Membrane Fusion Process Research Articles

Dynamic Fusion of Nucleic Acid Functionalized Nano-/Micro-Cell-Like Containments: From Basic Concepts to Applications.

Membrane fusion processes play key roles in biological transformations, such as endocytosis/exocytosis, signal transduction, neurotransmission, or viral infections, and substantial research efforts have been directed to emulate these functions by artificial means. The recognition and dynamic reconfiguration properties of nucleic acids provide a versatile means to induce membrane fusion. Here we address recent advances in the functionalization of liposomes or membranes with structurally engineered lipidated nucleic acids guiding the fusion of cell-like containments, and the biophysical and chemical parameters controlling the fusion of the liposomes will be discussed. Intermembrane bridging by duplex or triplex nucleic acids and light-induced activation of membrane-associated nucleic acid constituents provide the means for spatiotemporal fusion of liposomes or nucleic acid modified liposome fusion with native cell membranes. The membrane fusion processes lead to exchange of loads in the fused containments and are a means to integrate functional assemblies. This is exemplified with the operation of biocatalytic cascades and dynamic DNA polymerization/nicking or transcription machineries in fused protocell systems. Membrane fusion processes of protocell assemblies are found to have important drug-delivery, therapeutic, sensing, and biocatalytic applications. The future challenges and perspectives of DNA-guided fused containments and membranes are addressed.

Kaempferol inhibits SARS-CoV-2 invasion by impairing heptad repeats-mediated viral fusion

BackgroundThe continuous evolution of SARS-CoV-2 has underscored the development of broad-spectrum prophylaxis. Antivirals targeting the membrane fusion process represent promising paradigms. Kaempferol (Kae), an ubiquitous plant flavonol, has been shown efficacy against various enveloped viruses. However, its potential in anti-SARS-CoV-2 invasion remains obscure. PurposeTo evaluate capabilities and mechanisms of Kae in preventing SARS-CoV-2 invasion. MethodsTo avoid interference of viral replication, virus-like particles (VLPs) constructed with luciferase reporter were applied. To investigate the antiviral potency of Kae, human induced pluripotent stem cells (hiPSC)-derived alveolar epithelial cells type II (AECII) and human ACE2 (hACE2) transgenic mice were utilized as in vitro and in vivo models, respectively. Using dual split protein (DSP) assays, inhibitory activities of Kae in viral fusion were determined in Alpha, Delta and Omicron variants of SARS-CoV-2, as well as in SARS-CoV and MERS-CoV. To further reveal molecular determinants of Kae in restricting viral fusion, synthetic peptides corresponding to the conserved heptad repeat (HR) 1 and 2, involved in viral fusion, and the mutant form of HR2 were explored by circular dichroism and native polyacrylamide gel electrophoresis. ResultsKae inhibited SARS-CoV-2 invasion both in vitro and in vivo, which was mainly attributed to its suppressive effects on viral fusion, but not endocytosis, two pathways that mediate viral invasion. In accordance with the proposed model of anti-fusion prophylaxis, Kae functioned as a pan-inhibitor of viral fusion, including three emerged highly pathogenic coronaviruses, and the currently circulating Omicron BQ.1.1 and XBB.1 variants of SARS-CoV-2. Consistent with the typical target of viral fusion inhibitors, Kae interacted with HR regions of SARS-CoV-2 S2 subunits. Distinct from previous inhibitory fusion peptides which prevent the formation of six-helix bundle (6-HB) by competitively interacting with HRs, Kae deformed HR1 and directly reacted with lysine residues within HR2 region, the latter of which was considered critical for the preservation of stabilized S2 during SARS-CoV-2 invasion. ConclusionsKae prevents SARS-CoV-2 infection by blocking membrane fusion and possesses a broad-spectrum anti-fusion ability. These findings provide valuable insights into potential benefits of Kae-containing botanical products as a complementary prophylaxis, especially during the waves of breakthrough infections and re-infections.

