Cryo-EM structure of SARS-CoV-2 postfusion spike in membrane.

The entry of SARS-CoV-2 into host cells depends on the refolding of the virus-encoded spike protein from a prefusion conformation, which is metastable after cleavage, to a lower-energy stable postfusion conformation1,2. This transition overcomes kinetic barriers for fusion of viral and target cell membranes3,4. Here we report a cryogenic electron microscopy (cryo-EM) structure of the intact postfusion spike in a lipid bilayer that represents the single-membrane product of the fusion reaction. The structure provides structural definition of the functionally critical membrane-interacting segments, including the fusion peptide and transmembrane anchor. The internal fusion peptide forms a hairpin-like wedge that spans almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion. These results advance our understanding of the spike protein in a membrane environment and may guide development of intervention strategies.

Effect of Cholesterol on Biomimetic Membrane Curvature and Coronavirus Fusion Peptide Encapsulation.

Biomimetic cubic phases can be used for protein encapsulation in a variety of applications such as biosensors and drug delivery. Cubic phases with a high concentration of cholesterol and phospholipids were obtained herein. It is shown that the cubic phase structure can be maintained with a higher concentration of biomimetic membrane additives than has been reported previously. Opposing effects on the curvature of the membrane were observed upon the addition of phospholipids and cholesterol. Furthermore, the coronavirus fusion peptide significantly increased the negative curvature of the biomimetic membrane with cholesterol. We show that the viral fusion peptide can undergo structural changes leading to the formation of hydrophobic α-helices that insert into the lipid bilayer. This is of high importance, as a fusion peptide that induces increased negative curvature as shown by the formation of inverse hexagonal phases allows for greater contact area between two membranes, which is required for viral fusion to occur. The cytotoxicity assay showed that the toxicity toward HeLa cells was dramatically decreased when the cholesterol or peptide level in the nanoparticles increased. This suggests that the addition of cholesterol can improve the biocompatibility of the cubic phase nanoparticles, making them safer for use in biomedical applications. As the results, this work improves the potential for the biomedical end-use applications of the nonlamellar lipid nanoparticles and shows the need of systematic formulation studies due to the complex interplay of all components.

Comprehensive classification of proteins based on structures that engage lipids by COMPOSEL.

Structures can now be predicted for any protein using programs like AlphaFold and Rosetta, which rely on a foundation of experimentally determined structures of architecturally diverse proteins. The accuracy of such artificial intelligence and machine learning (AI/ML) approaches benefits from the specification of restraints which assist in navigating the universe of folds to converge on models most representative of a given protein's physiological structure. This is especially pertinent for membrane proteins, with structures and functions that depend on their presence in lipid bilayers. Structures of proteins in their membrane environments could conceivably be predicted from AI/ML approaches with user-specificized parameters that describe each element of the architecture of a membrane protein accompanied by its lipid environment. We propose the Classification Of Membrane Proteins based On Structures Engaging Lipids (COMPOSEL), which builds on existing nomenclature types for monotopic, bitopic, polytopic and peripheral membrane proteins as well as lipids. Functional and regulatory elements are also defined in the scripts, as shown with membrane fusing synaptotagmins, multidomain PDZD8 and Protrudin proteins that recognize phosphoinositide (PI) lipids, the intrinsically disordered MARCKS protein, caveolins, the ß barrel assembly machine (BAM), an adhesion G-protein coupled receptor (aGPCR) and two lipid modifying enzymes - diacylglycerol kinase DGKε and fatty aldehyde dehydrogenase FALDH. This demonstrates how COMPOSEL communicates lipid interactivity as well as signaling mechanisms and binding of metabolites, drug molecules, polypeptides or nucleic acids to describe the operations of any protein. Moreover COMPOSEL can be scaled to express how genomes encode membrane structures and how our organs are infiltrated by pathogens such as SARS-CoV-2.

Ionizable lipids penetrate phospholipid bilayers with high phase transition temperatures: perspectives from free energy calculations.

