Membrane Fusion Assay Research Articles

SARS-CoV-2 spike fusion peptide trans interaction with phosphatidylserine lipid triggers membrane fusion for viral entry.

The role of lipids in the host cell target membrane for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry is not clear. We do not know whether SARS-CoV-2 spike protein has any specificity in terms of lipid for membrane fusion reaction. Here, using in vitro reconstitution of membrane fusion assay and single-molecule fluorescence resonance energy transfer imaging of SARS-CoV-2 spike trimers on the surface of the virion, we have demonstrated that phosphatidylserine (PS) lipid plays a key role in SARS-CoV-2 spike-mediated membrane fusion reaction for entry. Membrane-externalized PS lipid strongly promotes spike-mediated membrane fusion and COVID-19 infection. Blocking externalized PS lipid with PS-binding protein or in the absence of PS, SARS-CoV-2 spike-mediated fusion is strongly inhibited. Therefore, PS is an important target for restricting viral entry and intervening spike, and PS interaction presents new targets for COVID-19 interventions.

Mutations of Single Residues in the Complexin N-terminus Exhibit Distinct Phenotypes in Synaptic Vesicle Fusion.

The release of neurotransmitters (NTs) at central synapses is dependent on a cascade of protein interactions, specific to the presynaptic compartment. Among those dedicated molecules, the cytosolic complexins play an incompletely defined role as synaptic transmission regulators. Complexins are multidomain proteins that bind soluble N-ethylmaleimide sensitive factor attachment protein receptor complexes, conferring both inhibitory and stimulatory functions. Using systematic mutagenesis and comparing reconstituted in vitro membrane fusion assays with electrophysiology in cultured neurons from mice of either sex, we deciphered the function of the N-terminus of complexin (Cpx) II. The N-terminus (amino acid 1-27) starts with a region enriched in hydrophobic amino acids (1-12), which binds lipids. Mutants maintaining this hydrophobic character retained the stimulatory function of Cpx, whereas exchanges introducing charged residues perturbed both spontaneous and evoked exocytosis. Mutants in the more distal region of the N-terminal domain (amino acid 11-18) showed a spectrum of effects. On the one hand, mutation of residue A12 increased spontaneous release without affecting evoked release. On the other hand, replacing D15 with amino acids of different shapes or hydrophobic properties (but not charge) not only increased spontaneous release but also impaired evoked release. Most surprising, this substitution reduced the size of the readily releasable pool, a novel function for Cpx at mammalian synapses. Thus, the exact amino acid composition of the Cpx N-terminus fine-tunes the degree of spontaneous and evoked NT release.

Hexestrol, an estrogen receptor agonist, inhibits Lassa virus entry.

Lassa virus (LASV) is the causative agent of human Lassa fever which in severe cases manifests as hemorrhagic fever leading to thousands of deaths annually. However, no approved vaccines or antiviral drugs are currently available. Recently, we screened approximately 2,500 compounds using a recombinant vesicular stomatitis virus (VSV) expressing LASV glycoprotein GP (VSV-LASVGP) and identified a P-glycoprotein inhibitor as a potential LASV entry inhibitor. Here, we show that another identified candidate, hexestrol (HES), an estrogen receptor agonist, is also a LASV entry inhibitor. HES inhibited VSV-LASVGP replication with a 50% inhibitory concentration (IC50) of 0.63 µM. Importantly, HES also inhibited authentic LASV replication with IC50 values of 0.31 µM-0.61 µM. Time-of-addition and cell-based membrane fusion assays suggested that HES inhibits the membrane fusion step during virus entry. Alternative estrogen receptor agonists did not inhibit VSV-LASVGP replication, suggesting that the estrogen receptor itself is unlikely to be involved in the antiviral activity of HES. Generation of a HES-resistant mutant revealed that the phenylalanine at amino acid position 446 (F446) of LASVGP, which is located in the transmembrane region, conferred resistance to HES. Although mutation of F446 enhanced the membrane fusion activity of LASVGP, it exhibited reduced VSV-LASVGP replication, most likely due to the instability of the pre-fusion state of LASVGP. Collectively, our results demonstrated that HES is a promising anti-LASV drug that acts by inhibiting the membrane fusion step of LASV entry. This study also highlights the importance of the LASVGP transmembrane region as a target for anti-LASV drugs.IMPORTANCELassa virus (LASV), the causative agent of Lassa fever, is the most devastating mammarenavirus with respect to its impact on public health in West Africa. However, no approved antiviral drugs or vaccines are currently available. Here, we identified hexestrol (HES), an estrogen receptor agonist, as the potential antiviral candidate drug. We showed that the estrogen receptor itself is not involved in the antiviral activity. HES directly bound to LASVGP and blocked membrane fusion, thereby inhibiting LASV infection. Through the generation of a HES-resistant virus, we found that phenylalanine at position 446 (F446) within the LASVGP transmembrane region plays a crucial role in the antiviral activity of HES. The mutation at F446 caused reduced virus replication, likely due to the instability of the pre-fusion state of LASVGP. These findings highlight the potential of HES as a promising candidate for the development of antiviral compounds targeting LASV.