Analyzing the role of ACE2, AR, MX1 and TMPRSS2 genetic markers for COVID-19 severity

BackgroundThe use of molecular biomarkers for COVID-19 remains unconclusive. The application of a molecular biomarker in combination with clinical ones that could help classifying aggressive patients in first steps of the disease could help clinician and sanitary system a better management of the disease. Here we characterize the role of ACE2, AR, MX1, ERG, ETV5 and TMPRSS2 for trying a better classification of COVID-19 through knowledge of the disease mechanisms.MethodsA total of 329 blood samples were genotyped in ACE2, MX1 and TMPRSS2. RNA analyses were also performed from 258 available samples using quantitative polymerase chain reaction for genes: ERG, ETV5, AR, MX1, ACE2, and TMPRSS2. Moreover, in silico analysis variant effect predictor, ClinVar, IPA, DAVID, GTEx, STRING and miRDB database was also performed. Clinical and demographic data were recruited from all participants following WHO classification criteria.ResultsWe confirm the use of ferritin (p < 0.001), D-dimer (p < 0.010), CRP (p < 0.001) and LDH (p < 0.001) as markers for distinguishing mild and severe cohorts. Expression studies showed that MX1 and AR are significantly higher expressed in mild vs severe patients (p < 0.05). ACE2 and TMPRSS2 are involved in the same molecular process of membrane fusion (p = 4.4 × 10–3), acting as proteases (p = 0.047).ConclusionsIn addition to the key role of TMPSRSS2, we reported for the first time that higher expression levels of AR are related with a decreased risk of severe COVID-19 disease in females. Moreover, functional analysis demonstrates that ACE2, MX1 and TMPRSS2 are relevant markers in this disease.Graphical abstract

Reversible binding of divalent cations to Ductin protein assemblies-A putative new regulatory mechanism of membrane traffic processes.

Ductins are a family of homologous and structurally similar membrane proteins with 2 or 4 trans-membrane alpha-helices. The active forms of the Ductins are membranous ring- or star-shaped oligomeric assemblies and they provide various pore, channel, gap-junction functions, assist in membrane fusion processes and also serve as the rotor c-ring domain of V-and F-ATPases. All functions of the Ductins have been reported to be sensitive to the presence of certain divalent metal cations (Me2+), most frequently Cu2+ or Ca2+ ions, for most of the better known members of the family, and the mechanism of this effect is not yet known. Given that we have earlier found a prominent Me2+ binding site in a well-characterised Ductin protein, we hypothesise that certain divalent cations can structurally modulate the various functions of Ductin assemblies via affecting their stability by reversible non-covalent binding to them. A fine control of the stability of the assembly ranging from separated monomers through a loosely/weakly to tightly/strongly assembled ring might render precise regulation of Ductin functions possible. The putative role of direct binding of Me2+ to the c-ring subunit of active ATP hydrolase in autophagy and the mechanism of Ca2+-dependent formation of the mitochondrial permeability transition pore are also discussed.

Inter-neuronal signaling mediated by small extracellular vesicles: wireless communication?

Conventional inter-neuronal communication conceptualizes the wired method of chemical synapses that physically connect pre-and post-synaptic neurons. In contrast, recent studies indicate that neurons also utilize synapse-independent, hence "wireless" broadcasting-type communications via small extracellular vesicles (EVs). Small EVs including exosomes are secreted vesicles released by cells and contain a variety of signaling molecules including mRNAs, miRNAs, lipids, and proteins. Small EVs are subsequently absorbed by local recipient cells via either membrane fusion or endocytic processes. Therefore, small EVs enable cells to exchange a "packet" of active biomolecules for communication purposes. It is now well established that central neurons also secrete and uptake small EVs, especially exosomes, a type of small EVs that are derived from the intraluminal vesicles of multivesicular bodies. Specific molecules carried by neuronal small EVs are shown to affect a variety of neuronal functions including axon guidance, synapse formation, synapse elimination, neuronal firing, and potentiation. Therefore, this type of volume transmission mediated by small EVs is thought to play important roles not only in activity-dependent changes in neuronal function but also in the maintenance and homeostatic control of local circuitry. In this review, we summarize recent discoveries, catalog neuronal small EV-specific biomolecules, and discuss the potential scope of small EV-mediated inter-neuronal signaling.

