Balancing Functional Tradeoffs between Protein Stability and ACE2 Binding in the SARS-CoV-2 Omicron BA.2, BA.2.75 and XBB Lineages: Dynamics-Based Network Models Reveal Epistatic Effects Modulating Compensatory Dynamic and Energetic Changes.

Evolutionary and functional studies suggested that the emergence of the Omicron variants can be determined by multiple fitness trade-offs including the immune escape, binding affinity for ACE2, conformational plasticity, protein stability and allosteric modulation. In this study, we systematically characterize conformational dynamics, structural stability and binding affinities of the SARS-CoV-2 Spike Omicron complexes with the host receptor ACE2 for BA.2, BA.2.75, XBB.1 and XBB.1.5 variants. We combined multiscale molecular simulations and dynamic analysis of allosteric interactions together with the ensemble-based mutational scanning of the protein residues and network modeling of epistatic interactions. This multifaceted computational study characterized molecular mechanisms and identified energetic hotspots that can mediate the predicted increased stability and the enhanced binding affinity of the BA.2.75 and XBB.1.5 complexes. The results suggested a mechanism driven by the stability hotspots and a spatially localized group of the Omicron binding affinity centers, while allowing for functionally beneficial neutral Omicron mutations in other binding interface positions. A network-based community model for the analysis of epistatic contributions in the Omicron complexes is proposed revealing the key role of the binding hotspots R498 and Y501 in mediating community-based epistatic couplings with other Omicron sites and allowing for compensatory dynamics and binding energetic changes. The results also showed that mutations in the convergent evolutionary hotspot F486 can modulate not only local interactions but also rewire the global network of local communities in this region allowing the F486P mutation to restore both the stability and binding affinity of the XBB.1.5 variant which may explain the growth advantages over the XBB.1 variant. The results of this study are consistent with a broad range of functional studies rationalizing functional roles of the Omicron mutation sites that form a coordinated network of hotspots enabling a balance of multiple fitness tradeoffs and shaping up a complex functional landscape of virus transmissibility.

Antigenic evolution of SARS-CoV-2 in immunocompromised hosts.

OBJECTIVES/AIMS: Prolonged infections of immunocompromised individuals have been proposed as a crucial source of new variants of SARS-CoV-2 during the COVID-19 pandemic. In principle, sustained within-host antigenic evolution in immunocompromised hosts could allow novel immune escape variants to emerge more rapidly, but little is known about how and when immunocompromised hosts play a critical role in pathogen evolution. MATERIALS AND METHODS: Here, we use a simple mathematical model to understand the effects of immunocompromised hosts on the emergence of immune escape variants in the presence and absence of epistasis. CONCLUSIONS: We show that when the pathogen does not have to cross a fitness valley for immune escape to occur (no epistasis), immunocompromised individuals have no qualitative effect on antigenic evolution (although they may accelerate immune escape if within-host evolutionary dynamics are faster in immunocompromised individuals). But if a fitness valley exists between immune escape variants at the between-host level (epistasis), then persistent infections of immunocompromised individuals allow mutations to accumulate, therefore, facilitating rather than simply speeding up antigenic evolution. Our results suggest that better genomic surveillance of infected immunocompromised individuals and better global health equality, including improving access to vaccines and treatments for individuals who are immunocompromised (especially in lower- and middle-income countries), may be crucial to preventing the emergence of future immune escape variants of SARS-CoV-2.

Coordinated evolution at amino acid sites of SARS-CoV-2 spike.

SARS-CoV-2 has adapted in a stepwise manner, with multiple beneficial mutations accumulating in a rapid succession at origins of VOCs, and the reasons for this are unclear. Here, we searched for coordinated evolution of amino acid sites in the spike protein of SARS-CoV-2. Specifically, we searched for concordantly evolving site pairs (CSPs) for which changes at one site were rapidly followed by changes at the other site in the same lineage. We detected 46 sites which formed 45 CSP. Sites in CSP were closer to each other in the protein structure than random pairs, indicating that concordant evolution has a functional basis. Notably, site pairs carrying lineage defining mutations of the four VOCs that circulated before May 2021 are enriched in CSPs. For the Alpha VOC, the enrichment is detected even if Alpha sequences are removed from analysis, indicating that VOC origin could have been facilitated by positive epistasis. Additionally, we detected nine discordantly evolving pairs of sites where mutations at one site unexpectedly rarely occurred on the background of a specific allele at another site, for example on the background of wild-type D at site 614 (four pairs) or derived Y at site 501 (three pairs). Our findings hint that positive epistasis between accumulating mutations could have delayed the assembly of advantageous combinations of mutations comprising at least some of the VOCs.