No evidence for a direct extracellular interaction between human Fc receptor-like 3 (MAIA) and the sperm ligand IZUMO1.

Fertilization involves the recognition and fusion of sperm and egg to form a previously unidentified organism. In mammals, surface molecules on the sperm and egg have central roles, and while adhesion is mediated by the IZUMO1-JUNO sperm-egg ligand-receptor pair, the molecule/s responsible for membrane fusion remain mysterious. Recently, MAIA/FCRL3 was identified as a mammalian egg receptor, which bound IZUMO1 and JUNO and might therefore have a bridging role in gamete recognition and fusion. Here, we use sensitive assays designed to detect extracellular protein binding to investigate the interactions between MAIA and both IZUMO1 and JUNO. Despite using reagents with demonstrable biochemical activity, we did not identify any direct binding between MAIA/FCRL3 and either IZUMO1 or JUNO. We also observed no fusogenic activity of MAIA/FCRL3 in a cell-based membrane fusion assay. Our findings encourage caution in further investigations on the role played by MAIA/FCRL3 in fertilization.

Differential Behavior of Eicosapentaenoic and Docosahexaenoic Acids on the Organization, Dynamics, and Fusion of Homogeneous and Heterogeneous Membranes.

Membrane fusion is a common course in innumerable biological processes that helps in the survival of eukaryotes. Enveloped viruses utilize this process to enter the host cells. Generally, the membrane lipid compositions play an important role in membrane fusion by modulating the membrane's physical properties and the behavior of membrane proteins in the cellular milieu. In this work, we have demonstrated the role of polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, on the organization, dynamics, and fusion of homogeneous and heterogeneous membranes. We have exploited arrays of steady-state and time-resolved fluorescence spectroscopic methods and polyethylene glycol-induced membrane fusion assay to elucidate the behavior of EPA and DHA on dioleoyl phosphatidylcholine (DOPC)/cholesterol (CH) homogeneous and DOPC/sphingomyelin/CH heterogeneous membranes. Our results suggest that EPA and DHA display differential effects on two different membranes. The effects of PUFAs in homogeneous membranes are majorly attributed to their flexible chain dynamics, whereas the ability of PUFA-induced cholesterol transfer from the lo to the ld phase rules their behavior in heterogeneous membranes. Overall, our results provide detailed information on the effect of PUFAs on homogeneous and heterogeneous membranes.

Lancemaside A from Codonopsis lanceolata: Studies on Antiviral Activity and Mechanism of Action against SARS-CoV-2 and Its Variants of Concern.

Several plant-derived natural products with anti-SARS-CoV-2 activity have been evaluated for the potential to serve as chemotherapeutic agents for the treatment of COVID-19. Codonopsis lanceolata (CL) has long been used as a medicinal herb in East Asian countries to treat inflammatory diseases of the respiratory system but its antiviral activity has not been investigated so far. Here, we showed that CL extract and its active compound lancemaside A (LA) displayed potent inhibitory activity against SARS-CoV-2 infection using a pseudotyped SARS-CoV-2 entry assay system. We demonstrated that this inhibitory effect of LA was due to the alteration of membrane cholesterol and blockade of the membrane fusion between SARS-CoV-2 and host cells by filipin staining and cell-based membrane fusion assays. Our findings also showed that LA, as a membrane fusion blocker, could impede the endosomal entry pathway of SARS-CoV-2 and its variants of concern (VOCs), including Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2), and Omicron (B.1.1.529), in Vero cells with similar of IC50 values ranging from 2.23 to 3.37 μM as well as the TMPRSS2-mediated viral entry pathway in A549 cells overexpressing ACE2 and TMPRSS2 with IC50 value of 3.92 μM. We further demonstrated that LA could prevent the formation of multinucleated syncytia arising from SARS-CoV-2 spike protein-mediated membrane fusion. Altogether, the findings reported here suggested that LA could be a broad-spectrum anti-SARS-CoV-2 therapeutic agent by targeting the fusion of viral envelope with the host cell membrane.

Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime Analysis.