The SARS-CoV-2 fusion domain preferentially initiates fusion in the late endosome.

SARS-CoV-2 can enter target cells via both the plasma and endosomal membranes. This ability to utilize either pathway increases the likelihood by which viral entry will succeed in delivering the viral genetic information, a vital component of the viral lifecycle. Most other viruses only utilize one or the other fusion pathway, partly due to vastly different environmental factors such as pH and membrane lipid composition. The coronavirus spike glycoprotein is responsible for facilitating viral entry by first binding to the receptor followed by membrane fusion, the latter of which is initiated by the fusion domain (FD). Improving our understanding of how the FD initiates fusion in both pathways, and whether or not one is preferred over the other, will help to elucidate the molecular mechanisms involved in the membrane fusion process of SARS-CoV-2. Through a FRET-based lipid mixing assay, the SARS-CoV-2 FD (S816-F855) displays a biphasic relationship between pH and fusion, with a pH similar to that of the late endosome (∼pH5.5-4.5) demonstrating the highest fusogenic activity. Intriguingly, when assessing the structure of the FD at pH7.4 and pH5.0 in dodecylphosphocholine, only minor conformational changes occur. This change in conformation results in the elongation of an α-helix within the fusion peptide, which alongside the fusion loop, are the two structural components of the FD that are found to be critical for full functionality of the peptide. Furthermore, the FD also favors the lipid environment of the late endosome to elicit fusion, with a specificity for the endosomal lipid BMP observed. In conclusion, the SARS-CoV-2 FD displays an α-helical extension at low pH when compared to neutral pH, that allows it to preferentially initiate fusion in a lipid and pH environment resembling that of the late endosome.

Study of fusion peptide release for the spike protein of SARS-CoV-2.

The spike protein of SARS-CoV-2 can recognize the ACE2 membrane protein on the host cell and plays a key role in the membrane fusion process between the virus envelope and the host cell membrane. However, to date, the mechanism for the spike protein recognizing host cells and initiating membrane fusion remains unknown. In this study, based on the general assumption that all three S1/S2 junctions of the spike protein are cleaved, structures with different forms of S1 subunit stripping and S2' site cleavage were constructed. Then, the minimum requirement for the release of the fusion peptide was studied by all-atom structure-based MD simulations. The results from simulations showed that stripping an S1 subunit from the A-, B- or C-chain of the spike protein and cleaving the specific S2' site on the B-chain (C-chain or A-chain) may result in the release of the fusion peptide, suggesting that the requirement for the release of FP may be more relaxed than previously expected.

Complexins: Ubiquitously Expressed Presynaptic Regulators of SNARE-Mediated Synaptic Vesicle Fusion.

Neurotransmitter release is a spatially and temporally tightly regulated process, which requires assembly and disassembly of SNARE complexes to enable the exocytosis of transmitter-loaded synaptic vesicles (SVs) at presynaptic active zones (AZs). While the requirement for the core SNARE machinery is shared by most membrane fusion processes, SNARE-mediated fusion at AZs is uniquely regulated to allow very rapid Ca2+-triggered SV exocytosis following action potential (AP) arrival. To enable a sub-millisecond time course of AP-triggered SV fusion, synapse-specific accessory SNARE-binding proteins are required in addition to the core fusion machinery. Among the known SNARE regulators specific for Ca2+-triggered SV fusion are complexins, which are almost ubiquitously expressed in neurons. This chapter summarizes the structural features of complexins, models for their molecular interactions with SNAREs, and their roles in SV fusion.

Cell-Cell Fusion Assays to Study Henipavirus Entry and Evaluate Therapeutics.

