SARS-CoV-2 Omicron Variant: ACE2 Binding, Cryo-EM Structure of Spike Protein-ACE2 Complex and Antibody Evasion (preprint)

The newly reported Omicron variant is poised to replace Delta as the most rapidly spread 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, explaining our finding of similar biochemical ACE2 binding affinities for Delta and Omicron variants. Neutralization assays show that pseudoviruses displaying the Omicron spike protein exhibit increased antibody evasion, with greater evasion observed in sera obtained from unvaccinated convalescent patients as compared to doubly vaccinated individuals (8- vs 3-fold). The retention of strong interactions at the ACE2 interface and the increase in antibody evasion are molecular factors that likely contribute to the increased transmissibility of the Omicron variant.

Emerging SARS-CoV-2 Variants of Concern: Spike Protein Mutational Analysis and Epitope for Broad Neutralization (preprint)

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 report the discovery of a novel antibody fragment (V H ab6) that neutralizes all major variants, with a unique mode of binding revealed by cryo-EM studies. Further, we provide a comparative analysis of the mutational effects within 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.

Structural and Biochemical Rationale for Enhanced Spike Protein Fitness in Delta and Kappa SARS-CoV-2 Variants (preprint)

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 novel head-to-head dimer-of-trimers assembly, which we demonstrate is a result of the E484Q mutation. The combination of increased antibody escape and enhanced ACE2 binding provides an explanation, in part, for the rapid global dominance of the Delta variant.

Structural Analysis of Receptor Binding Domain Mutations in SARS-CoV-2 Variants of Concern that Modulate ACE2 and Antibody Binding (preprint)

The recently emerged SARS-CoV-2 South African (B. 1.351) and Brazil/Japan (P.1) variants of concern (VoCs) include a key mutation (N501Y) found in the UK variant that enhances affinity of the spike protein for its receptor, ACE2. Additional mutations are found in these variants at residues 417 and 484 that appear to promote antibody evasion. In contrast, the Californian VoCs (B.1.427/429) lack the N501Y mutation, yet exhibit antibody evasion. We engineered spike proteins to express these RBD VoC mutations either in isolation, or in different combinations, and analyzed the effects using biochemical assays and 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 either increased ACE2 affinity, increased antibody evasion, or both, providing a framework to dissect the molecular factors that drive VoC evolution.

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

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. Here, we report cryo-EM structures of SARS-CoV-2 spike protein ectodomains with and without the N501Y mutation, in complex with the VH fragment of the potent neutralizing antibody, VH -Fc ab8. The mutation results in localized structural perturbations near Y501, but VH -Fc ab8 retains the ability to bind and neutralize pseudotyped viruses expressing the N501Y mutant with efficiencies comparable to that of unmutated viruses. Our results show that despite the higher affinity of ACE2 for the N501Y mutant, it can still be neutralized efficiently by an antibody that binds epitopes in the receptor binding domain of the SARS-CoV-2 spike protein.

Snake venom phospholipases A2 possess a strong virucidal activity against SARS-CoV-2 in vitro and block the cell fusion mediated by spike glycoprotein interaction with the ACE2 receptor (preprint)

A new coronavirus was recently discovered and named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In the absence of specific therapeutic and prophylactic agents, the virus has infected almost hundred million people, of whom nearly two million have died from the viral disease COVID-19. The ongoing COVID-19 pandemic is a global threat requiring new therapeutic strategies. Among them, antiviral studies based on natural molecules are a promising approach. The superfamily of phospholipases A2 (PLA2s) consists of a large number of members that catalyze the hydrolysis of phospholipids at a specific position. Here we show that secreted PLA2s from the venom of various snakes protect to varying degrees the Vero E6 cells widely used for the replication of viruses with evident cytopathic action, from SARS-CoV-2 infection PLA2s showed low cytotoxicity to Vero E6 cells and the high antiviral activity against SARS-CoV-2 with IC50 values ranged from 0.06 to 7.71 ug/ml. Dimeric PLA2 HDP-2 from the viper Vipera nikolskii, as well as its catalytic and inhibitory subunits, had potent virucidal (neutralizing) activity against SARS-CoV-2. Inactivation of the enzymatic activity of the catalytic subunit of dimeric PLA2 led to a significant decrease in antiviral activity. In addition, dimeric PLA2 inhibited cell-cell fusion mediated by SARS-CoV-2 spike glycoprotein. These results suggest that snake PLA2s, in particular dimeric ones, are promising candidates for the development of antiviral drugs that target lipid bilayers of the viral envelope and may be good tools to study the interaction of viruses with host cell membranes.

Novel RT-ddPCR assays for determining the transcriptional profile of SARS-CoV-2 (preprint)

The exact mechanism of coronavirus replication and transcription is not fully understood; however, a hallmark of coronavirus transcription is the generation of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and an array of subgenomic mRNAs (sgRNAs) encompassing sequences arising from discontinuous transcription. Existing PCR-based diagnostic assays for SAR-CoV-2 are qualitative or semi-quantitative and do not provide the resolution needed to assess the complex transcription dynamics of SARS-CoV-2 over the course of infection. We developed and validated a novel panel of specially designed SARS-CoV-2 ddPCR-based assays to map the viral transcription profile. Application of these assays to clinically relevant samples will enhance our understanding of SARS-CoV-2 replication and transcription and may also inform the development of improved diagnostic tools and therapeutics.