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a well-established and useful tool in physics and chemistry. Furthermore, due to its hybrid character of being a bulk assay at a single molecular level, it found many applications in biophysics and molecular biochemistry. Examples may be investigating kinetics and dynamics of chemical and biochemical reactions suchas protein-ligand-, protein-protein-binding, fast conformational changes, and intracellular transportation. Also, it was utilized to characterize larger structures suchas lipid vesicles and multi-protein complexes. A two-photon excitation source makes FCCS relatively easy-to-use and easy-to-maintain. Combining this technique with fluorescence lifetime analysis results in a versatile biophysical tool that can be used to solve many biological problems, as even small changes in the local environment, like pH or salt concentration, can be monitored if appropriate fluorophores are used. An example of its use for membrane docking and fusion assays is described in Chap. 13 . In this chapter, we want to give the reader a simple, detailed step-by-step guideof how to set up such a tool.

Structural basis for broad coronavirus neutralization.

Three highly pathogenic β-coronaviruses have crossed the animal-to-human species barrier in the past two decades: SARS-CoV, MERS-CoV and SARS-CoV-2. To evaluate the possibility of identifying antibodies with broad neutralizing activity, we isolated a monoclonal antibody, termed B6, that cross-reacts with eight β-coronavirus spike glycoproteins, including all five human-infecting β-coronaviruses. B6 broadly neutralizes entry of pseudotyped viruses from lineages A and C, but not from lineage B, and the latter includes SARS-CoV and SARS-CoV-2. Cryo-EM, X-ray crystallography and membrane fusion assays reveal that B6 binds to a conserved cryptic epitope located in the fusion machinery. The data indicate that antibody binding sterically interferes with the spike conformational changes leading to membrane fusion. Our data provide a structural framework explaining B6 cross-reactivity with β-coronaviruses from three lineages, along with a proof of concept for antibody-mediated broad coronavirus neutralization elicited through vaccination. This study unveils an unexpected target for next-generation structure-guided design of a pan-β-coronavirus vaccine.

First Molecular Evidence and Genetic Characterization of Ovine Herpesvirus 2 in Multiple Animal Species in India.

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), a highly fatal disease syndrome that predominantly affects susceptible hosts of the order Artiodactyla. In this study, an in-depth clinico-molecular investigation of SA-MCF disease in a morbid 50-days-old cattle calf (Bos taurus indicus) and asymptomatic infection in the in-contact reservoir hosts, sheep (Ovis aries), and goat (Capra hircus) housed on a farm located in the Southern India is reported. An OIE recommended SA-MCF type-specific PCR confirmed the etiological agent as OvHV-2. The genetic characterization and phylogenetic analyses based on the glycoprotein B (gB) gene indicate that three genetic variants of OvHV-2 had infected the animal cluster of this study. As the OvHV-2 infection eventually lead to the death of the cattle calf, and the fact that its gB sequence carried four unique amino acid substitutions (N169S, L594P, I645V, and V730A), an investigation of these substitutions impact on its stability and molecular flexibility was carried out. The mapping of these amino acid substitutions on the three-dimensional structure of gB coupled with supplementary investigations showed that these substitutions conveyed the molecular flexibility to the gB, at the cost of its stability. Future studies would be to investigate whether these gB substitutions have any impact on membrane fusion activity using a virus-free cell-to-cell membrane fusion assay. The study also highlights the importance of adopting stringent biosecurity measures where mixed animal farming is a common practice.

Cell-based membrane fusion assays with viral fusion proteins for identification of entry inhibitors

Cell-based membrane fusion assays with viral fusion proteins for identification of entry inhibitors

The Antimalarial Compound Atovaquone Inhibits Zika and Dengue Virus Infection by Blocking E Protein-Mediated Membrane Fusion.

Flaviviruses bear class II fusion proteins as their envelope (E) proteins. Here, we describe the development of an in vitro quantitative mosquito-cell-based membrane-fusion assay for the E protein using dual split proteins (DSPs). The assay does not involve the use of live viruses and allows the analysis of a membrane-fusion step independent of other events in the viral lifecycle, such as endocytosis. The progress of membrane fusion can be monitored continuously by measuring the activities of Renilla luciferase derived from the reassociation of DSPs during cell fusion. We optimized the assay to screen an FDA-approved drug library for a potential membrane fusion inhibitor using the E protein of Zika virus. Screening results identified atovaquone, which was previously described as an antimalarial agent. Atovaquone potently blocked the in vitro Zika virus infection of mammalian cells with an IC90 of 2.1 µM. Furthermore, four distinct serotypes of dengue virus were also inhibited by atovaquone with IC90 values of 1.6–2.5 µM, which is a range below the average blood concentration of atovaquone after its oral administration in humans. These findings make atovaquone a likely candidate drug to treat illnesses caused by Zika as well as dengue viruses. Additionally, the DSP assay is useful to study the mechanism of membrane fusion in Flaviviruses.