The efficacies of modern gene-therapies strongly depend on their contents. At the same time the most potent formulations might not contain the best compounds. In this work we investigated the effect of phospholipids and their saturation on the binding ability of (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino) butanoate (DLin-MC3-DMA) to model membranes at the neutral pH. We discovered that DLin-MC3-DMA has affinity to the most saturated monocomponent lipid bilayer 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and an aversion to the unsaturated one 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). The preference to a certain membrane was also well-correlated to the phase transition temperatures of phospholipid bilayers, and to their structural and dynamical properties. Additionally, in the case of the presence of DLin-MC3-DMA in the membrane with DOPC the ionizable lipid penetrated it, which indicates possible synergistic effects. Comparisons with other ionizable lipids were performed using a model lipid bilayer of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC). Particularly, the lipids heptadecan-9-yl 8-[2-hydroxyethyl-(6-oxo-6-undecoxyhexyl)amino]octanoate (SM-102) and [(4-hydroxybutyl) azanediyl] di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315) from modern mRNA-vaccines against COVID-19 were investigated and force fields parameters were derived for those new lipids. It was discovered that ALC-0315 binds strongest to the membrane, while DLin-MC3-DMA is not able to reside in the bilayer center. The ability to penetrate the membrane POPC by SM-102 and ALC-0315 can be related to their saturation, comparing to DLin-MC3-DMA.

SARS-CoV-2 Omicron Subvariants Balance Host Cell Membrane, Receptor, and Antibody Docking via an Overlapping Target Site.

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are emerging rapidly and offer surfaces that are optimized for recognition of host cell membranes while also evading antibodies arising from vaccinations and previous infections. Host cell infection is a multi-step process in which spike heads engage lipid bilayers and one or more angiotensin-converting enzyme 2 (ACE-2) receptors. Here, the membrane binding surfaces of Omicron subvariants are compared using cryo-electron microscopy (cEM) structures of spike trimers from BA.2, BA.2.12.1, BA.2.13, BA.2.75, BA.3, BA.4, and BA.5 viruses. Despite significant differences around mutated sites, they all maintain strong membrane binding propensities that first appeared in BA.1. Both their closed and open states retain elevated membrane docking capacities, although the presence of more closed than open states diminishes opportunities to bind receptors while enhancing membrane engagement. The electrostatic dipoles are generally conserved. However, the BA.2.75 spike dipole is compromised, and its ACE-2 affinity is increased, and BA.3 exhibits the opposite pattern. We propose that balancing the functional imperatives of a stable, readily cleavable spike that engages both lipid bilayers and receptors while avoiding host defenses underlies betacoronavirus evolution. This provides predictive criteria for rationalizing future pandemic waves and COVID-19 transmissibility while illuminating critical sites and strategies for simultaneously combating multiple variants.

The Cytoplasmic Domain of the SARS-CoV-2 Envelope Protein Assembles into a ß-Sheet Bundle in Lipid Bilayers.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) envelope (E) protein forms a pentameric ion channel in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment (ERGIC) of the infected cell. The cytoplasmic domain of E interacts with host proteins to cause virus pathogenicity and may also mediate virus assembly and budding. To understand the structural basis of these functions, here we investigate the conformation and dynamics of an E protein construct (residues 8-65) that encompasses the transmembrane domain and the majority of the cytoplasmic domain using solid-state NMR. 13C and 15N chemical shifts indicate that the cytoplasmic domain adopts a ß-sheet-rich conformation that contains three ß-strands separated by turns. The five subunits associate into an umbrella-shaped bundle that is attached to the transmembrane helices by a disordered loop. Water-edited NMR spectra indicate that the third ß-strand at the C terminus of the protein is well hydrated, indicating that it is at the surface of the ß-bundle. The structure of the cytoplasmic domain cannot be uniquely determined from the inter-residue correlations obtained here due to ambiguities in distinguishing intermolecular and intramolecular contacts for a compact pentameric assembly of this small domain. Instead, we present four structural topologies that are consistent with the measured inter-residue contacts. These data indicate that the cytoplasmic domain of the SARS-CoV-2 E protein has a strong propensity to adopt ß-sheet conformations when the protein is present at high concentrations in lipid bilayers. The equilibrium between the ß-strand conformation and the previously reported α-helical conformation may underlie the multiple functions of E in the host cell and in the virion.

