G protein-coupled receptors (GPCRs) form the largest family of cell-surface receptors and remain prime targets in drug discovery. A central challenge in modern GPCR drug discovery is understanding and exploiting biased agonism: the ability of ligands to favor signaling via therapeutically beneficial pathways while avoiding those that trigger side effects. Therefore, pinpointing the structural determinants of signaling bias is crucial for rational drug design. Biased agonists are particularly compelling for targeting opioid receptors, as in this family, ligands that limit B-arrestin (B-arr) recruitment are believed to preserve analgesia while reducing respiratory depression and addiction liabilities. Here, we use extensive all-atom molecular dynamics (MD) simulations to dissect signaling bias in the d-opioid receptor (dOR). Focusing on a receptor mutant with a strong B-arr bias, we employed a reverse-engineering approach to reveal the conformational mechanisms that promote B-arr recruitment. Building on these insights, engineer new mutations that reshape the receptor signaling profile. Importantly, this approach allowed us to pinpoint signaling bias to motions of a single microswitch and identify how structural receptor motions induced by the mutations and ligand contacts cooperate to promote a specific functional response. In this proof-of-concept study, we not only provide structural insights into dOR pharmacology but also demonstrate how computational methods can be leveraged to probe structural mechanisms of signaling specificity across GPCRs, paving the way for the rational design of tailored receptor variants and novel, safer, and more effective therapeutics. ### Competing Interest Statement The authors have declared no competing interest. MICIU/AEI /10.13039/501100011033 and FEDER, PID2023-146609OA-I00 National Science Centre, 2017/27/N/NZ2/02571 Canadian Institutes of Health Research and the Natural Sciences and Engineering Research Council of Canada

Recent models of GPCR signaling extend beyond the canonical paradigm of ligand-induced activation primarily mediated by G-proteins and β-arrestins. Indeed, the various outcomes of GPCR activation are precisely regulated by several factors, including GPCR-interacting proteins, isoform diversity, and GPCR phosphorylation patterns catalyzed by kinases. One underexplored source contributing to the pleiotropic activities of GPCRs is the dynamic recruitment and dissociation of 14-3-3 proteins, a family of ubiquitously expressed adaptor proteins that modulate key cellular processes. Recent studies have shown that GPCR-14-3-3 protein interactions are isoform-specific and can be modulated in a spatiotemporal manner by ligands; however, an investigation of 14-3-3 dynamics across all seven human isoforms and throughout a large panel of GPCRs has yet to be conducted. Thus, this work provides the first characterization of proximal GPCR-14-3-3 protein interactions at a GPCRome scale, achieved by constructing stable reporter cell lines expressing all human 14-3-3 isoforms and performing cell-based high-throughput screening to probe 82 GPCR interactomes. Among the notable findings from this screen, the NK3 receptor emerged as a promising candidate for an in-depth examination of 14-3-3 protein modulation of GPCR activity, especially given the limited literature regarding NK3 signaling, including its relation to G-proteins or β-arrestins. Our findings suggest that different combinations of hetero- and homodimers of 14-3-3 isoforms result in varying functional outcomes at NK3, such as upregulation of NK3 surface expression by 14-3-3γ. Furthermore, 14-3-3γ was found to modulate canonical signaling pathways by attenuating G-protein dissociation and enhancing β-arrestin recruitment signals.

G protein–coupled receptors (GPCRs) form the largest family of cell-surface receptors and remain prime targets in drug discovery. A central challenge in modern GPCR drug discovery is understanding and exploiting biased agonism: the ability of ligands to favor signaling via therapeutically beneficial pathways while avoiding those that trigger side effects. Therefore, pinpointing the structural determinants of signaling bias is crucial for rational drug design. Biased agonists are particularly compelling for targeting opioid receptors, as in this family, ligands that limit β-arrestin (β-arr) recruitment are believed to preserve analgesia while reducing respiratory depression and addiction liabilities. Here, we use extensive all-atom molecular dynamics (MD) simulations to dissect signaling bias in the δ-opioid receptor (δOR). Focusing on a receptor mutant with a strong β-arr bias, we employed a reverse-engineering approach to reveal the conformational mechanisms that promote β-arr recruitment. Building on these insights, engineer new mutations that reshape the receptor’s signaling profile. Importantly, this approach allowed us to pinpoint signaling bias to motions of a single microswitch and identify how structural receptor motions induced by the mutations and ligand contacts cooperate to promote a specific functional response. In this proof-of-concept study, we not only provide structural insights into δOR pharmacology but also demonstrate how computational methods can be leveraged to probe structural mechanisms of signaling specificity across GPCRs, paving the way for the rational design of tailored receptor variants and novel, safer, and more effective therapeutics.