Hemagglutinin Traits Determine Transmission of Avian A/H10N7 Virus between Mammals

An outbreak of A/H10N7 influenza virus among seals in 2014 provided a unique opportunity to study cross-species transmission and mammalian adaptation of avian influenza A virus in nature. The seal A/H10N7 virus was aerosol or respiratory droplet-transmissible between ferrets. Its H10 hemagglutinin showed stronger binding to human-type receptor compared to avian H10 hemagglutinin, with preferential binding to α2,6-linked sialic acids on long extended branches. In X-ray structures, changes in the 220-loop of the receptor-binding pocket resulted in similar interaction with human receptor as seen for pandemic strains. In a membrane fusion assay, two substitutions made the seal H10 hemagglutinin more stable than the avian H10 hemagglutinin and similar to human hemagglutinin. Consequently, identification of avian-origin influenza viruses with increased aerosol or respiratory droplet transmissibility between mammals, through HA stability and human-type receptor preference, appears critical to detect influenza A viruses that pose a major threat to humans and other mammals.

Guanidine modifications enhance the anti‐herpes simplex virus activity of (E,E)‐4,6‐bis(styryl)‐pyrimidine derivatives in vitro and in vivo

The worldwide prevalence of herpes simplex virus (HSV) and shortage of efficient therapeutic strategies to counteract it are global concerns. In terms of treatment, the widely utilized anti-HSV drugs such as acyclovir have serious limitations, for example, drug resistance and side effects. Here, we have identified the guanidine-modified (E,E)-4,6-bis(styryl)-pyrimidine (BS-pyrimidine) derivative compound 5d as an inhibitor of HSV and further elucidated the anti-HSV mechanisms of compound 5d both in vitro and in vivo. Cytopathic effect inhibition assay, plaque assay, and immunofluorescence assay were used to evaluate the anti-HSV effects of compound 5d in vitro. Membrane fusion assays, immunofluorescence assays, Western blotting assays, and pull-down assays were used to explore the anti-HSV mechanisms of compound 5d. HSV-1-infected mice, combined with haematoxylin-eosin staining and quantitative RT-PCR, were used to study the anti-HSV effects of compound 5d in vivo. The guanidine-modified compound 5d rather than the un-modified compound 3a effectively inhibited both HSV-1 and HSV-2 multiplication in different cell lines, more effectively than acyclovir. Compound 5d may block virus binding and post-binding processes such as membrane fusion, by targeting virus gB protein. In addition, compound 5d may also down-regulate the cellular PI3K/Akt signalling pathway to interfere with HSV replication. Treatment with compound 5d also markedly improved survival and decreased viral titres in HSV-infected mice. Thus, the guanidine-modified BS-pyrimidine derivatives have the potential to be developed into novel anti-HSV agents targeting both virus gB protein and cellular PI3K/Akt signalling pathways.

Real-time assay for exosome membrane fusion with an artificial lipid membrane based on enhancement of gramicidin A channel conductance

We report that the channel activities of gramicidin A in a supported lipid bilayer (SLB) were modulated by membrane fusion with exosomes. The mechanism of the modulation was an increase in the number of exosomes inserting into the SLB membrane, rather than enhancements of the single channel activity of gramicidin A. The modulation of apparent channel activities was applicable to the exosome fusion assay. This assay revealed that the membrane fusion of HEK-293 and MCF-7 exosomes was enhanced at pH 6.0, and the initial rates of membrane fusion for MCF-7 exosomes were higher than those for HEK-293 cells.

In vitro fusion of single synaptic and dense core vesicles reproduces key physiological properties

Regulated exocytosis of synaptic vesicles is substantially faster than of endocrine dense core vesicles despite similar molecular machineries. The reasons for this difference are unknown and could be due to different regulatory proteins, different spatial arrangements, different vesicle sizes, or other factors. To address these questions, we take a reconstitution approach and compare regulated SNARE-mediated fusion of purified synaptic and dense core chromaffin and insulin vesicles using a single vesicle-supported membrane fusion assay. In all cases, Munc18 and complexin are required to restrict fusion in the absence of calcium. Calcium triggers fusion of all docked vesicles. Munc13 (C1C2MUN domain) is required for synaptic and enhanced insulin vesicle fusion, but not for chromaffin vesicles, correlating inversely with the presence of CAPS protein on purified vesicles. Striking disparities in calcium-triggered fusion rates are observed, increasing with curvature with time constants 0.23 s (synaptic vesicles), 3.3 s (chromaffin vesicles), and 9.1 s (insulin vesicles) and correlating with rate differences in cells.

