DGCddG: Deep Graph Convolution for Predicting Protein-Protein Binding Affinity Changes Upon Mutations.

Effectively and accurately predicting the effects of interactions between proteins after amino acid mutations is a key issue for understanding the mechanism of protein function and drug design. In this study, we present a deep graph convolution (DGC) network-based framework, DGCddG, to predict the changes of protein-protein binding affinity after mutation. DGCddG incorporates multi-layer graph convolution to extract a deep, contextualized representation for each residue of the protein complex structure. The mined channels of the mutation sites by DGC is then fitted to the binding affinity with a multi-layer perceptron. Experiments with results on multiple datasets show that our model can achieve relatively good performance for both single and multi-point mutations. For blind tests on datasets related to angiotensin-converting enzyme 2 binding with the SARS-CoV-2 virus, our method shows better results in predicting ACE2 changes, may help in finding favorable antibodies. Code and data availability: https://github.com/lennylv/DGCddG.

Interactions of angiotensin-converting enzyme-2 (ACE2) and SARS-CoV-2 spike receptor-binding domain (RBD): a structural perspective.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has caused millions of infections and deaths worldwide since its discovery in late 2019 in Wuhan, China. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein binds to the human angiotensin-converting enzyme-2 (ACE2) receptor, a critical component of the renin-angiotensin system (RAS) that initiates the viral transmission. Most of the critical mutations found in SARS-CoV-2 are associated with the RBD of the spike protein. These mutations have the potential to reduce the efficacy of vaccines and neutralizing antibodies. METHODS: In this review, the structural details of ACE2, RBD and their interactions are discussed. In addition, some critical mutations of RBD and their impact on ACE2-RBD interactions are also discussed. CONCLUSION: Preventing the interaction between Spike RBD and ACE2 is considered a viable therapeutic strategy since ACE2 binding by RBD is the first step in virus infection. Because the interactions between the two entities are critical for both viral transmission and therapeutic development, it is essential to understand their interactions in detail.

Experimental and clinical data analysis for identification of COVID-19 resistant ACE2 mutations.

The high magnitude zoonotic event has caused by Severe Acute Respitarory Syndrome CoronaVirus-2 (SARS-CoV-2) is Coronavirus Disease-2019 (COVID-19) epidemics. This disease has high rate of spreading than mortality in humans. The human receptor, Angiotensin-Converting Enzyme 2 (ACE2), is the leading target site for viral Spike-protein (S-protein) that function as binding ligands and are responsible for their entry in humans. The patients infected with COVID-19 with comorbidities, particularly cancer patients, have a severe effect or high mortality rate because of the suppressed immune system. Nevertheless, there might be a chance wherein cancer patients cannot be infected with SARS-CoV-2 because of mutations in the ACE2, which may be resistant to the spillover between species. This study aimed to determine the mutations in the sequence of the human ACE2 protein and its dissociation with SARS-CoV-2 that might be rejecting viral transmission. The in silico approaches were performed to identify the impact of SARS-CoV-2 S-protein with ACE2 mutations, validated experimentally, occurred in the patient, and reported in cell lines. The identified changes significantly affect SARS-CoV-2 S-protein interaction with ACE2, demonstrating the reduction in the binding affinity compared to SARS-CoV. The data presented in this study suggest ACE2 mutants have a higher and lower affinity with SARS-Cov-2 S-protein to the wild-type human ACE2 receptor. This study would likely be used to report SARS-CoV-2 resistant ACE2 mutations and can be used to design active peptide development to inactivate the viral spread of SARS-CoV-2 in humans.

SARS-CoV-2 Delta Variant: Interplay between Individual Mutations and Their Allosteric Synergy.

