A pseudovirus system enables deep mutational scanning of the full SARS-CoV-2 spike

Cell, Год журнала: 2023, Номер 186(6), С. 1263 - 1278.e20

Опубликована: Фев. 13, 2023

A major challenge in understanding SARS-CoV-2 evolution is interpreting the antigenic and functional effects of emerging mutations viral spike protein. Here, we describe a deep mutational scanning platform based on non-replicative pseudotyped lentiviruses that directly quantifies how large numbers impact antibody neutralization pseudovirus infection. We apply this to produce libraries Omicron BA.1 Delta spikes. These each contain ∼7,000 distinct amino acid context up ∼135,000 unique mutation combinations. use these map escape from neutralizing antibodies targeting receptor-binding domain, N-terminal S2 subunit spike. Overall, work establishes high-throughput safe approach measure ∼10

Язык: Английский

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Deep mutational scans of XBB.1.5 and BQ.1.1 reveal ongoing epistatic drift during SARS-CoV-2 evolution

PLoS Pathogens, Год журнала: 2023, Номер 19(12), С. e1011901 - e1011901

Опубликована: Дек. 29, 2023

Substitutions that fix between SARS-CoV-2 variants can transform the mutational landscape of future evolution via epistasis. For example, large epistatic shifts in effects caused by N501Y underlied original emergence Omicron, but whether such saltations continue to define ongoing remains unclear. We conducted deep scans measure impacts all single amino acid mutations and single-codon deletions spike receptor-binding domain (RBD) on ACE2-binding affinity protein expression recent Omicron BQ.1.1 XBB.1.5 variants, we compared patterns earlier viral strains have previously profiled. As with previous scans, find many are tolerated or even enhance binding ACE2 receptor. The tolerance sites deletion largely conforms mutation. Though RBD not yet been seen dominant lineages, observe including at positions exhibit indel variation across broader sarbecovirus emerging interest, most notably well-tolerated Δ483 BA.2.86. substitutions distinguish induced as dramatic perturbations N501Y, identify drift interaction R493Q reversions 453, 455, 456, F456L defines XBB.1.5-derived EG.5 lineage. Our results highlight due epistasis, which may direct into new regions sequence space.

Язык: Английский

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High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike

Nature Communications, Год журнала: 2023, Номер 14(1)

Опубликована: Апрель 10, 2023

Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All vaccines in US encode with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering immunogens involve tedious experimental work and heavily rely structural information. Here, we establish a systematic unbiased method identifying that concomitantly improve expression conformation spike. Our integrates fluorescence-based fusion assay, mammalian cell display technology, deep mutational scanning. As proof-of-concept, apply this region S2 domain includes first heptad repeat central helix. results reveal besides K986P V987P, several simultaneously significantly lower fusogenicity stabilization common challenge viral immunogen design, will help accelerate vaccine development against different viruses.

Язык: Английский

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Deep mutational scanning of SARS-CoV-2 Omicron BA.2.86 and epistatic emergence of the KP.3 variant

Virus Evolution, Год журнала: 2024, Номер 10(1)

Опубликована: Янв. 1, 2024

Deep mutational scanning experiments aid in the surveillance and forecasting of viral evolution by providing prospective measurements effects on traits, but epistatic shifts impacts mutations can hinder when were made outdated strain backgrounds. Here, we report impact all single amino acid ACE2-binding affinity protein folding expression SARS-CoV-2 Omicron BA.2.86 spike receptor-binding domain. As with other variants, find a plastic evolvable basis for receptor binding, many at ACE2 interface maintaining or even improving affinity. Despite its large genetic divergence, have not diverged greatly from those measured BA.2 ancestor. However, do identify strong positive epistasis among subsequent that accrued descendants. Specifically, Q493E mutation decreased previous backgrounds is reversed sign to enhance human coupled L455S F456L currently emerging KP.3 variant. Our results point modest degree drift during recent highlight how these small important consequences emergence new variants.

Язык: Английский

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SARS-CoV-2 resistance to monoclonal antibodies and small-molecule drugs

Cell chemical biology, Год журнала: 2024, Номер 31(4), С. 632 - 657

Опубликована: Апрель 1, 2024

Over four years have passed since the beginning of COVID-19 pandemic. The scientific response has been rapid and effective, with many therapeutic monoclonal antibodies small molecules developed for clinical use. However, given ability viruses to become resistant antivirals, it is perhaps no surprise that field identified resistance nearly all these compounds. Here, we provide a comprehensive review profile each therapeutics. We hope this resource provides an atlas mutations be aware agent, particularly as springboard considerations next generation antivirals. Finally, discuss outlook thoughts moving forward in how continue manage this, next,