Membrane Fusion Assays for Studying Entry Hepatitis C Virus into Cells.

The membrane fusion properties of HCV envelope glycoproteins can be evaluated using several assays. Fusion assays generally require contacts between glycoproteins expressed on a donor membrane, such as those from a cell or a viral particle, and an acceptor membrane that may or may not express cognate viral receptor, such as those from an indicator cell or a liposome. In this chapter, we describe three well-established methods in the field that use either cell surface expression of glycoproteins, HCV pseudoparticles (HCVpp), or cell culture-grown HCV (HCVcc) particles for donor membrane and cells or liposomes as acceptor membrane in which specific components can be included to monitor and quantify fusion. We provide details of cell-cell fusion assay, virus-liposome fusion assay, and finally virus-plasma membrane fusion assay. We also describe inhibitors that can block HCV envelope membrane fusion.

The N-peptide–binding mode is critical to Munc18-1 function in synaptic exocytosis

Sec1/Munc18 (SM) proteins promote intracellular vesicle fusion by binding to N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). A key SNARE-binding mode of SM proteins involves the N-terminal peptide (N-peptide) motif of syntaxin, a SNARE subunit localized to the target membrane. In in vitro membrane fusion assays, inhibition of N-peptide motif binding previously has been shown to abrogate the stimulatory function of Munc18-1, a SM protein involved in synaptic exocytosis in neurons. The physiological role of the N-peptide-binding mode, however, remains unclear. In this work, we addressed this key question using a "clogged" Munc18-1 protein, in which an ectopic copy of the syntaxin N-peptide motif was directly fused to Munc18-1. We found that the ectopic N-peptide motif blocks the N-peptide-binding pocket of Munc18-1, preventing the latter from binding to the native N-peptide motif on syntaxin-1. In a reconstituted system, we observed that clogged Munc18-1 is defective in promoting SNARE zippering. When introduced into induced neuronal cells (iN cells) derived from human pluripotent stem cells, clogged Munc18-1 failed to mediate synaptic exocytosis. As a result, both spontaneous and evoked synaptic transmission was abolished. These genetic findings provide direct evidence for the crucial role of the N-peptide-binding mode of Munc18-1 in synaptic exocytosis. We suggest that clogged SM proteins will also be instrumental in defining the physiological roles of the N-peptide-binding mode in other vesicle-fusion pathways.

A Cell-Free Content Mixing Assay for SNARE-Mediated Multivesicular Body-Vacuole Membrane Fusion.

Endocytosis is a fundamental process underlying diverse eukaryotic physiology. The terminal stage of this process is membrane fusion between the perimeter membrane of a late endosome filled with intraluminal vesicles, or multivesicular body (MVB), and the lysosome membrane to facilitate catabolism of internalized biomaterials or surface polytopic proteins. To comprehensively understand the mechanisms underlying MVB-lysosome membrane fusion, we developed a quantitative, cell-free assay to study this SNARE-mediated event in molecular detail using Saccharomyces cerevisiae and its vacuolar lysosome, or vacuole, as models. This involves separately isolating organelles from two yeast strains each expressing a different complementary fusion probe targeted to the lumen of either MVBs or vacuoles. Isolated organelles are mixed in vitro under fusogenic conditions. Upon MVB-vacuole membrane fusion, luminal contents mix to facilitate probe interaction, reconstituting β-lactamase activity recorded by a colorimetric enzyme activity assay. This method accommodates a multitude of approaches (e.g., genetics, addition of purified protein reagents) to study this process in isolation, and in theory could be repurposed to study other SNARE-mediated fusion events within cells.

Analysis of the Role of Sec3 in SNARE Assembly and Membrane Fusion.

Intracellular membrane fusion is mediated by the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins that are highly conserved and tightly regulated by a variety of factors. The exocyst complex is one of the multi-subunit tethering complexes and functions in the tethering of the secretory vesicles to the plasma membrane. We have found that the yeastSec3, a subunit of the exocyst, binds to the t-SNARE protein Sso2 and promotes its interaction with another t-SNARE protein, Sec9. Here, we describe the structural analysis and in vitro membrane fusion assays, by which we found that Sec3 binding leads to a conformational change within Sso2, and facilitates SNARE assembly and the membrane fusion.

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion.

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.