Since its first appearance in April 2021, B.1.617.2, also termed variant Delta, catalyzed one major worldwide wave dominating the second year of coronavirus disease 2019 (COVID-19) pandemic. Despite its quick disappearance worldwide, the strong virulence caused by a few point mutations remains an unsolved problem largely. Along with the other two sublineages, the Delta variant harbors an accumulation of Spike protein mutations, including the previously identified L452R, E484Q, and the newly emerged T478K on its receptor binding domain (RBD). We used molecular dynamics (MD) simulations, in combination with free energy perturbation (FEP) calculations, to examine the effects of two combinative mutation sets, L452R + E484Q and L452R + T478K. Our dynamic trajectories reveal an enhancement in binding affinity between mutated RBD and the common receptor protein angiotensin converting enzyme 2 (ACE2) through a net increase in the buried molecular surface area of the binary complex. This enhanced binding, mediated through Gln493, sets the same stage for all three sublineages due to the presence of L452R mutation. The other mutation component, E484Q or T478K, was found to impact the RBD-ACE2 binding and help the variant to evade several monoclonal antibodies (mAbs) in a distinct manner. Especially for L452R + T478K, synergies between mutations are mediated through a complex residual and water interaction network and further enhance its binding to ACE2. Taking together, this study demonstrates that new variants of SARS-CoV-2 accomplish both "attack" (infection) and "defense" (antibody neutralization escape) with the same "polished sword" (mutated Spike RBD).

Structural bases for the higher adherence to ACE2 conferred by the SARS-CoV-2 spike Q498Y substitution.

A remarkable number of SARS-CoV-2 variants and other as yet unmonitored lineages harbor amino-acid substitutions with the potential to modulate the interface between the spike receptor-binding domain (RBD) and its receptor ACE2. The naturally occurring Q498Y substitution, which is present in currently circulating SARS-CoV-2 variants, has drawn the attention of several investigations. While computational predictions and in vitro binding studies suggest that Q498Y increases the binding affinity of the spike protein for ACE2, experimental in vivo models of infection have shown that a triple mutant carrying the Q498Y replacement is fatal in mice. To accurately characterize the binding kinetics of the RBD Q498Y-ACE2 interaction, biolayer interferometry analyses were performed. A significant enhancement of the RBD-ACE2 binding affinity relative to a reference SARS-CoV-2 variant of concern carrying three simultaneous replacements was observed. In addition, the RBD Q498Y mutant bound to ACE2 was crystallized. Compared with the structure of its wild-type counterpart, the RBD Q498Y-ACE2 complex reveals the conservation of major hydrogen-bond interactions and a more populated, nonpolar set of contacts mediated by the bulky side chain of Tyr498 that collectively lead to this increase in binding affinity. In summary, these studies contribute to a deeper understanding of the impact of a relevant mutation present in currently circulating SARS-CoV-2 variants which might lead to stronger host-pathogen interactions.

Physicochemical effect of the N501Y, E484K/Q, K417N/T, L452R and T478K mutations on the SARS-CoV-2 spike protein RBD and its influence on agent fitness and on attributes developed by emerging variants of concern.

The spike protein comprises one of the main structural components of SARS-CoV-2 because it is directly involved in the infection process and viral transmission, and also because of its immunogenic properties, as an inducer of the protective antibodies production and as a vaccine component. The occurrence of mutations in this region or in other the virus genome regions, comprises a natural phenomenon in its evolution. However, they also occur due to the selective immune pressure, to which the agent is continuously subjected, especially in the spike protein immunodominant regions, such as the RBD. Mutations in the spike protein can change the virus' fitness, increasing its affinity for target cells, its transmissibility and its virulence. In addition, these mutations can giving it the potential ability to evade the protective antibodies action obtained from convalescent sera or vaccine origin, as well as those used in therapy, which may favor the virus expansion and compromise the infection control. Five mutations N501Y, E484K/Q, K417N/T, L452R and T478K, located in the spike protein RBD, have had a greater impact because they are associated with new attributes developed by the virus, which characterize the emerging variants of concern (VOCs) of SARS-Cov-2 identified so far. The occurrence of these mutations induces complex physicochemical effects that can alter the spike protein's structure and its function, which in turn, lead to changes in the agents' fitness. This manuscript discusses the attributes of VOCs associated with the physicochemical effects caused by the aforementioned mutations.