Epistasis-Driven Evolution of the SARS-CoV-2 Secondary Structure.

Epistasis is an evolutionary phenomenon whereby the fitness effect of a mutation depends on the genetic background in which it arises. A key source of epistasis in an RNA molecule is its secondary structure, which contains functionally important topological motifs held together by hydrogen bonds between Watson-Crick (WC) base pairs. Here we study epistasis in the secondary structure of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by examining properties of derived alleles arising from substitution mutations at ancestral WC base-paired and unpaired (UP) sites in 15 conserved topological motifs across the genome. We uncover fewer derived alleles and lower derived allele frequencies at WC than at UP sites, supporting the hypothesis that modifications to the secondary structure are often deleterious. At WC sites, we also find lower derived allele frequencies for mutations that abolish base pairing than for those that yield G·U "wobbles," illustrating that weak base pairing can partially preserve the integrity of the secondary structure. Last, we show that WC sites under the strongest epistatic constraint reside in a three-stemmed pseudoknot motif that plays an essential role in programmed ribosomal frameshifting, whereas those under the weakest epistatic constraint are located in 3' UTR motifs that regulate viral replication and pathogenicity. Our findings demonstrate the importance of epistasis in the evolution of the SARS-CoV-2 secondary structure, as well as highlight putative structural and functional targets of different forms of natural selection.

The Spike-Stabilizing D614G Mutation Interacts with S1/S2 Cleavage Site Mutations To Promote the Infectious Potential of SARS-CoV-2 Variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remained genetically stable during the first 3 months of the pandemic, before acquiring a D614G spike mutation that rapidly spread worldwide and then generating successive waves of viral variants with increasingly high transmissibility. We set out to evaluate possible epistatic interactions between the early-occurring D614G mutation and the more recently emerged cleavage site mutations present in spike of the Alpha, Delta, and Omicron variants of concern. The P681H/R mutations at the S1/S2 cleavage site increased spike processing and fusogenicity but limited its incorporation into pseudoviruses. In addition, the higher cleavage rate led to higher shedding of the spike S1 subunit, resulting in a lower infectivity of the P681H/R-carrying pseudoviruses compared to those expressing the Wuhan wild-type spike. The D614G mutation increased spike expression at the cell surface and limited S1 shedding from pseudovirions. As a consequence, the D614G mutation preferentially increased the infectivity of P681H/R-carrying pseudoviruses. This enhancement was more marked in cells where the endosomal route predominated, suggesting that more stable spikes could better withstand the endosomal environment. Taken together, these findings suggest that the D614G mutation stabilized S1/S2 association and enabled the selection of mutations that increased S1/S2 cleavage, leading to the emergence of SARS-CoV-2 variants expressing highly fusogenic spikes. IMPORTANCE The first SARS-CoV-2 variant that spread worldwide in early 2020 carried a D614G mutation in the viral spike, making this protein more stable in its cleaved form at the surface of virions. The Alpha and Delta variants, which spread in late 2020 and early 2021, respectively, proved increasingly transmissible and pathogenic compared to the original strain. Interestingly, Alpha and Delta both carried the mutations P681H/R in a cleavage site that made the spike more cleaved and more efficient at mediating viral fusion. We show here that variants with increased spike cleavage due to P681H/R were even more dependent on the stabilizing effect of the D614G mutation, which limited the shedding of cleaved S1 subunits from viral particles. These findings suggest that the worldwide spread of the D614G mutation was a prerequisite for the emergence of more pathogenic SARS-CoV-2 variants with highly fusogenic spikes.

Unique mutations in SARS-CoV-2 Omicron subvariants' non-spike proteins: Potential impacts on viral pathogenesis and host immune evasion.

