A pan-variant miniprotein inhibitor protects against SARS-CoV-2 variants.

The continued evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has compromised neutralizing antibody responses elicited by prior infection or vaccination and abolished the utility of most monoclonal antibody therapeutics. We previously described a computationally-designed, homotrimeric miniprotein inhibitor, designated TRI2-2, that protects mice against pre-Omicron SARS-CoV-2 variants. Here, we show that TRI2-2 exhibits pan neutralization of variants that evolved during the 4.5 years since the emergence of SARS-CoV-2 and protects mice against BQ.1.1, XBB.1.5 and BA.2.86 challenge when administered post-exposure by an intranasal route. The resistance of TRI2-2 to viral escape and its direct delivery to the upper airways rationalize a path toward clinical advancement.

Spike deep mutational scanning helps predict success of SARS-CoV-2 clades.

SARS-CoV-2 variants acquire mutations in the spike protein that promote immune evasion1 and affect other properties that contribute to viral fitness, such as ACE2 receptor binding and cell entry2,3. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning4 to measure how more than 9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully affected ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456 and 473; however, the antigenic effects of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however, many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.

A systematic search for RNA structural switches across the human transcriptome.

RNA structural switches are key regulators of gene expression in bacteria, but their characterization in Metazoa remains limited. Here, we present SwitchSeeker, a comprehensive computational and experimental approach for systematic identification of functional RNA structural switches. We applied SwitchSeeker to the human transcriptome and identified 245 putative RNA switches. To validate our approach, we characterized a previously unknown RNA switch in the 3' untranslated region of the RORC (RAR-related orphan receptor C) transcript. In vivo dimethyl sulfate (DMS) mutational profiling with sequencing (DMS-MaPseq), coupled with cryogenic electron microscopy, confirmed its existence as two alternative structural conformations. Furthermore, we used genome-scale CRISPR screens to identify trans factors that regulate gene expression through this RNA structural switch. We found that nonsense-mediated messenger RNA decay acts on this element in a conformation-specific manner. SwitchSeeker provides an unbiased, experimentally driven method for discovering RNA structural switches that shape the eukaryotic gene expression landscape.

A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines.

Evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S2 subunit, which folds as a spring-loaded fusion machinery. We describe a strategy for prefusion-stabilization and high yield recombinant production of SARS-CoV-2 S2 trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S2 subunits. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S2 trimer elicits broadly reactive sarbecovirus antibodies and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines.

Broadly neutralizing antibodies against emerging delta-coronaviruses.

Porcine deltacoronavirus (PDCoV) spillovers were recently detected in children with acute undifferentiated febrile illness, underscoring recurrent zoonoses of divergent coronaviruses. To date, no vaccines or specific therapeutics are approved for use in humans against PDCoV. To prepare for possible future PDCoV epidemics, we isolated human spike (S)-directed monoclonal antibodies from transgenic mice and found that two of them, designated PD33 and PD41, broadly neutralized a panel of PDCoV variants. Cryo-electron microscopy structures of PD33 and PD41 in complex with the PDCoV receptor-binding domain and S ectodomain trimer provide a blueprint of the epitopes recognized by these mAbs, rationalizing their broad inhibitory activity. We show that both mAbs inhibit PDCoV by competitively interfering with host APN binding to the PDCoV receptor-binding loops, explaining the mechanism of viral neutralization. PD33 and PD41 are candidates for clinical advancement, which could be stockpiled to prepare for possible future PDCoV outbreaks.

Full-spike deep mutational scanning helps predict the evolutionary success of SARS-CoV-2 clades.

SARS-CoV-2 variants acquire mutations in spike that promote immune evasion and impact other properties that contribute to viral fitness such as ACE2 receptor binding and cell entry. Knowledge of how mutations affect these spike phenotypes can provide insight into the current and potential future evolution of the virus. Here we use pseudovirus deep mutational scanning to measure how >9,000 mutations across the full XBB.1.5 and BA.2 spikes affect ACE2 binding, cell entry, or escape from human sera. We find that mutations outside the receptor-binding domain (RBD) have meaningfully impacted ACE2 binding during SARS-CoV-2 evolution. We also measure how mutations to the XBB.1.5 spike affect neutralization by serum from individuals who recently had SARS-CoV-2 infections. The strongest serum escape mutations are in the RBD at sites 357, 420, 440, 456, and 473-however, the antigenic impacts of these mutations vary across individuals. We also identify strong escape mutations outside the RBD; however many of them decrease ACE2 binding, suggesting they act by modulating RBD conformation. Notably, the growth rates of human SARS-CoV-2 clades can be explained in substantial part by the measured effects of mutations on spike phenotypes, suggesting our data could enable better prediction of viral evolution.