A Facile Method to Coat Nanoparticles with Lipid Bilayer Membrane: Hybrid Silica Nanoparticles Disguised as Biomembrane Vesicles by Particle Penetration of Concentrated Lipid Layers.

Natural membrane vesicles, including extracellular vesicles and enveloped viruses, participate in various events in vivo. To study and manipulate these events, biomembrane-coated nanoparticles inspired by natural membrane vesicles are developed. Herein, an efficient method is presented to prepare organic-inorganic hybrid materials in high yields that can accommodate various lipid compositions and particle sizes. To demonstrate this method, silica nanoparticles are passed through concentrated lipid layers prepared using density gradient centrifugation, followed by purification, to obtain lipid membrane-coated nanoparticles. Various lipids, including neutral, anionic, and cationic lipids, are used to prepare concentrated lipid layers. Single-particle analysis by imaging flow cytometry determines that silica nanoparticles are uniformly coated with a single lipid bilayer. Moreover, cellular uptake of silica nanoparticles is enhanced when covered with a lipid membrane containing cationic lipids. Finally, cell-free protein expression is applied to embed a membrane protein, namely the Spike protein of severe acute respiratory syndrome coronavirus 2, into the coating of the nanoparticles, with the correct orientation. Therefore, this method can be used to develop organic-inorganic hybrid nanomaterials with an inorganic core and a virus-like coating, serving as carriers for targeted delivery of cargos such as proteins, DNA, and drugs.

Oligomerization-Dependent Beta-Structure Formation in SARS-CoV-2 Envelope Protein.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the current COVID-19 pandemic. In SARS-CoV-2, the channel-forming envelope (E) protein is almost identical to the E protein in SARS-CoV, and both share an identical α-helical channel-forming domain. Structures for the latter are available in both detergent and lipid membranes. However, models of the extramembrane domains have only been obtained from solution NMR in detergents, and show no ß-strands, in contrast to secondary-structure predictions. Herein, we have studied the conformation of purified SARS-CoV-2 E protein in lipid bilayers that mimic the composition of ER-Golgi intermediate compartment (ERGIC) membranes. The full-length E protein at high protein-to-lipid ratios produced a clear shoulder at 1635 cm-1, consistent with the ß-structure, but this was absent when the E protein was diluted, which instead showed a band at around 1688 cm-1, usually assigned to ß-turns. The results were similar with a mixture of POPC:POPG (2-oleoyl-1-palmitoyl-sn-glycero-3-phosphocholine/3-glycerol) and also when using an E-truncated form (residues 8-65). However, the latter only showed ß-structure formation at the highest concentration tested, while having a weaker oligomerization tendency in detergents than in full-length E protein. Therefore, we conclude that E monomer-monomer interaction triggers formation of the ß-structure from an undefined structure (possibly ß-turns) in at least about 15 residues located at the C-terminal extramembrane domain. Due to its proximity to the channel, this ß-structure domain could modulate channel activity or modify membrane structure at the time of virion formation inside the cell.

Proviral role of human respiratory epithelial cell-derived small extracellular vesicles in SARS-CoV-2 infection.

Small Extracellular Vesicles (sEVs) are 50-200 nm in diameter vesicles delimited by a lipid bilayer, formed within the endosomal network or derived from the plasma membrane. They are secreted in various biological fluids, including airway nasal mucus. The goal of this work was to understand the role of sEVs present in the mucus (mu-sEVs) produced by human nasal epithelial cells (HNECs) in SARS-CoV-2 infection. We show that uninfected HNECs produce mu-sEVs containing SARS-CoV-2 receptor ACE2 and activated protease TMPRSS2. mu-sEVs cleave prefusion viral Spike proteins at the S1/S2 boundary, resulting in higher proportions of prefusion S proteins exposing their receptor binding domain in an 'open' conformation, thereby facilitating receptor binding at the cell surface. We show that the role of nasal mu-sEVs is to complete prefusion Spike priming performed by intracellular furin during viral egress from infected cells. This effect is mediated by vesicular TMPRSS2 activity, rendering SARS-CoV-2 virions prone to entry into target cells using the 'early', TMPRSS2-dependent pathway instead of the 'late', cathepsin-dependent route. These results indicate that prefusion Spike priming by mu-sEVs in the nasal cavity plays a role in viral tropism. They also show that nasal mucus does not protect from SARS-CoV-2 infection, but instead facilitates it.