Язык: Английский

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Advancing Antibody Engineering through Synthetic Evolution and Machine Learning

The Journal of Immunology, Год журнала: 2024, Номер 212(2), С. 235 - 243

Опубликована: Янв. 2, 2024

Abstract Abs are versatile molecules with the potential to achieve exceptional binding target Ags, while also possessing biophysical properties suitable for therapeutic drug development. Protein display and directed evolution systems have transformed synthetic Ab discovery, engineering, optimization, vastly expanding number of clones able be experimentally screened binding. Moreover, burgeoning integration high-throughput screening, deep sequencing, machine learning has further augmented in vitro promising accelerate design process massively expand sequence space interrogated. In this Brief Review, we discuss experimental computational tools employed engineering optimization. We explore challenges posed by developing infectious diseases, prospects leveraging learning–guided protein prospectively resistant viral escape.

Язык: Английский

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Functional and antigenic characterization of SARS-CoV-2 spike fusion peptide by deep mutational scanning

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Май 14, 2024

Abstract The fusion peptide of SARS-CoV-2 spike protein is functionally important for membrane during virus entry and part a broadly neutralizing epitope. However, sequence determinants at the its adjacent regions pathogenicity antigenicity remain elusive. In this study, we perform series deep mutational scanning (DMS) experiments on an S2 region spanning authentic in different cell lines presence antibodies. We identify mutations residue 813 that reduced TMPRSS2-mediated with decreased virulence. addition, show F823Y mutation, present bat betacoronavirus HKU9 protein, confers resistance to Our findings provide mechanistic insights into also highlight potential challenge developing protective S2-based coronavirus vaccines.

Язык: Английский

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Deep mutagenesis scanning using whole trimeric SARS-CoV-2 spike highlights the importance of NTD-RBD interactions in determining spike phenotype

PLoS Pathogens, Год журнала: 2023, Номер 19(8), С. e1011545 - e1011545

Опубликована: Авг. 3, 2023

New variants of SARS-CoV-2 are continually emerging with mutations in spike associated increased transmissibility and immune escape. Phenotypic maps can inform the prediction concerning from genomic surveillance, however most these currently derive studies using monomeric RBD, while is trimeric, contains additional domains. These may fail to reflect interdomain interactions phenotypes. To try improve on this, we developed a platform for deep mutational scanning whole trimeric spike. We confirmed previously reported epistatic effect within RBD affecting ACE2 binding, that highlights importance updating base sequence future studies. Using post vaccine sera, found response vaccinated individuals was highly focused one or two epitopes single point at positions account escape mediated by Omicron BA.1 RBD. However, unexpectedly alone does not high level antigenic show NTD amplifies evasion its reduces neutralistion directed monoclonal antibodies, impacts interaction. variation thus an important mechanism SARS-CoV-2. Such effects seen when pre-stabilized proteins used, suggesting require protein mobility express their phenotype.

Язык: Английский

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Towards Quantum-Chemical Level Calculations of SARS-CoV-2 Spike Protein Variants of Concern by First Principles Density Functional Theory

Biomedicines, Год журнала: 2023, Номер 11(2), С. 517 - 517

Опубликована: Фев. 10, 2023

The spike protein (S-protein) is a crucial part of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), with its many domains responsible for binding, fusion, and host cell entry. In this review we use density functional theory (DFT) calculations to analyze atomic-scale interactions investigate consequences mutations in S-protein domains. We specifically describe key amino acids functions each domain, which are essential structural stability as well recognition fusion processes cell; addition, speculate on how affect these properties. Such unprecedented large-scale ab initio calculations, up 5000 atoms system, based novel concept acid–amino acid-bond pair unit (AABPU) that allows an alternative description proteins, providing valuable information partial charge, interatomic bonding hydrogen bond (HB) formation. general, our results show different variants foster increased positive alter interactions, disrupt HB networks. conclude by outlining roadmap future computational research biomolecular virus-related systems.

Язык: Английский

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Deep mutational scanning of proteins in mammalian cells

Cell Reports Methods, Год журнала: 2023, Номер 3(11), С. 100641 - 100641

Опубликована: Ноя. 1, 2023

Protein mutagenesis is essential for unveiling the molecular mechanisms underlying protein function in health, disease, and evolution. In past decade, deep mutational scanning methods have evolved to support functional analysis of nearly all possible single-amino acid changes a interest. While historically these were developed lower organisms such as E. coli yeast, recent technological advancements resulted increased use mammalian cells, particularly studying proteins involved human disease. These will aid significantly classification interpretation variants unknown significance, which are being discovered at large scale due current surge whole-genome sequencing clinical contexts. Here, we explore experimental aspects studies cells report different used each step workflow, ultimately providing useful guide toward design studies.

Язык: Английский

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