Recent advances in infectious disease research using cryo-electron tomography.

With the increasing spread of infectious diseases worldwide, there is an urgent need for novel strategies to combat them. Cryogenic sample electron microscopy (cryo-EM) techniques, particularly electron tomography (cryo-ET), have revolutionized the field of infectious disease research by enabling multiscale observation of biological structures in a near-native state. This review highlights the recent advances in infectious disease research using cryo-ET and discusses the potential of this structural biology technique to help discover mechanisms of infection in native environments and guiding in the right direction for future drug discovery.

A broadly generalizable stabilization strategy for sarbecovirus fusion machinery vaccines.

Continuous evolution of SARS-CoV-2 alters the antigenicity of the immunodominant spike (S) receptor-binding domain and N-terminal domain, undermining the efficacy of vaccines and monoclonal antibody therapies. To overcome this challenge, we set out to develop a vaccine focusing antibody responses on the highly conserved but metastable S2 subunit, which folds as a spring-loaded fusion machinery. Here, we describe a protein design strategy enabling prefusion-stabilization of the SARS-CoV-2 S2 subunit and high yield recombinant expression of trimers with native structure and antigenicity. We demonstrate that our design strategy is broadly generalizable to all sarbecoviruses, as exemplified with the SARS-CoV-1 (clade 1a) and PRD-0038 (clade 3) S2 fusion machineries. Immunization of mice with a prefusion-stabilized SARS-CoV-2 S2 trimer vaccine elicits broadly reactive sarbecovirus antibody responses and neutralizing antibody titers of comparable magnitude against Wuhan-Hu-1 and the immune evasive XBB.1.5 variant. Vaccinated mice were protected from weight loss and disease upon challenge with SARS-CoV-2 XBB.1.5, providing proof-of-principle for fusion machinery sarbecovirus vaccines motivating future development.

Classifying liganded states in heterogeneous single-particle cryo-EM datasets.

A powerful aspect of single-particle cryogenic electron microscopy is its ability to determine high-resolution structures from samples containing heterogeneous mixtures of the same macromolecule in different conformational or compositional states. Beyond determining structures at higher resolutions, one outstanding question is if macromolecules with only subtle conformation differences, such as the same protein bound with different ligands in the same binding pocket, can be separated reliably, and if information concerning binding kinetics can be derived from the particle distributions of different conformations obtained in classification. In this study, we address these questions by assessing the classification of synthetic heterogeneous datasets of Transient Receptor Potential Vanilloid 1 generated by combining different homogeneous experimental datasets. Our results indicate that classification can isolate highly homogeneous subsets of particle for calculating high-resolution structures containing individual ligands, but with limitations.

Dispatched uses Na+ flux to power release of lipid-modified Hedgehog.

The Dispatched protein, which is related to the NPC1 and PTCH1 cholesterol transporters1,2 and to H+-driven transporters of the RND family3,4, enables tissue-patterning activity of the lipid-modified Hedgehog protein by releasing it from tightly -localized sites of embryonic expression5-10. Here we determine a cryo-electron microscopy structure of the mouse protein Dispatched homologue 1 (DISP1), revealing three Na+ ions coordinated within a channel that traverses its transmembrane domain. We find that the rate of Hedgehog export is dependent on the Na+ gradient across the plasma membrane. The transmembrane channel and Na+ binding are disrupted in DISP1-NNN, a variant with asparagine substitutions for three intramembrane aspartate residues that each coordinate and neutralize the charge of one of the three Na+ ions. DISP1-NNN and variants that disrupt single Na+ sites retain binding to, but are impaired in export of the lipid-modified Hedgehog protein to the SCUBE2 acceptor. Interaction of the amino-terminal signalling domain of the Sonic hedgehog protein (ShhN) with DISP1 occurs via an extensive buried surface area and contacts with an extended furin-cleaved DISP1 arm. Variability analysis reveals that ShhN binding is restricted to one extreme of a continuous series of DISP1 conformations. The bound and unbound DISP1 conformations display distinct Na+-site occupancies, which suggests a mechanism by which transmembrane Na+ flux may power extraction of the lipid-linked Hedgehog signal from the membrane. Na+-coordinating residues in DISP1 are conserved in PTCH1 and other metazoan RND family members, suggesting that Na+ flux powers their conformationally driven activities.