Investigation of the ionic conditions in SiRNA-mediated delivery through its carriers in the cell membrane: a molecular dynamic simulation.

SiRNA is a new generation of drug molecules and a new approach for treating a variety of diseases such as cancer and viral infections. SiRNA delivery to cells and translocation into cytoplasm are the main challenges in the clinical application of siRNA. Lipid carriers are one of the most successful carriers for siRNA delivery. In this study, we investigated the interaction of siRNA with a zwitterionic bilayer and how ion concentration and lipid conjugation can affect it. The divalent cation such as Mg2+ ions could promote the siRNA adsorption on the bilayer surface. The cation ions can bind to the head groups of lipids and the grooves of siRNA molecules and form bridges between the siRNA and bilayer surface. Our findings demonstrated the bridges formed by divalent ions could facilitate the attachment of siRNA to the membrane surface. We showed that the divalent cations can regulate the bridging-driven membrane attachment and it seems the result of this modulation can be used for designing biomimetic devices. In the following, we examined the effect of cations on the interaction between siRNA modified by cholesterol and the membrane surface. Our MD simulations showed that in the presence of Mg2+, the electrostatic and vdW energy between the membrane and siRNA were higher compared to those in the presence of NA+. We showed that the electrostatic interaction between membrane and siRNA cannot be facilitated only by cholesterol conjugated. Indeed, cations are essential to create coulomb repulsion and enable membrane attachment. This study provides important insight into liposome carriers for siRNA delivery and could help us in the development of siRNA-based therapeutics. Due to the coronavirus pandemic outbreak, these results may shed light on the new approach for treating these diseases and their molecular details.

SARS-CoV-2 Envelope Protein Forms Clustered Pentamers in Lipid Bilayers.

The SARS-CoV-2 envelope (E) protein is a viroporin associated with the acute respiratory symptoms of COVID-19. E forms cation-selective ion channels that assemble in the lipid membrane of the endoplasmic reticulum Golgi intermediate compartment. The channel activity of E is linked to the inflammatory response of the host cell to the virus. Like many viroporins, E is thought to oligomerize with a well-defined stoichiometry. However, attempts to determine the E stoichiometry have led to inconclusive results and suggested mixtures of oligomers whose exact nature might vary with the detergent used. Here, we employ 19F solid-state nuclear magnetic resonance and the centerband-only detection of exchange (CODEX) technique to determine the oligomeric number of E's transmembrane domain (ETM) in lipid bilayers. The CODEX equilibrium value, which corresponds to the inverse of the oligomeric number, indicates that ETM assembles into pentamers in lipid bilayers, without any detectable fraction of low-molecular-weight oligomers. Unexpectedly, at high peptide concentrations and in the presence of the lipid phosphatidylinositol, the CODEX data indicate that more than five 19F spins are within a detectable distance of about 2 nm, suggesting that the ETM pentamers cluster in the lipid bilayer. Monte Carlo simulations that take into account peptide-peptide and peptide-lipid interactions yielded pentamer clusters that reproduced the CODEX data. This supramolecular organization is likely important for E-mediated virus assembly and budding and for the channel function of the protein.

mRNA lipid nanoparticle phase transition.