SARS-CoV-2 is the causative agent behind the ongoing COVID-19 pandemic. This virus is a cumulative outcome of mutations, leading to frequent emergence of new variants and their subvariants. Some of them are a matter of high concern, while others are variants of interest for studying the mutational effect. The major five variants of concern (VOCs) are Alpha (B.1.1.7), Beta (B.1.315), Gamma (P.1), Delta (B.1.617.2), and Omicron (B.1.1.529.*/BA.*). Omicron itself has >100 subvariants at present, among which BA.1 (21K), BA.2 (21L), BA.4 (22A), BA.5 (22B), and BA.2.12.1 (22C) are the dominant ones. Undoubtedly, these variants and sometimes their progeny subvariants have significant differences in their spike region that impart them the unique properties they harbor. But alongside, the mutations in their non-spike regions could also be responsible elements behind their characteristics, such as replication time, virulence, survival, host immune evasion, and such. There exists a probability that these mutations of non-spike proteins may also impart epistatic effects that are yet to be brought to light. The focus of this review encompasses the non-spike mutations of Omicron, especially in its widely circulating subvariants (BA.1, BA.2, BA.4, BA.5, and BA.2.12.1). The mutations such as in NSP3, NSP6, NSP13, M protein, ORF7b, and ORF9b are mentioned few of all, which might have led to the varying properties, including growth advantages, higher transmission rate, lower infectivity, and most importantly better host immune evasion through natural killer cell inactivation, autophagosome-lysosome fusion prevention, host protein synthesis disruption, and so on. This aspect of Omicron subvariants has not yet been explored. Further study of alteration of expression or interaction profile of these non-spike mutations bearing proteins, if present, can add a great deal of knowledge to the current understanding of the viral properties and thus effective prevention strategies.

Selection Analysis Identifies Clusters of Unusual Mutational Changes in Omicron Lineage BA.1 That Likely Impact Spike Function.

Among the 30 nonsynonymous nucleotide substitutions in the Omicron S-gene are 13 that have only rarely been seen in other SARS-CoV-2 sequences. These mutations cluster within three functionally important regions of the S-gene at sites that will likely impact (1) interactions between subunits of the Spike trimer and the predisposition of subunits to shift from down to up configurations, (2) interactions of Spike with ACE2 receptors, and (3) the priming of Spike for membrane fusion. We show here that, based on both the rarity of these 13 mutations in intrapatient sequencing reads and patterns of selection at the codon sites where the mutations occur in SARS-CoV-2 and related sarbecoviruses, prior to the emergence of Omicron the mutations would have been predicted to decrease the fitness of any virus within which they occurred. We further propose that the mutations in each of the three clusters therefore cooperatively interact to both mitigate their individual fitness costs, and, in combination with other mutations, adaptively alter the function of Spike. Given the evident epidemic growth advantages of Omicron overall previously known SARS-CoV-2 lineages, it is crucial to determine both how such complex and highly adaptive mutation constellations were assembled within the Omicron S-gene, and why, despite unprecedented global genomic surveillance efforts, the early stages of this assembly process went completely undetected.

Epistasis at the SARS-CoV-2 Receptor-Binding Domain Interface and the Propitiously Boring Implications for Vaccine Escape.

At the time of this writing, December 2021, potential emergence of vaccine escape variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a grave global concern. The interface between the receptor-binding domain (RBD) of SARS-CoV-2 spike (S) protein and the host receptor (ACE2) overlaps the binding site of principal neutralizing antibodies (NAb), limiting the repertoire of viable mutations. Nonetheless, variants with multiple RBD mutations have risen to dominance. Nonadditive, epistatic relationships among RBD mutations are apparent, and assessing the impact of such epistasis on the mutational landscape, particularly the risk of vaccine escape, is crucial. We employed protein structure modeling using Rosetta to compare the effects of all single mutants at the RBD-NAb and RBD-ACE2 interfaces for the wild type and Delta, Gamma, and Omicron variants. Overall, epistasis at the RBD interface appears to be limited, and the effects of most multiple mutations are additive. Epistasis at the Delta variant interface weakly stabilizes NAb interaction relative to ACE2 interaction, whereas in Gamma, epistasis more substantially destabilizes NAb interaction. Despite bearing many more RBD mutations, the epistatic landscape of Omicron closely resembles that of Gamma. Thus, although Omicron poses new risks not observed with Delta, structural constraints on the RBD appear to hamper continued evolution toward more complete vaccine escape. The modest ensemble of mutations relative to the wild type that are currently known to reduce vaccine efficacy is likely to contain the majority of all possible escape mutations for future variants, predicting the continued efficacy of the existing vaccines. IMPORTANCE Emergence of vaccine escape variants of SARS-CoV-2 is arguably the most pressing problem during the COVID-19 pandemic as vaccines are distributed worldwide. We employed a computational approach to assess the risk of antibody escape resulting from mutations in the receptor-binding domain of the spike protein of the wild-type SARS-CoV-2 virus as well as the Delta, Gamma, and Omicron variants. The efficacy of the existing vaccines against Omicron could be substantially reduced relative to the wild type, and the potential for vaccine escape is of grave concern. Our results suggest that although Omicron poses new evolutionary risks not observed for Delta, structural constraints on the RBD make continued evolution toward more complete vaccine escape from either Delta or Omicron unlikely. The modest set of escape-enhancing mutations already identified for the wild type likely include the majority of all possible mutations with this effect.