Risk of rapid evolutionary escape from biomedical interventions targeting SARS-CoV-2 spike protein.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is the molecular target for many vaccines and antibody-based prophylactics aimed at bringing COVID-19 under control. Such a narrow molecular focus raises the specter of viral immune evasion as a potential failure mode for these biomedical interventions. With the emergence of new strains of SARS-CoV-2 with altered transmissibility and immune evasion potential, a critical question is this: how easily can the virus escape neutralizing antibodies (nAbs) targeting the spike RBD? To answer this question, we combined an analysis of the RBD structure-function with an evolutionary modeling framework. Our structure-function analysis revealed that epitopes for RBD-targeting nAbs overlap one another substantially and can be evaded by escape mutants with ACE2 affinities comparable to the wild type, that are observed in sequence surveillance data and infect cells in vitro. This suggests that the fitness cost of nAb-evading mutations is low. We then used evolutionary modeling to predict the frequency of immune escape before and after the widespread presence of nAbs due to vaccines, passive immunization or natural immunity. Our modeling suggests that SARS-CoV-2 mutants with one or two mildly deleterious mutations are expected to exist in high numbers due to neutral genetic variation, and consequently resistance to vaccines or other prophylactics that rely on one or two antibodies for protection can develop quickly -and repeatedly- under positive selection. Predicted resistance timelines are comparable to those of the decay kinetics of nAbs raised against vaccinal or natural antigens, raising a second potential mechanism for loss of immunity in the population. Strategies for viral elimination should therefore be diversified across molecular targets and therapeutic modalities.

Convergent Evolution of Multiple Mutations Improves the Viral Fitness of SARS-CoV-2 Variants by Balancing Positive and Negative Selection.

Multiple mutations have been seen to undergo convergent evolution in SARS-CoV-2 variants of concern. One such evolution occurs in Beta, Gamma, and Omicron variants at three amino acid positions K417, E484, and N501 in the receptor binding domain of the spike protein. We examined the physical mechanisms underlying the convergent evolution of three mutations K417T/E484K/N501Y by delineating the individual and collective effects of mutations on binding to angiotensin converting enzyme 2 receptor, immune escape from neutralizing antibodies, protein stability, and expression. Our results show that each mutation serves a distinct function that improves virus fitness supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. Compared to the wild-type, K417T escapes Class 1 antibodies and has increased stability and expression; however, it has decreased receptor binding. E484K escapes Class 2 antibodies; however, it has decreased receptor binding, stability, and expression. N501Y increases receptor binding; however, it has decreased stability and expression. When these mutations come together, the deleterious effects are mitigated due to the presence of compensatory effects. Triple mutant K417T/E484K/N501Y has increased receptor binding, escapes both Class 1 and Class 2 antibodies, and has similar stability and expression as that of the wild-type. These results show that the convergent evolution of multiple mutations enhances viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together.

A computational protein design protocol for optimization of the SARS-CoV-2 receptor-binding-motif affinity for human ACE2.

The present protocol describes the computational design of the SARS-CoV-2 receptor binding motif (RBD) to identify mutations that can potentially improve binding affinity for the human ACE2 (hACE2) receptor. We focus on four positions located at the interface with the hACE2 receptor in the RBD:hACE2 complex. We conduct the design with a high-throughput computational protein design (CPD) program, Proteus, incorporating an adaptive Monte Carlo (MC) protocol that promotes the selection of sequences with good binding affinities. For complete details on the use and execution of this protocol, please refer to Polydorides and Archontis (2021).

Molecular Dynamics and MM-PBSA Analysis of the SARS-CoV-2 Gamma Variant in Complex with the hACE-2 Receptor.

The SARS-CoV-2 virus, since its appearance in 2019, has caused millions of cases and deaths. To date, there is no effective treatment or a vaccine that is fully protective. Despite the efforts made by governments and health institutions around the globe to control its propagation, the evolution of the virus has accelerated, diverging into hundreds of variants. However, not all of them are variants of concern (VoC's). VoC's have appeared in different regions and throughout the two years of the pandemic they have spread around the world. Specifically, in South America, the gamma variant (previously known as P.1) appeared in early 2021, bringing with it a second wave of infections. This variant contains the N501Y, E484K and K417T mutations in the receptor binding domain (RBD) of the spike protein. Although these mutations have been described experimentally, there is still no clarity regarding their role in the stabilization of the complex with the human angiotensin converting enzyme 2 (hACE-2) receptor. In this article we dissect the influence of mutations on the interaction with the hACE-2 receptor using molecular dynamics and estimations of binding affinity through a screened version of the molecular mechanics Poisson Boltzmann surface area (MM-PBSA) and interaction entropy. Our results indicate that mutations E484K and K417T compensate each other in terms of binding affinity, while the mutation N501Y promotes a more convoluted effect. This effect consists in the adoption of a cis configuration in the backbone of residue Y495 within the RBD, which in turn promotes polar interactions with the hACE-2 receptor. These results not only correlate with experimental observations and complement previous knowledge, but also expose new features associated with the specific contribution of concerned mutations. Additionally, we propose a recipe to assess the residue-specific contribution to the interaction entropy.