Crucial for mRNA-based vaccines are the composition, structure, and properties of lipid nanoparticles (LNPs) as their delivery vehicle. Using all-atom molecular dynamics simulations as a computational microscope, we provide an atomistic view of the structure of the Comirnaty vaccine LNP, its molecular organization, physicochemical properties, and insight in its pH-driven phase transition enabling mRNA release at atomistic resolution. At physiological pH, our simulations suggest an oil-like LNP core that is composed of the aminolipid ALC-0315 and cholesterol (ratio 72:28). It is surrounded by a lipid monolayer formed by distearoylphosphatidylcholine, ALC-0315, PEGylated lipids, and cholesterol at a ratio of 22:9:6:63. Protonated aminolipids enveloping mRNA formed inverted micellar structures that provide a shielding and likely protection from environmental factors. In contrast, at low pH, the Comirnaty lipid composition instead spontaneously formed lipid bilayers that display a high degree of elasticity. These pH-dependent lipid phases suggest that a change in pH of the environment upon LNP transfer to the endosome likely acts as trigger for cargo release from the LNP core by turning aminolipids inside out, thereby destabilizing both the LNP shell and the endosomal membrane.

Cryo-electron tomography of enveloped viruses.

Viruses are macromolecular machineries that hijack cellular metabolism for replication. Enveloped viruses comprise a large variety of RNA and DNA viruses, many of which are notorious human or animal pathogens. Despite their importance, the presence of lipid bilayers in their assembly has made most enveloped viruses too pleomorphic to be reconstructed as a whole by traditional structural biology methods. Furthermore, structural biology of the viral lifecycle was hindered by the sample thickness. Here, I review the recent advances in the applications of cryo-electron tomography (cryo-ET) on enveloped viral structures and intracellular viral activities.

Continuum Models of Membrane Fusion: Evolution of the Theory.

Starting from fertilization, through tissue growth, hormone secretion, synaptic transmission, and sometimes morbid events of carcinogenesis and viral infections, membrane fusion regulates the whole life of high organisms. Despite that, a lot of fusion processes still lack well-established models and even a list of main actors. A merger of membranes requires their topological rearrangements controlled by elastic properties of a lipid bilayer. That is why continuum models based on theories of membrane elasticity are actively applied for the construction of physical models of membrane fusion. Started from the view on the membrane as a structureless film with postulated geometry of fusion intermediates, they developed along with experimental and computational techniques to a powerful tool for prediction of the whole process with molecular accuracy. In the present review, focusing on fusion processes occurring in eukaryotic cells, we scrutinize the history of these models, their evolution and complication, as well as open questions and remaining theoretical problems. We show that modern approaches in this field allow continuum models of membrane fusion to stand shoulder to shoulder with molecular dynamics simulations, and provide the deepest understanding of this process in multiple biological systems.

A Supported Lipid Bilayer-Based Lab-on-a-Chip Biosensor for the Rapid Electrical Screening of Coronavirus Drugs.

With the rapid spread and multigeneration variation of coronavirus, rapid drug development has become imperative. A major obstacle to addressing this issue is adequately constructing the cell membrane at the molecular level, which enables in vitro observation of the cell response to virus and drug molecules quantitatively, shortening the drug experiment cycle. Herein, we propose a rapid and label-free supported lipid bilayer-based lab-on-a-chip biosensor for the screening of effective inhibition drugs. An extended gate electrode was prepared and functionalized by an angiotensin-converting enzyme II (ACE2) receptor-incorporated supported lipid bilayer (SLB). Such an integrated system can convert the interactions of targets and membrane receptors into real-time charge signals. The platform can simulate the cell membrane microenvironment in vitro and accurately capture the interaction signal between the target and the cell membrane with minimized interference, thus observing the drug action pathway quantitatively and realizing drug screening effectively. Due to these label-free, low-cost, convenient, and integrated advantages, it is a suitable candidate method for the rapid drug screening for the early treatment and prevention of worldwide spread of coronavirus.

Drug-Membrane Interactions: Effects of Virus-Specific RNA-Dependent RNA Polymerase Inhibitors Remdesivir and Favipiravir on the Structure of Lipid Bilayers.