Roth and colleagues have developed PRESTO-Tango, a new open-source platform for high-throughput screening of the entire human nonolfactory GPCRome, and they show how it can be used to identify new ligands for orphan human GPCRs. G protein–coupled receptors (GPCRs) are essential mediators of cellular signaling and are important targets of drug action. Of the approximately 350 nonolfactory human GPCRs, more than 100 are still considered to be 'orphans' because their endogenous ligands remain unknown. Here, we describe a unique open-source resource that allows interrogation of the druggable human GPCRome via a G protein–independent β-arrestin–recruitment assay. We validate this unique platform at more than 120 nonorphan human GPCR targets, demonstrate its utility for discovering new ligands for orphan human GPCRs and describe a method (parallel receptorome expression and screening via transcriptional output, with transcriptional activation following arrestin translocation (PRESTO-Tango)) for the simultaneous and parallel interrogation of the entire human nonolfactory GPCRome.

Protein-protein interactions (PPIs) form the foundation of any cell signaling network. Considering that PPIs are highly dynamic processes, cellular assays are often essential for their study because they closely mimic the biological complexities of cellular environments. However, incongruity may be observed across different PPI assays when investigating a protein partner of interest; these discrepancies can be partially attributed to the fusion of different large functional moieties, such as fluorescent proteins or enzymes, which can yield disparate perturbations to the protein's stability, subcellular localization, and interaction partners depending on the given cellular assay. Owing to their smaller size, epitope tags may exhibit a diminished susceptibility to instigate such perturbations. However, while they have been widely used for detecting or manipulating proteins in vitro, epitope tags lack the in vivo traceability and functionality needed for intracellular biosensors. Herein, we develop NbV5, an intracellular nanobody binding the V5-tag, which is suitable for use in cellular assays commonly used to study PPIs such as BRET, NanoBiT, and Tango. The NbV5:V5 tag system has been applied to interrogate G protein-coupled receptor signaling, specifically by replacing larger functional moieties attached to the protein interactors, such as fluorescent or luminescent proteins (∼30 kDa), by the significantly smaller V5-tag peptide (1.4 kDa), and for microscopy imaging which is successfully detected by NbV5-based biosensors. Therefore, the NbV5:V5 tag system presents itself as a versatile tool for live-cell imaging and a befitting adaptation to existing cellular assays dedicated to probing PPIs.

Representing the most attractive and successful druggable receptors of the proteome, GPCRs regulate a myriad of physiological and pathophysiological functions. Although over half of present pharmaceuticals target GPCRs, the advancement of drug discovery is hampered by a lack of adequate screening tools, the majority of which are limited to probing agonist-induced G-protein and β-arrestin-2-mediated events as a measure of receptor activation. Here, we develop Tango-Trio, a comprehensive cell-based high-throughput platform comprising cumate-inducible expression of transducers, capable of the parallelized profiling of both basal and agonist-dependent GPCR activities. We capture the functional diversity of GPCRs, reporting β-arrestin-1/2 couplings, selectivities, and receptor internalization signatures across the GPCRome. Moreover, we present the construction of cumate-induced basal activation curves at approximately 200 receptors, including over 50 orphans. Overall, Tango-Trio's robustness is well-suited for the functional characterization and screening of GPCRs, especially for parallel interrogation, and is a valuable addition to the pharmacological toolbox.

ABSTRACT/SUMMARY Protein-protein interactions (PPIs) form the underpinnings of any cellular signaling network. PPIs are highly dynamic processes and often, cell-based assays can be essential for their study as they closely mimic the biological intricacies of cellular environments. Since no sole platform can perform all needed experiments to gain a thoroughly comprehensive understanding into these processes, developing a versatile toolkit is much needed to address this longstanding gap. The use of small peptide tags, such as the V5-tag, has been extensively used in biological and biomedical research, including labeling the C-termini of one of the largest human genome-wide open-reading frame collections. However, these small peptide tags have been primarily used in vitro and lack the in vivo traceability and functionality of larger specialized tags. In this study, we combined structural studies and computer-aided maturation to generate an intracellular nanobody, interacting with the V5-tag. Suitable for assays commonly used to study protein-protein interactions, our nanobody has been applied herein to interrogate G protein-coupled receptor signalling. This novel serviceable intrabody is the cornerstone of a multipurpose intracellular nanobody-based biosensors toolkit, named iBodyV5, which will be available for the scientific community at large.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spike glycoprotein (S) binds to angiotensin-converting enzyme 2 (ACE2) to mediate membrane fusion via two distinct pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In this study, we found that SARS-CoV-2 S has a wider protease usage and can also be activated by TMPRSS13 and matrix metalloproteinases (MMPs). We found that MMP-2 and MMP-9 played roles in SARS-CoV-2 S cell-cell fusion and TMPRSS2- and cathepsin-independent viral entry in cells expressing high MMP levels. MMPdependent viral entry required cleavage at the S1/S2 junction in viral producer cells, and differential processing of variants of concern S dictated its usage; the efficiently processed Delta S preferred metalloproteinase-dependent entry when available, and less processed Omicron S was unable to us metalloproteinases for entry. As MMP- 2/9 are released during inflammation, they may play roles in S-mediated cytopathic effects, tropism, and disease outcome.

