Virion morphology and on-virus spike protein structures of diverse SARS-CoV-2 variants (preprint)

The evolution of SARS-CoV-2 variants with increased fitness has been accompanied by structural changes in the spike (S) proteins that are the major target for the adaptive immune response. Single-particle cryo-EM analysis of soluble S from SARS-CoV-2 variants has revealed this structural adaptation at high-resolution. The analysis of S trimers in situ on intact virions has the potential to provide more functionally relevant insights into S structure and virion morphology. Here, we characterized B.1, Alpha, Beta, Gamma, Delta, Kappa, and Mu variants by cryo-electron microscopy and tomography, assessing S cleavage, virion morphology, S incorporation, "in-situ" high-resolution S structures and the range of S conformational states. We found no evidence for adaptive changes in virion morphology, but describe multiple different positions in the S protein where amino acid changes alter local protein structure. Considered together, our data is consistent with a model where amino acid changes at multiple positions from the top to the base of the spike cause structural changes that can modulate the conformational dynamics of S.

Antibodies to the SARS-CoV-2 receptor-binding domain that maximize breadth and resistance to viral escape (preprint)

An ideal anti-SARS-CoV-2 antibody would resist viral escape, have activity against diverse SARS-related coronaviruses, and be highly protective through viral neutralization and effector functions. Understanding how these properties relate to each other and vary across epitopes would aid development of antibody therapeutics and guide vaccine design. Here, we comprehensively characterize escape, breadth, and potency across a panel of SARS-CoV-2 antibodies targeting the receptor-binding domain (RBD), including S309, the parental antibody of the late-stage clinical antibody VIR-7831. We observe a tradeoff between SARS-CoV-2 in vitro neutralization potency and breadth of binding across SARS-related coronaviruses. Nevertheless, we identify several neutralizing antibodies with exceptional breadth and resistance to escape, including a new antibody (S2H97) that binds with high affinity to all SARS-related coronavirus clades via a unique RBD epitope centered on residue E516. S2H97 and other escape-resistant antibodies have high binding affinity and target functionally constrained RBD residues. We find that antibodies targeting the ACE2 receptor binding motif (RBM) typically have poor breadth and are readily escaped by mutations despite high neutralization potency, but we identify one potent RBM antibody (S2E12) with breadth across sarbecoviruses closely related to SARS-CoV-2 and with a high barrier to viral escape. These data highlight functional diversity among antibodies targeting the RBD and identify epitopes and features to prioritize for antibody and vaccine development against the current and potential future pandemics.

Structure, Dynamics, Receptor Binding, and Antibody Binding of Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane (preprint)

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mediates host cell entry by binding to angiotensin-converting enzyme 2 (ACE2), and is considered the major target for drug and vaccine development. We previously built fully-glycosylated full-length SARS-CoV-2 S protein models in a viral membrane including both open and closed conformations of receptor binding domain (RBD) and different templates for the stalk region. In this work, multiple s-long all-atom molecular dynamics simulations were performed to provide deeper insight into the structure and dynamics of S protein, and glycan functions. Our simulations reveal that the highly flexible stalk is composed of two independent joints and most probable S protein orientations are competent for ACE2 binding. We identify multiple glycans stabilizing the open and/or closed states of RBD, and demonstrate that the exposure of antibody epitopes can be captured by detailed antibody-glycan clash analysis instead of a commonly-used accessible surface area analysis that tends to overestimate the impact of glycan shielding and neglect possible detailed interactions between glycan and antibody. Overall, our observations offer structural and dynamic insight into SARS-CoV-2 S protein and potentialize for guiding the design of effective antiviral therapeutics.

Modeling and Simulation of a Fully-glycosylated Full-length SARS-CoV-2 Spike Protein in a Viral Membrane (preprint)

This technical study describes all-atom modeling and simulation of a fully-glycosylated full-length SARS-CoV-2 spike (S) protein in a viral membrane. First, starting from PDB:6VSB and 6VXX, full-length S protein structures were modeled using template-based modeling, de-novo protein structure prediction, and loop modeling techniques in GALAXY modeling suite. Then, using the recently-determined most occupied glycoforms, 22 N-glycans and 1 O-glycan of each monomer were modeled using Glycan Reader & Modeler in CHARMM-GUI. These fully-glycosylated full-length S protein model structures were assessed and further refined against the low-resolution data in their respective experimental maps using ISOLDE. We then used CHARMM-GUI Membrane Builder to place the S proteins in a viral membrane and performed all-atom molecular dynamics simulations. All structures are available in CHARMM-GUI COVID-19 Archive (http://www.charmm-gui.org/docs/archive/covid19), so researchers can use these models to carry out innovative and novel modeling and simulation research for the prevention and treatment of COVID-19.