Potential inhibitor for blocking binding between ACE2 and SARS-CoV-2 spike protein with mutations.

At the time of writing, more than 440 million confirmed coronavirus disease 2019 (COVID-19) cases and more than 5.97 million COVID-19 deaths worldwide have been reported by the World Health Organization since the start of the outbreak of the pandemic in Wuhan, China. During the COVID-19 pandemic, many variants of SARS-CoV-2 have arisen because of high mutation rates. N501Y, E484K, K417N, K417T, L452R and T478K in the receptor binding domain (RBD) region may increase the infectivity in several variants of SARS-CoV-2. In this study, we discovered that GB-1, developed from Chiehyuan herbal formula which obtained from Tian Shang Sheng Mu of Chiayi Puzi Peitian Temple, can inhibit the binding between ACE2 and RBD with Wuhan type, K417N-E484K-N501Y and L452R-T478K mutation. In addition, GB-1 inhibited the binding between ACE2 and RBD with a single mutation (E484K or N501Y), except the K417N mutation. In the compositions of GB-1, glycyrrhizic acid can inhibit the binding between ACE2 and RBD with Wuhan type, except K417N-E484K-N501Y mutation. Our results suggest that GB-1 could be a potential candidate for the prophylaxis of different variants of SARS-CoV-2 infection because of its inhibition of binding between ACE2 and RBD with different mutations (L452R-T478K, K417N-E484K-N501Y, N501Y or E484K).

Characterization of the novel SARS-CoV-2 Omicron (B.1.1.529) variant of concern and its global perspective.

As the latest identified novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variant of concern (VOC), the influence of Omicron on our globe grows promptly. Compared with the last VOC (Delta variant), more mutations were identified, which may address the characteristics of Omicron. Considering these crucial mutations and their implications including an increase in transmissibility, COVID-19 severity, and reduction of efficacy of currently available diagnostics, vaccines, and therapeutics, Omicron has been classified as one of the VOC. Notably, 15 of these mutations reside in the receptor-binding domain of spike glycoprotein, which may alter transmissibility, infectivity, neutralizing antibody escape, and vaccine breakthrough cases of COVID-19. Therefore, our present study characterizes the mutational hotspots of the Omicron variant in comparison with the Delta variant of SARS-CoV-2. Furthermore, detailed information was analyzed to characterize the global perspective of Omicron, including transmission dynamic, effect on testing, and immunity, which shall promote the progress of the clinical application and basic research. Collectively, our data suggest that due to continuous variation in the spike glycoprotein sequences, the use of coronavirus-specific attachment inhibitors may not be the current choice of therapy for emerging SARS-CoV-2 VOCs. Hence, we need to proceed with a sense of urgency in this matter.

Structural basis for SARS-CoV-2 Delta variant recognition of ACE2 receptor and broadly neutralizing antibodies.

The SARS-CoV-2 Delta variant is currently the dominant circulating strain in the world. Uncovering the structural basis of the enhanced transmission and altered immune sensitivity of Delta is particularly important. Here we present cryo-EM structures revealing two conformational states of Delta spike and S/ACE2 complex in four states. Our cryo-EM analysis suggests that RBD destabilizations lead to population shift towards the more RBD-up and S1 destabilized fusion-prone state, beneficial for engagement with ACE2 and shedding of S1. Noteworthy, we find the Delta T478K substitution plays a vital role in stabilizing and reshaping the RBM loop473-490, enhancing interaction with ACE2. Collectively, increased propensity for more RBD-up states and the affinity-enhancing T478K substitution together contribute to increased ACE2 binding, providing structural basis of rapid spread of Delta. Moreover, we identify a previously generated MAb 8D3 as a cross-variant broadly neutralizing antibody and reveal that 8D3 binding induces a large K478 side-chain orientation change, suggesting 8D3 may use an "induced-fit" mechanism to tolerate Delta T478K mutation. We also find that all five RBD-targeting MAbs tested remain effective on Delta, suggesting that Delta well preserves the neutralizing antigenic landscape in RBD. Our findings shed new lights on the pathogenicity and antibody neutralization of Delta.