Existing pharmacotherapies acting on the opioid receptor system have been extensively used to treat chronic pain and addictive disorders. Nevertheless, the adverse side effects associated with opioid therapy underscore the need for concerted measures to develop safer analgesics. A promising avenue of research stems from the characterization of a sodium-dependent allosteric regulation site housed within the delta-opioid receptor and several other G protein-coupled receptors (GPCRs), thereby revealing the presence of a cluster of sodium and water molecules lodged in a cavity thought to be present only in the inactive conformation of the receptor. Studies into the structure-function relationship of said pocket demonstrated its critical involvement in the functional control of GPCR signaling. While the sodium pocket has been proposed to be present in the majority of class A GPCRs, the shape of this allosteric cavity appears to have significant structural variation among crystallographically solved GPCRs, making this site optimal for the design of new allosteric modulators that will be selective for opioid receptors. The size of the sodium pocket supports the accommodation of small molecules, and it has been speculated that promiscuous amiloride and 5'-substituted amiloride-related derivatives could target this cavity within many GPCRs, including opioid receptors. Using pharmacological approaches, we have described the selectivities of 5'-substituted amiloride-related derivatives, as well as the hitherto undescribed activity of the NHE1 inhibitor zoniporide toward class A GPCRs. Our investigations into the structural features of the delta-opioid receptor and its ensuing signaling activities suggest a bitopic mode of overlapping interactions involving the orthosteric site and the juxtaposed Na+ pocket, but only at the active or partially active opioid receptor.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

ABSTRACTTo infect cells, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) binds to angiotensin converting enzyme 2 (ACE2) via its spike glycoprotein (S), delivering its genome upon S-mediated membrane fusion. SARS-CoV-2 uses two distinct entry pathways: 1) a surface, serine protease-dependent or 2) an endosomal, cysteine protease-dependent pathway. In investigating serine protease-independent cell-cell fusion, we found that the matrix metalloproteinases (MMPs), MMP2/9, can activate SARS-CoV-2 S fusion activity, but not that of SARS-CoV-1. Importantly, metalloproteinase activation of SARS-CoV-2 S represents a third entry pathway in cells expressing high MMP levels. This route of entry required cleavage at the S1/S2 junction in viral producer cells and differential processing of variants of concern S dictated its usage. In addition, metalloproteinase inhibitors reduced replicative Alpha infection and abrogated syncytia formation. Finally, we found that the Omicron S exhibit reduced metalloproteinase-dependent fusion and viral entry. Taken together, we identified a MMP2/9-dependent mode of activation of SARS-CoV-2 S. As MMP2/9 are released during inflammation and severe COVID-19, they may play important roles in SARS-CoV-2 S-mediated cytopathic effects, tropism, and disease outcome.

Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H2S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment.

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The nanobodies were collectively shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across existing VoCs; wide-ranging epitopic and mechanistic diversity and high and broad in vitro neutralization potencies. A select set of Fc-fused nanobodies showed high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a potential therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to combat multiple SARS-CoV-2 variants.

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function.

The mutation rate and evolution of RNA viruses correlate with viral adaptation. While most mutations do not make significant contributions to viral molecular evolution, some are naturally selected and produce variants through positive selection.