Structural insight into SARS-CoV-2 neutralizing antibodies and modulation of syncytia.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by binding of the viral Spike protein to host receptor angiotensin-converting enzyme 2 (ACE2), followed by fusion of viral and host membranes. Although antibodies that block this interaction are in emergency use as early coronavirus disease 2019 (COVID-19) therapies, the precise determinants of neutralization potency remain unknown. We discovered a series of antibodies that potently block ACE2 binding but exhibit divergent neutralization efficacy against the live virus. Strikingly, these neutralizing antibodies can inhibit or enhance Spike-mediated membrane fusion and formation of syncytia, which are associated with chronic tissue damage in individuals with COVID-19. As revealed by cryoelectron microscopy, multiple structures of Spike-antibody complexes have distinct binding modes that not only block ACE2 binding but also alter the Spike protein conformational cycle triggered by ACE2 binding. We show that stabilization of different Spike conformations leads to modulation of Spike-mediated membrane fusion with profound implications for COVID-19 pathology and immunity.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

CryoEM and AI reveal a structure of SARS-CoV-2 Nsp2, a multifunctional protein involved in key host processes.

The SARS-CoV-2 protein Nsp2 has been implicated in a wide range of viral processes, but its exact functions, and the structural basis of those functions, remain unknown. Here, we report an atomic model for full-length Nsp2 obtained by combining cryo-electron microscopy with deep learning-based structure prediction from AlphaFold2. The resulting structure reveals a highly-conserved zinc ion-binding site, suggesting a role for Nsp2 in RNA binding. Mapping emerging mutations from variants of SARS-CoV-2 on the resulting structure shows potential host-Nsp2 interaction regions. Using structural analysis together with affinity tagged purification mass spectrometry experiments, we identify Nsp2 mutants that are unable to interact with the actin-nucleation-promoting WASH protein complex or with GIGYF2, an inhibitor of translation initiation and modulator of ribosome-associated quality control. Our work suggests a potential role of Nsp2 in linking viral transcription within the viral replication-transcription complexes (RTC) to the translation initiation of the viral message. Collectively, the structure reported here, combined with mutant interaction mapping, provides a foundation for functional studies of this evolutionary conserved coronavirus protein and may assist future drug design.

Enhancing the signal-to-noise ratio and generating contrast for cryo-EM images with convolutional neural networks.

In cryogenic electron microscopy (cryo-EM) of radiation-sensitive biological samples, both the signal-to-noise ratio (SNR) and the contrast of images are critically important in the image-processing pipeline. Classic methods improve low-frequency image contrast experimentally, by imaging with high defocus, or computationally, by applying various types of low-pass filter. These contrast improvements typically come at the expense of the high-frequency SNR, which is suppressed by high-defocus imaging and removed by low-pass filtration. Recently, convolutional neural networks (CNNs) trained to denoise cryo-EM images have produced impressive gains in image contrast, but it is not clear how these algorithms affect the information content of the image. Here, a denoising CNN for cryo-EM images was implemented and a quantitative evaluation of SNR enhancement, induced bias and the effects of denoising on image processing and three-dimensional reconstructions was performed. The study suggests that besides improving the visual contrast of cryo-EM images, the enhanced SNR of denoised images may be used in other parts of the image-processing pipeline, such as classification and 3D alignment. These results lay the groundwork for the use of denoising CNNs in the cryo-EM image-processing pipeline beyond particle picking.

Bivalent binding of a fully human IgG to the SARS-CoV-2 spike proteins reveals mechanisms of potent neutralization.