Rapid characterization of spike variants via mammalian cell surface display.

The SARS-CoV-2 spike protein is a critical component of vaccines and a target for neutralizing monoclonal antibodies (nAbs). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. Here, we describe Spike Display, a high-throughput platform to rapidly characterize glycosylated spike ectodomains across multiple coronavirus-family proteins. We assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in variants of concern B.1.1.7 (alpha), B.1.351 (beta), B.1.1.28 (gamma), B.1.427/B.1.429 (epsilon), and B.1.617.2 (delta) impact spike expression and escape most NTD-targeting nAbs. Finally, B.1.351 and B.1.1.28 completely escape a potent ACE2 mimic. We anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.

The glycosylation in SARS-CoV-2 and its receptor ACE2.

Coronavirus disease 2019 (COVID-19), a highly infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 235 million individuals and led to more than 4.8 million deaths worldwide as of October 5 2021. Cryo-electron microscopy and topology show that the SARS-CoV-2 genome encodes lots of highly glycosylated proteins, such as spike (S), envelope (E), membrane (M), and ORF3a proteins, which are responsible for host recognition, penetration, binding, recycling and pathogenesis. Here we reviewed the detections, substrates, biological functions of the glycosylation in SARS-CoV-2 proteins as well as the human receptor ACE2, and also summarized the approved and undergoing SARS-CoV-2 therapeutics associated with glycosylation. This review may not only broad the understanding of viral glycobiology, but also provide key clues for the development of new preventive and therapeutic methodologies against SARS-CoV-2 and its variants.

The Role of Spike Protein Mutations in the Infectious Power of SARS-COV-2 Variants: A Molecular Interaction Perspective.

Specific S477N, N501Y, K417N, K417T, E484K mutations in the receptor binding domain (RBD) of the spike protein in the wild type SARS-COV-2 virus have resulted, among others, in the following variants: B.1.160 (20A or EU2, first reported in continental Europe), B1.1.7 (α or 20I501Y.V1, first reported in the United Kingdom), B.1.351 (ß or 20H/501Y.V2, first reported in South Africa), B.1.1.28.1 (γ or P.1 or 20J/501Y.V3, first reported in Brazil), and B.1.1.28.2 (ζ, or P.2 or 20B/S484K, also first reported in Brazil). From the analysis of a set of bonding descriptors firmly rooted in the formalism of quantum mechanics, including Natural Bond Orbitals (NBO), Quantum Theory of Atoms In Molecules (QTAIM) and highly correlated energies within the Domain Based Local Pair Natural Orbital Coupled Cluster Method (DLPNO-CCSD(T)), and from a set of computed electronic spectral patterns with environmental effects, we show that the new variants improve their ability to recognize available sites to either hydrogen bond or to form salt bridges with residues in the ACE2 receptor of the host cells. This results in significantly improved initial virus⋅⋅⋅cell molecular recognition and attachment at the microscopic level, which trigger the infectious cycle.

Function Is More Reliable than Quantity to Follow Up the Humoral Response to the Receptor-Binding Domain of SARS-CoV-2-Spike Protein after Natural Infection or COVID-19 Vaccination.

Both the SARS-CoV-2 pandemic and emergence of variants of concern have highlighted the need for functional antibody assays to monitor the humoral response over time. Antibodies directed against the spike (S) protein of SARS-CoV-2 are an important component of the neutralizing antibody response. In this work, we report that in a subset of patients-despite a decline in total S-specific antibodies-neutralizing antibody titers remain at a similar level for an average of 98 days in longitudinal sampling of a cohort of 59 Hispanic/Latino patients exposed to SARS-CoV-2. Our data suggest that 100% of seroconverting patients make detectable neutralizing antibody responses which can be quantified by a surrogate viral neutralization test. Examination of sera from ten out of the 59 subjects which received mRNA-based vaccination revealed that both IgG titers and neutralizing activity of sera were higher after vaccination compared to a cohort of 21 SARS-CoV-2 naïve subjects. One dose was sufficient for the induction of a neutralizing antibody, but two doses were necessary to reach 100% surrogate virus neutralization in subjects irrespective of previous SARS-CoV-2 natural infection status. Like the pattern observed after natural infection, the total anti-S antibodies titers declined after the second vaccine dose; however, neutralizing activity remained relatively constant for more than 80 days after the first vaccine dose. Furthermore, our data indicates that-compared with mRNA vaccination-natural infection induces a more robust humoral immune response in unexposed subjects. This work is an important contribution to understanding the natural immune response to the novel coronavirus in a population severely impacted by SARS-CoV-2. Furthermore, by comparing the dynamics of the immune response after the natural infection vs. the vaccination, these findings suggest that functional neutralizing antibody tests are more relevant indicators than the presence or absence of binding antibodies.