Background Antibodies raised against human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 severity. However, the relationship between sCoVs exposure and SARS-CoV-2 correlates of protection are not clearly identified. Methods We performed a cross-sectional analysis of cross-reactivity and cross-neutralization to SARS-CoV-2 antigens (S-RBD, S-trimer, N) using pre-pandemic sera from four different groups: pediatrics and adolescents, individuals 21 to 70 years of age, older than 70 years of age, and individuals living with HCV or HIV. Data was then further analysed using machine learning to identify predictive patterns of neutralization based on sCoVs serology. Findings Antibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also show a range of neutralizing activity (0-45%) with median inhibition ranging from 17.6 % to 23.3 % in serum that interferes with SARS-CoV-2 spike attachment to ACE2 independently of age group. While the abundance of sCoV antibodies did not directly correlate with neutralization, we show that neutralizing activity is rather dependent on relative ratios of IgGs in sera directed to all four sCoV spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the most important predictors of neutralization. Interpretation Our data support the concept that exposure to sCoVs triggers antibody responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, which may potentially impact COVID-19 disease severity through other latent variables. Funding This study was supported by a grant by the CIHR (VR2 -172722) and by a grant supplement by the CITF, and by a NRC Collaborative R&D Initiative Grant (PR031-1).

Filoviruses, such as the Ebola virus (EBOV) and Marburg virus (MARV), are causative agents of sporadic outbreaks of hemorrhagic fevers in humans. To infect cells, filoviruses are internalized via macropinocytosis and traffic through the endosomal pathway where host cathepsin-dependent cleavage of the viral glycoproteins occurs. Subsequently, the cleaved viral glycoprotein interacts with the late endosome/lysosome resident host protein, Niemann-Pick C1 (NPC1). This interaction is hypothesized to trigger viral and host membrane fusion, which results in the delivery of the viral genome into the cytoplasm and subsequent initiation of replication. Some studies suggest that EBOV viral particles activate signaling cascades and host-trafficking factors to promote their localization with host factors that are essential for entry. However, the mechanism through which these activating signals are initiated remains unknown. By screening a kinase inhibitor library, we found that receptor tyrosine kinase inhibitors potently block EBOV and MARV GP-dependent viral entry. Inhibitors of epidermal growth factor receptor (EGFR), tyrosine protein kinase Met (c-Met), and the insulin receptor (InsR)/insulin like growth factor 1 receptor (IGF1R) blocked filoviral GP-mediated entry and prevented growth of replicative EBOV in Vero cells. Furthermore, inhibitors of c-Met and InsR/IGF1R also blocked viral entry in macrophages, the primary targets of EBOV infection. Interestingly, while the c-Met and InsR/IGF1R inhibitors interfered with EBOV trafficking to NPC1, virus delivery to the receptor was not impaired in the presence of the EGFR inhibitor. Instead, we observed that the NPC1 positive compartments were phenotypically altered and rendered incompetent to permit viral entry. Despite their different mechanisms of action, all three RTK inhibitors tested inhibited virus-induced Akt activation, providing a possible explanation for how EBOV may activate signaling pathways during entry. In sum, these studies strongly suggest that receptor tyrosine kinases initiate signaling cascades essential for efficient post-internalization entry steps.

ABSTRACTNanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent β-arrestin2 recruitment assay, the universality of β-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango's sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has a genome comprised of a ~30K nucleotides non-segmented, positive single-stranded RNA. Although its RNA-dependent RNA polymerase exhibits exonuclease proofreading activity, viral sequence diversity can be induced by replication errors and host factors. These variations can be observed in the population of viral sequences isolated from infected host cells and are not necessarily reflected in the genome of transmitted founder viruses. We profiled intra-sample genetic diversity of SARS-CoV-2 variants using 15,289 high-throughput sequencing datasets from infected individuals and infected cell lines. Most of the genetic variations observed, including C->U and G->U, were consistent with errors due to heat-induced DNA damage during sample processing, and/or sequencing protocols. Despite high mutational background, we confidently identified intra-variable positions recurrent in the samples analyzed, including several positions at the end of the gene encoding the viral S protein. Notably, most of the samples possesses a C->A missense mutation resulting in the S protein lacking the last 20 amino acids (S{Delta}20). Here we demonstrate that S{Delta}20 exhibits increased cell-to-cell fusion and syncytia formations. Our findings are suggestive of the consistent emergence of high-frequency viral quasispecies that are not horizontally transmitted but involved in intra-host infection and spread. Author summaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated disease, COVID-19, has caused significant worldwide mortality and unprecedented economic burden. Here we studied the intra-host genetic diversity of SARS-CoV-2 genomes and identified a high-frequency and recurrent non-sense mutation yielding a truncated form of the viral spike protein, in both human COVID-19 samples and in cell culture experiments. Through the use of a functional assay, we observed that this truncated spike protein displays an elevated fusogenic potential and forms syncytia. Given the high frequency at which this mutation independently arises across various samples, it can be hypothesized that this deletion mutation provides a selective advantage to viral replication and may also have a role in pathogenesis in humans.