Insights on the mutational landscape of the SARS-CoV-2 Omicron variant receptor-binding domain.

The Omicron variant features enhanced transmissibility and antibody escape. Here, we describe the Omicron receptor-binding domain (RBD) mutational landscape using amino acid interaction (AAI) networks, which are well suited for interrogating constellations of mutations that function in an epistatic manner. Using AAI, we map Omicron mutations directly and indirectly driving increased escape breadth and depth in class 1-4 antibody epitopes. Further, we present epitope networks for authorized therapeutic antibodies and assess perturbations to each antibody's epitope. Since our initial modeling following the identification of Omicron, these predictions have been realized by experimental findings of Omicron neutralization escape from therapeutic antibodies ADG20, AZD8895, and AZD1061. Importantly, the AAI predicted escape resulting from indirect epitope perturbations was not captured by previous sequence or point mutation analyses. Finally, for several Omicron RBD mutations, we find evidence for a plausible role in enhanced transmissibility via disruption of RBD-down conformational stability at the RBDdown-RBDdown interface.

Epistatic models predict mutable sites in SARS-CoV-2 proteins and epitopes.

The emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major concern given their potential impact on the transmissibility and pathogenicity of the virus as well as the efficacy of therapeutic interventions. Here, we predict the mutability of all positions in SARS-CoV-2 protein domains to forecast the appearance of unseen variants. Using sequence data from other coronaviruses, preexisting to SARS-CoV-2, we build statistical models that not only capture amino acid conservation but also more complex patterns resulting from epistasis. We show that these models are notably superior to conservation profiles in estimating the already observable SARS-CoV-2 variability. In the receptor binding domain of the spike protein, we observe that the predicted mutability correlates well with experimental measures of protein stability and that both are reliable mutability predictors (receiver operating characteristic areas under the curve ∼0.8). Most interestingly, we observe an increasing agreement between our model and the observed variability as more data become available over time, proving the anticipatory capacity of our model. When combined with data concerning the immune response, our approach identifies positions where current variants of concern are highly overrepresented. These results could assist studies on viral evolution and future viral outbreaks and, in particular, guide the exploration and anticipation of potentially harmful future SARS-CoV-2 variants.

The Genomic Physics of COVID-19 Pathogenesis and Spread.

Coronavirus disease (COVID-19) spreads mainly through close contact of infected persons, but the molecular mechanisms underlying its pathogenesis and transmission remain unknown. Here, we propose a statistical physics model to coalesce all molecular entities into a cohesive network in which the roadmap of how each entity mediates the disease can be characterized. We argue that the process of how a transmitter transforms the virus into a recipient constitutes a triad unit that propagates COVID-19 along reticulate paths. Intrinsically, person-to-person transmissibility may be mediated by how genes interact transversely across transmitter, recipient, and viral genomes. We integrate quantitative genetic theory into hypergraph theory to code the main effects of the three genomes as nodes, pairwise cross-genome epistasis as edges, and high-order cross-genome epistasis as hyperedges in a series of mobile hypergraphs. Charting a genome-wide atlas of horizontally epistatic hypergraphs can facilitate the systematic characterization of the community genetic mechanisms underlying COVID-19 spread. This atlas can typically help design effective containment and mitigation strategies and screen and triage those more susceptible persons and those asymptomatic carriers who are incubation virus transmitters.

Potential molecular link between the ß-amyloid precursor protein (APP) and hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme in Lesch-Nyhan disease and cancer.