Characterization and structural basis of a lethal mouse-adapted SARS-CoV-2.

There is an urgent need for animal models to study SARS-CoV-2 pathogenicity. Here, we generate and characterize a novel mouse-adapted SARS-CoV-2 strain, MASCp36, that causes severe respiratory symptoms, and mortality. Our model exhibits age- and gender-related mortality akin to severe COVID-19. Deep sequencing identified three amino acid substitutions, N501Y, Q493H, and K417N, at the receptor binding domain (RBD) of MASCp36, during in vivo passaging. All three RBD mutations significantly enhance binding affinity to its endogenous receptor, ACE2. Cryo-electron microscopy analysis of human ACE2 (hACE2), or mouse ACE2 (mACE2), in complex with the RBD of MASCp36, at 3.1 to 3.7 Å resolution, reveals the molecular basis for the receptor-binding switch. N501Y and Q493H enhance the binding affinity to hACE2, whereas triple mutations at N501Y/Q493H/K417N decrease affinity and reduce infectivity of MASCp36. Our study provides a platform for studying SARS-CoV-2 pathogenesis, and unveils the molecular mechanism for its rapid adaptation and evolution.

Neutral evolution test of the spike protein of SARS-CoV-2 and its implications in the binding to ACE2.

As the SARS-CoV-2 has spread and the pandemic has dragged on, the virus continued to evolve rapidly resulting in the emergence of new highly transmissible variants that can be of public health concern. The evolutionary mechanisms that drove this rapid diversity are not well understood but neutral evolution should open the first insight. The neutral theory of evolution states that most mutations in the nucleic acid sequences are random and they can be fixed or disappear by purifying selection. Herein, we performed a neutrality test to better understand the selective pressures exerted over SARS-CoV-2 spike protein from homologue proteins of Betacoronavirus, as well as to the spikes from human clinical isolates of the virus. Specifically, Tyr and Asn have higher occurrence rates on the Receptor Binding Domain (RBD) and in the overall sequence of spike proteins of Betacoronavirus, whereas His and Arg have lower occurrence rates. The in vivo evolutionary phenomenon of SARS-CoV-2 shows that Glu, Lys, Phe, and Val have the highest probability of occurrence in the emergent viral particles. Amino acids that have higher occurrence than the expected by the neutral control, are favorable and are fixed in the sequence while the ones that have lower occurrence than expected, influence the stability and/or functionality of the protein. Our results show that most unique mutations either for SARS-CoV-2 or its variants of health concern are under selective pressures, which could be related either to the evasion of the immune system, increasing the virus' fitness or altering protein - protein interactions with host proteins. We explored the consequences of those selected mutations in the structure and protein - protein interaction with the receptor. Altogether all these forces have shaped the spike protein and the continually evolving variants.

A molecular sensor determines the ubiquitin substrate specificity of SARS-CoV-2 papain-like protease.

The SARS-CoV-2 papain-like protease (PLpro) is a target for antiviral drug development. It is essential for processing viral polyproteins for replication and functions in host immune evasion by cleaving ubiquitin (Ub) and ubiquitin-like protein (Ubl) conjugates. While highly conserved, SARS-CoV-2 and SARS-CoV PLpro have contrasting Ub/Ubl substrate preferences. Using a combination of structural analyses and functional assays, we identify a molecular sensor within the S1 Ub-binding site of PLpro that serves as a key determinant of substrate specificity. Variations within the S1 sensor specifically alter cleavage of Ub substrates but not of the Ubl interferon-stimulated gene 15 protein (ISG15). Significantly, a variant of concern associated with immune evasion carries a mutation in the S1 sensor that enhances PLpro activity on Ub substrates. Collectively, our data identify the S1 sensor region as a potential hotspot of variability that could alter host antiviral immune responses to newly emerging SARS-CoV-2 lineages.