In vitro antibody selection against pathogens from naïve combinatorial libraries can yield various classes of antigen-specific binders that are distinct from those evolved from natural infection1-4. Also, rapid neutralizing antibody discovery can be made possible by a strategy that selects for those interfering with pathogen and host interaction5. Here we report the discovery of antibodies that neutralize SARS-CoV-2, the virus responsible for the COVID-19 pandemic, from a highly diverse naïve human Fab library. Lead antibody 5A6 blocks the receptor binding domain (RBD) of the viral spike from binding to the host receptor angiotensin converting enzyme 2 (ACE2), neutralizes SARS-CoV-2 infection of Vero E6 cells, and reduces viral replication in reconstituted human nasal and bronchial epithelium models. 5A6 has a high occupancy on the viral surface and exerts its neutralization activity via a bivalent binding mode to the tip of two neighbouring RBDs at the ACE2 interaction interface, one in the "up" and the other in the "down" position, explaining its superior neutralization capacity. Furthermore, 5A6 is insensitive to several spike mutations identified in clinical isolates, including the D614G mutant that has become dominant worldwide. Our results suggest that 5A6 could be an effective prophylactic and therapeutic treatment of COVID-19.

Structural insight into TRPV5 channel function and modulation.

TRPV5 (transient receptor potential vanilloid 5) is a unique calcium-selective TRP channel essential for calcium homeostasis. Unlike other TRPV channels, TRPV5 and its close homolog, TRPV6, do not exhibit thermosensitivity or ligand-dependent activation but are constitutively open at physiological membrane potentials and modulated by calmodulin (CaM) in a calcium-dependent manner. Here we report high-resolution electron cryomicroscopy structures of truncated and full-length TRPV5 in lipid nanodiscs, as well as of a TRPV5 W583A mutant and TRPV5 in complex with CaM. These structures highlight the mechanism of calcium regulation and reveal a flexible stoichiometry of CaM binding to TRPV5.

Structural Basis for Cholesterol Transport-like Activity of the Hedgehog Receptor Patched.

Hedgehog protein signals mediate tissue patterning and maintenance by binding to and inactivating their common receptor Patched, a 12-transmembrane protein that otherwise would suppress the activity of the 7-transmembrane protein Smoothened. Loss of Patched function, the most common cause of basal cell carcinoma, permits unregulated activation of Smoothened and of the Hedgehog pathway. A cryo-EM structure of the Patched protein reveals striking transmembrane domain similarities to prokaryotic RND transporters. A central hydrophobic conduit with cholesterol-like contents courses through the extracellular domain and resembles that used by other RND proteins to transport substrates, suggesting Patched activity in cholesterol transport. Cholesterol activity in the inner leaflet of the plasma membrane is reduced by PTCH1 expression but rapidly restored by Hedgehog stimulation, suggesting that PTCH1 regulates Smoothened by controlling cholesterol availability.

Extending chemical perturbations of the ubiquitin fitness landscape in a classroom setting reveals new constraints on sequence tolerance.

Although the primary protein sequence of ubiquitin (Ub) is extremely stable over evolutionary time, it is highly tolerant to mutation during selection experiments performed in the laboratory. We have proposed that this discrepancy results from the difference between fitness under laboratory culture conditions and the selective pressures in changing environments over evolutionary timescales. Building on our previous work (Mavor et al., 2016), we used deep mutational scanning to determine how twelve new chemicals (3-Amino-1,2,4-triazole, 5-fluorocytosine, Amphotericin B, CaCl2, Cerulenin, Cobalt Acetate, Menadione, Nickel Chloride, p-Fluorophenylalanine, Rapamycin, Tamoxifen, and Tunicamycin) reveal novel mutational sensitivities of ubiquitin residues. Collectively, our experiments have identified eight new sensitizing conditions for Lys63 and uncovered a sensitizing condition for every position in Ub except Ser57 and Gln62. By determining the ubiquitin fitness landscape under different chemical constraints, our work helps to resolve the inconsistencies between deep mutational scanning experiments and sequence conservation over evolutionary timescales.

Structure of the human TRPM4 ion channel in a lipid nanodisc.

Transient receptor potential (TRP) melastatin 4 (TRPM4) is a widely expressed cation channel associated with a variety of cardiovascular disorders. TRPM4 is activated by increased intracellular calcium in a voltage-dependent manner but, unlike many other TRP channels, is permeable to monovalent cations only. Here we present two structures of full-length human TRPM4 embedded in lipid nanodiscs at ~3-angstrom resolution, as determined by single-particle cryo-electron microscopy. These structures, with and without calcium bound, reveal a general architecture for this major subfamily of TRP channels and a well-defined calcium-binding site within the intracellular side of the S1-S4 domain. The structures correspond to two distinct closed states. Calcium binding induces conformational changes that likely prime the channel for voltage-dependent opening.