The two RNA-dependent RNA polymerase inhibitors remdesivir and favipiravir were originally developed and approved as broad-spectrum antiviral drugs for the treatment of harmful viral infections such as Ebola and influenza. With the outbreak of the global SARS-CoV-2 pandemic, the two drugs were repurposed for the treatment of COVID-19 patients. Clinical studies suggested that the efficacy of the drugs is enhanced in the case of an early or even prophylactic application. Because the contact between drug molecules and the plasma membrane is essential for a successful permeation process of the substances and therefore for their intracellular efficiency, drug-induced effects on the membrane structure are likely and have already been shown for other substances. We investigated the impact of remdesivir and favipiravir on lipid bilayers in model and cell membranes via several biophysical approaches. The measurements revealed that the embedding of remdesivir molecules in the lipid bilayer results in a disturbance of the membrane structure of the tested phospholipid vesicles. Nevertheless, in a cell-based assay, the presence of remdesivir induced only weak hemolysis of the treated erythrocytes. In contrast, no experimental indication for an effect on the structure and integrity of the membrane was detected in the case of favipiravir. Regarding potential prophylactic or accompanying use of the drugs in the therapy of COVID-19, the physiologically relevant impacts associated with the drug-induced structural modifications of the membrane might be important to understand side effects and/or low effectivities.

Membrane Cholesterol Content and Lipid Organization Influence Melittin and Pneumolysin Pore-Forming Activity.

Melittin, the main toxic component in the venom of the European honeybee, interacts with natural and artificial membranes due to its amphiphilic properties. Rather than interacting with a specific receptor, melittin interacts with the lipid components, disrupting the lipid bilayer and inducing ion leakage and osmotic shock. This mechanism of action is shared with pneumolysin and other members of the cholesterol-dependent cytolysin family. In this manuscript, we investigated the inverse correlation for cholesterol dependency of these two toxins. While pneumolysin-induced damage is reduced by pretreatment with the cholesterol-depleting agent methyl-ß-cyclodextrin, the toxicity of melittin, after cholesterol depletion, increased. A similar response was also observed after a short incubation with lipophilic simvastatin, which alters membrane lipid organization and structure, clustering lipid rafts. Therefore, changes in toxin sensitivity can be achieved in cells by depleting cholesterol or changing the lipid bilayer organization.

Delivery of RNA Therapeutics: The Great Endosomal Escape!

RNA therapeutics, including siRNAs, antisense oligonucleotides, and other oligonucleotides, have great potential to selectively treat a multitude of human diseases, from cancer to COVID to Parkinson's disease. RNA therapeutic activity is mechanistically driven by Watson-Crick base pairing to the target gene RNA without the requirement of prior knowledge of the protein structure, function, or cellular location. However, before widespread use of RNA therapeutics becomes a reality, we must overcome a billion years of evolutionary defenses designed to keep invading RNAs from entering cells. Unlike small-molecule therapeutics that are designed to passively diffuse across the cell membrane, macromolecular RNA therapeutics are too large, too charged, and/or too hydrophilic to passively diffuse across the cellular membrane and are instead taken up into cells by endocytosis. However, similar to the cell membrane, endosomes comprise a lipid bilayer that entraps 99% or more of RNA therapeutics, even in semipermissive tissues such as the liver, central nervous system, and muscle. Consequently, before RNA therapeutics can achieve their ultimate clinical potential to treat widespread human disease, the rate-limiting delivery problem of endosomal escape must be solved in a clinically acceptable manner.

Circularized fluorescent nanodiscs for probing protein-lipid interactions.

Protein-lipid interactions are vital for numerous transmembrane signaling pathways. However, simple tools to characterize these interactions remain scarce and are much needed to advance our understanding of signal transduction across lipid bilayers. To tackle this challenge, we herein engineer nanodisc as a robust fluorescent sensor for reporting membrane biochemical reactions. We circularize nanodiscs via split GFP and thereby create an intensity-based fluorescent sensor (isenND) for detecting membrane binding and remodeling events. We show that isenND responds robustly and specifically to the action of a diverse array of membrane-interacting proteins and peptides, ranging from synaptotagmin and synuclein involved in neurotransmission to viral fusion peptides of HIV-1 and SARS-CoV-2. Together, isenND can serve as a versatile biochemical reagent useful for basic and translational research of membrane biology.

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission.

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.