Structural analysis of receptor engagement and antigenic drift within the BA.2 spike protein.

The BA.2 sub-lineage of the Omicron (B.1.1.529) severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant rapidly supplanted the original BA.1 sub-lineage in early 2022. Both lineages threatened the efficacy of vaccine-elicited antibodies and acquired increased binding to several mammalian ACE2 receptors. Cryoelectron microscopy (cryo-EM) analysis of the BA.2 spike (S) glycoprotein in complex with mouse ACE2 (mACE2) identifies BA.1- and BA.2-mutated residues Q493R, N501Y, and Y505H as complementing non-conserved residues between human and mouse ACE2, rationalizing the enhanced S protein-mACE2 interaction for Omicron variants. Cryo-EM structures of the BA.2 S-human ACE2 complex and of the extensively mutated BA.2 amino-terminal domain (NTD) reveal a dramatic reorganization of the highly antigenic N1 loop into a ß-strand, providing an explanation for decreased binding of the BA.2 S protein to antibodies isolated from BA.1-convalescent patients. Our analysis reveals structural mechanisms underlying the antigenic drift in the rapidly evolving Omicron variant landscape.

SARS-CoV-2 variants of concern: spike protein mutational analysis and epitope for broad neutralization.

Mutations in the spike glycoproteins of SARS-CoV-2 variants of concern have independently been shown to enhance aspects of spike protein fitness. Here, we describe an antibody fragment (VH ab6) that neutralizes all major variants including the recently emerged BA.1 and BA.2 Omicron subvariants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within previously emerged variant spikes and identify the structural role of mutations within the NTD and RBD in evading antibody neutralization. Our analysis shows that the highly mutated Gamma N-terminal domain exhibits considerable structural rearrangements, partially explaining its decreased neutralization by convalescent sera. Our results provide mechanistic insights into the structural, functional, and antigenic consequences of SARS-CoV-2 spike mutations and highlight a spike protein vulnerability that may be exploited to achieve broad protection against circulating variants.

Three-Dimensional Visualization of Viral Structure, Entry, and Replication Underlying the Spread of SARS-CoV-2.

The global spread of SARS-CoV-2 has proceeded at an unprecedented rate. Remarkably, characterization of the virus using modern tools in structural biology has also progressed at exceptional speed. Advances in electron-based imaging techniques, combined with decades of foundational studies on related viruses, have enabled the research community to rapidly investigate structural aspects of the novel coronavirus from the level of individual viral proteins to imaging the whole virus in a native context. Here, we provide a detailed review of the structural biology and pathobiology of SARS-CoV-2 as it relates to all facets of the viral life cycle, including cell entry, replication, and three-dimensional (3D) packaging based on insights obtained from X-ray crystallography, cryo-electron tomography, and single-particle cryo-electron microscopy. The structural comparison between SARS-CoV-2 and the related earlier viruses SARS-CoV and MERS-CoV is a common thread throughout this review. We conclude by highlighting some of the outstanding unanswered structural questions and underscore areas that are under rapid current development such as the design of effective therapeutics that block viral infection.

Potent and broad neutralization of SARS-CoV-2 variants of concern (VOCs) including omicron sub-lineages BA.1 and BA.2 by biparatopic human VH domains.

The emergence of SARS-CoV-2 variants of concern (VOCs) requires the development of next-generation biologics with high neutralization breadth. Here, we characterized a human VH domain, F6, which we generated by sequentially panning large phage-displayed VH libraries against receptor binding domains (RBDs) containing VOC mutations. Cryo-EM analyses reveal that F6 has a unique binding mode that spans a broad surface of the RBD and involves the antibody framework region. Attachment of an Fc region to a fusion of F6 and ab8, a previously characterized VH domain, resulted in a construct (F6-ab8-Fc) that broadly and potently neutralized VOCs including Omicron. Additionally, prophylactic treatment using F6-ab8-Fc reduced live Beta (B.1.351) variant viral titers in the lungs of a mouse model. Our results provide a new potential therapeutic against SARS-CoV-2 variants including Omicron and highlight a vulnerable epitope within the spike that may be exploited to achieve broad protection against circulating variants.

Structural and biochemical rationale for enhanced spike protein fitness in delta and kappa SARS-CoV-2 variants.

The Delta and Kappa variants of SARS-CoV-2 co-emerged in India in late 2020, with the Delta variant underlying the resurgence of COVID-19, even in countries with high vaccination rates. In this study, we assess structural and biochemical aspects of viral fitness for these two variants using cryo-electron microscopy (cryo-EM), ACE2-binding and antibody neutralization analyses. Both variants demonstrate escape of antibodies targeting the N-terminal domain, an important immune hotspot for neutralizing epitopes. Compared to wild-type and Kappa lineages, Delta variant spike proteins show modest increase in ACE2 affinity, likely due to enhanced electrostatic complementarity at the RBD-ACE2 interface, which we characterize by cryo-EM. Unexpectedly, Kappa variant spike trimers form a structural head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation and with unknown biological implications. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

SARS-CoV-2 Omicron variant: Antibody evasion and cryo-EM structure of spike protein-ACE2 complex.

The newly reported Omicron variant is poised to replace Delta as the most prevalent SARS-CoV-2 variant across the world. Cryo-EM structural analysis of the Omicron variant spike protein in complex with human ACE2 reveals new salt bridges and hydrogen bonds formed by mutated residues R493, S496 and R498 in the RBD with ACE2. These interactions appear to compensate for other Omicron mutations such as K417N known to reduce ACE2 binding affinity, resulting in similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion. The increase in antibody evasion, together with retention of strong interactions at the ACE2 interface, thus represent important molecular features that likely contribute to the rapid spread of the Omicron variant.

Structural analysis of receptor binding domain mutations in SARS-CoV-2 variants of concern that modulate ACE2 and antibody binding.

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Beta (B.1.351) and Gamma (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the Alpha (B.1.1.7) variant that enhances affinity of the spike protein for its receptor, angiotensin-converting enzyme 2 (ACE2). Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Epsilon variants (B.1.427/429) lack the N501Y mutation yet exhibit antibody evasion. We have engineered spike proteins to express these receptor binding domain (RBD) VoC mutations either in isolation or in different combinations and analyze the effects using biochemical assays and cryoelectron microscopy (cryo-EM) structural analyses. Overall, our findings suggest that the emergence of new SARS-CoV-2 variant spikes can be rationalized as the result of mutations that confer increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

Cryo-electron microscopy structures of the N501Y SARS-CoV-2 spike protein in complex with ACE2 and 2 potent neutralizing antibodies.

The recently reported "UK variant" (B.1.1.7) 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-Å resolution cryo-electron microscopy (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. This additional interaction provides a structural explanation for the increased ACE2 affinity of the N501Y mutant, and likely contributes to 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 2 representative potent neutralizing antibody fragments.