Lesch-Nyhan disease (LND) is a rare X-linked inherited neurogenetic disorders of purine metabolic in which the cytoplasmic enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGprt) is defective. Despite having been characterized over 60 years ago, however, up to now, there is no satisfactory explanation of how deficits in enzyme HGprt can lead to LND with the development of the persistent and severe self-injurious behavior. Recently, a role for epistasis between the mutated hypoxanthine phosphoribosyltransferase 1 (HPRT1) and the ß-amyloid precursor protein (APP) genes affecting the regulation of alternative APP pre-mRNA splicing in LND has been demonstrated. Furthermore, there were also some reported cases of LND developing thrombosis while APP is an important regulator of vein thrombosis and controls coagulation. Otherwise, the surface expression of HGprt enzyme was also observed in several somatic tissue cancers while APP and the APP-like protein-2 (APLP2) are deregulated in cancer cells and linked to increased tumor cell proliferation, migration, and invasion. The present review provides a discussion about these findings and suggests a potential molecular link between APP and HGprt via epistasis between HPRT1 and APP genes affecting the regulation of alternative APP pre-mRNA splicing. As a perspective, expression vectors for HGprt enzyme and APP are constructed as described in Ref. # 24 (Nguyen KV, Naviaux RK, Nyhan WL (2020) Lesch-Nyhan disease: I. Construction of expression vectors for hypoxanthine-guanine phosphoribosyltransferase (HGprt) enzyme and amyloid precursor protein (APP). Nucleosides Nucleotides Nucleic Acids 39: 905-922), and they could be used as tools for clarification of these issues. In addition, these expression vectors, especially the one with the glycosyl-phosphatidylinositol (GPI) anchor can be used as a model for the construction of expression vectors for any protein targeting to the cell plasma membrane for studying intermolecular interactions and could be therefore useful in the vaccines as well as antiviral drugs development (studying intermolecular interactions between the spike glycoprotein of the severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, as well as its variants and the angiotensin-converting enzyme 2, ACE2, in coronavirus disease 2019 (COVID-19) [43],[44], for example).

Ongoing global and regional adaptive evolution of SARS-CoV-2.

Understanding the trends in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evolution is paramount to control the COVID-19 pandemic. We analyzed more than 300,000 high-quality genome sequences of SARS-CoV-2 variants available as of January 2021. The results show that the ongoing evolution of SARS-CoV-2 during the pandemic is characterized primarily by purifying selection, but a small set of sites appear to evolve under positive selection. The receptor-binding domain of the spike protein and the region of the nucleocapsid protein associated with nuclear localization signals (NLS) are enriched with positively selected amino acid replacements. These replacements form a strongly connected network of apparent epistatic interactions and are signatures of major partitions in the SARS-CoV-2 phylogeny. Virus diversity within each geographic region has been steadily growing for the entirety of the pandemic, but analysis of the phylogenetic distances between pairs of regions reveals four distinct periods based on global partitioning of the tree and the emergence of key mutations. The initial period of rapid diversification into region-specific phylogenies that ended in February 2020 was followed by a major extinction event and global homogenization concomitant with the spread of D614G in the spike protein, ending in March 2020. The NLS-associated variants across multiple partitions rose to global prominence in March to July, during a period of stasis in terms of interregional diversity. Finally, beginning in July 2020, multiple mutations, some of which have since been demonstrated to enable antibody evasion, began to emerge associated with ongoing regional diversification, which might be indicative of speciation.

Global analysis of more than 50,000 SARS-CoV-2 genomes reveals epistasis between eight viral genes.

Genome-wide epistasis analysis is a powerful tool to infer gene interactions, which can guide drug and vaccine development and lead to deeper understanding of microbial pathogenesis. We have considered all complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes deposited in the Global Initiative on Sharing All Influenza Data (GISAID) repository until four different cutoff dates, and used direct coupling analysis together with an assumption of quasi-linkage equilibrium to infer epistatic contributions to fitness from polymorphic loci. We find eight interactions, of which three are between pairs where one locus lies in gene ORF3a, both loci holding nonsynonymous mutations. We also find interactions between two loci in gene nsp13, both holding nonsynonymous mutations, and four interactions involving one locus holding a synonymous mutation. Altogether, we infer interactions between loci in viral genes ORF3a and nsp2, nsp12, and nsp6, between ORF8 and nsp4, and between loci in genes nsp2, nsp13, and nsp14. The paper opens the prospect to use prominent epistatically linked pairs as a starting point to search for combinatorial weaknesses of recombinant viral pathogens.

Positive Selection of ORF1ab, ORF3a, and ORF8 Genes Drives the Early Evolutionary Trends of SARS-CoV-2 During the 2020 COVID-19 Pandemic.

In this study, we analyzed full-length SARS-CoV-2 genomes from multiple countries to determine early trends in the evolutionary dynamics of the novel COVID-19 pandemic. Results indicated SARS-CoV-2 evolved early into at least three phylogenetic groups, characterized by positive selection at specific residues of the accessory proteins ORF3a and ORF8. Also, we are reporting potential relevant sites under positive selection at specific sites of non-structural proteins nsp6 and helicase. Our analysis of co-evolution showed evidence of epistatic interactions among sites in the genome that may be important in the generation of variants adapted to humans. These observations might impact not only public health but also suggest that more studies are needed to understand the genetic mechanisms that may affect the development of therapeutic and preventive tools, like antivirals and vaccines. Collectively, our results highlight the identification of ongoing selection even in a scenario of conserved sequences collected over the first 3 months of this pandemic.