associated with muscle/SC ageing and the declined regeneration potential. Muscle-specific deletion of Tfr1 results in the growth retardation, metabolic disorder and lethality, shedding light on the importance of Tfr1 in skeletal muscle physiology. Here, our investigation reported that conditional SC-ablation of Tfr1 leads to the SCs inactivation and skeletal muscle regeneration defects, followed by the labile iron accumulation, de novo lipogenesis via fibroadipogenic progenitors (FAPs) and Gpx4/Nrf2-mediated ROS-scavenger defects. These abnormal phenomena, such as Hmox1-mediated myoglobin degradation, Tfr1-Slc39a14 functional switch and the activation of unsaturated fatty acid biosynthesis pathway are orchestrated with the occurrence of ferroptosis in skeletal muscle. Ferroptosis may further prevent SC proliferation and skeletal muscle regeneration. Ferrostatin-1, a ferroptosis inhibitor could not rescue Tfr1-ablation induced ferroptosis. However, intramuscular administration of lentivirus expressing Tfr1 could partially reduce labile iron accumulation, decrease de novo lipogenesis and promote skeletal muscle regeneration. Most importantly, Tfr1/Slc39a14 functional switch, labile iron accumulation and fatty acid biosynthesis are recapitulated in aged skeletal muscle of rodents, indicating that ferroptosis occurs in the skeletal muscles of aged rodents. The present study also bridges the gap between pathogenesis of iron and functional defects in the skeletal muscle, providing mechanistic information to develop anti-aging strategies. One Sentence SummaryConditional ablation of Tfr1 in satellite cells (SCs) results in the SC inactivation, skeletal muscle regeneration defects, labile iron accumulation, and unsaturated fatty acid biosynthesis, leading to the activation of ferroptosis, which is recapitulated in skeletal muscles of aged rodents to be a new cell death form identified in skeletal muscle and sheds light on the development of novel anti-ageing strategies.

cell biology↗

bioRxiv2020

Effects of N-linked glycan of Lassa Virus Envelope Glycoprotein on the Immune Response

Lassa virus (LASV) belongs to the Mammarenavirus genus (family Arenaviridae) and causes severe hemorrhagic fever in humans. The glycoprotein precursor (GPC) contains eleven N-linked glycans that play essential roles in GPC functionalities such as cleavage, transport, receptor recognition, epitope shielding, and immune response. We used three mutagenesis strategies to abolish the individual glycan chains on the GPC and found that all three mutations led to cleavage inefficiency on the 2nd, 5th, and 8th glycosylation motifs. To evaluate N to Q mutagenesis for further research, it was found that deletion of the 2nd and 8th glycans completely inhibited the infectivity. We further investigated the role of glycans on GPC-mediated immune response by DNA immunization of mice. Deletion of the individual 1st, 3rd, 5th and 6th glycans significantly enhanced the proportion of effector CD4+ cells, whereas deletion of the 1st, 2nd, 3rd, 4th 5th, 6th, and 9th glycans enhanced the proportion of CD8+ effector T cells. Deletion of specific glycans improves the Th1-type immune response, and abolishment of glycan on GPC generally increases the antibody titer to the glycan-deficient GPC. However, the antibodies from either the mutant or WT GPC-immunized mice show little neutralization effect on wild-type LASV. The glycan residues on GPC provide an immune shield for the virus, and thus represent a target for the design and development of a vaccine. ImportanceAt present, there are no Food and Drug Administration-approved drugs or vaccines specific for LASV. Similar to other enveloped viruses with a heavy glycan shield, the N-linked glycans of LASV make it difficult for effector T cells and neutralization antibodies to access the glycoprotein epitope. In this study, we evaluated the effect of the individual glycan chains on GPC-mediated immune response, and found that deletion of the glycan improves the proportion of effector T cells, improving the Th1-type immune response, and increasing the antibody titer to the WT and mutant GPC, which may be beneficial to vaccine design and development.

microbiology↗

bioRxiv2020

A natural mutation between SARS-CoV-2 and SARS-CoV determines neutralization by a cross-reactive antibody

Epitopes that are conserved among SARS-like coronaviruses are attractive targets for design of cross-reactive vaccines and therapeutics. CR3022 is a SARS-CoV neutralizing antibody to a highly conserved epitope on the receptor binding domain (RBD) on the spike protein that can cross-react with SARS-CoV-2, but with lower affinity. Using x-ray crystallography, mutagenesis, and binding experiments, we illustrate that of four amino acid differences in the CR3022 epitope between SARS-CoV-2 and SARS-CoV, a single mutation P384A fully determines the affinity difference. CR3022 does not neutralize SARS-CoV-2, but the increased affinity to SARS-CoV-2 P384A mutant now enables neutralization with a similar potency to SARS-CoV. We further investigated CR3022 interaction with the SARS-CoV spike protein by negative-stain EM and cryo-EM. Three CR3022 Fabs bind per trimer with the RBD observed in different up-conformations due to considerable flexibility of the RBD. In one of these conformations, quaternary interactions are made by CR3022 to the N-terminal domain (NTD) of an adjacent subunit. Overall, this study provides insights into antigenic variation and potential for cross-neutralizing epitopes on SARS-like viruses.

microbiology↗