Henipaviruses include the deadly zoonotic Nipah (NiV) and Hendra (HeV) paramyxoviruses, which have caused recurring outbreaks in human populations. A hallmark of henipavirus infection is the induction of cell-cell fusion (syncytia), caused by the expression of the attachment (G) and fusion (F) glycoproteins on the surface of infected cells. The interactions of G and F with each other and with receptors on cellular plasma membranes drive both viral entry and syncytia formation and are thus of great interest. While F shares structural and functional homologies with class I fusion proteins of other viruses such as influenza and human immunodeficiency viruses, the intricate interactions between the G and F glycoproteins allow for unique approaches to studying the class I membrane fusion process. This allows us to study cell-cell fusion and viral entry kinetics for BSL-4 pathogens such as NiV and HeV under BSL-2 conditions using recombinant DNA techniques. Here, we present approaches to studying henipavirus-induced membrane fusion for currently identified and emerging henipaviruses, including more traditional syncytia counting-based cell-cell fusion assay and a new heterologous fluorescent dye exchange cell-cell fusion assay.

LY6E level associated with smoking as risk for lung cancer patients susceptible to COVID-19

Studies revealed that cancer patients seemed more susceptible to COVID-19 and the clinical symptoms were serious. A recent study depicted that lymphocyte antigen 6 complex, locus E (LY6E) might inhibit coronavirus entry into cells by interfering with the membrane fusion process mediated through spike protein, and potently restricting the SARS-CoV-2 (Severe acute respiratory syndrome-associated coronavirus 2) infection. LY6E mRNA level in lung cancer was detected by publicly available datasets. Patient-specific features were used to analyze the potential factors that could affect LY6E level. Analysis of association between LY6E level and immune infiltration was also performed. In present study, it was found that smoker with lung adenocarcinoma showed lower LY6E level than non-smoker (p &lt; 0.05). In LUSC (lung squamous cell carcinoma) patients, reformed smokers showed higher LY6E than smokers (p &lt; 0.05). These results suggested that smoke can be a risk susceptible to COVID-19 in lung cancer patients. Further studies exhibited that LY6E was positively associated with immune cell infiltration in lung adenocarcinoma, indicating that LY6E may influence the infection severity of COVID-19 in lung cancer patients. In summary, smoke may downregulate LY6E level and exacerbate infection and deterioration of COVID-19 in lung cancer patients.

Efficient liposome fusion to phase-separated giant vesicles

Lateral phase heterogeneity in biomembranes can govern cellular functions and may serve as a platform for enrichment or depletion of membrane-anchored molecules. In this work, we address the question of how the process of membrane fusion is affected by the membrane phase state (fluid or gel) and by phase coexistence, as well as the effects of fusion-mediated incorporation of exogeneous lipids on phase separation. Our system is based on the fusion of cationic fluid large unilamellar vesicles (LUVs) composed of dioleoyl trimethylammonium propane (DOTAP) and dioleoyl phosphoethanolamine (DOPE) with neutral and anionic giant unilamellar vesicles (GUVs) composed of phosphatidylcholine and phosphatidylglycerol. By changing the lipid composition of the GUVs, we modulated the phase state and charge of the different phases (charged or neutral, fluid or gel) and identified systems in which we can target fusion to specific domains on phase-separated membranes. Fusion efficiency was quantified using fluorescence microscopy-based lipid and content mixing assays, and flow chamber devices were used to assess the real-time sequence of events of the fusion process. To investigate the bilayer thermal behavior, differential scanning calorimetry (DSC) experiments were performed on LUVs. The results show that fusion is extensive in single-component GUVs only for fluid and negatively charged acceptor membranes. On the other hand, in phase-separated GUVs, high fusion efficiency was observed even when the gel phase was anionic and phase separation somewhat increased the fusion efficiency. Extensive fusion led to dissolution of the gel domains as a result of extensive incorporation of lipids in the fluid state from the fusogenic liposomes. Altogether, these findings have the potential to unravel the important role of membrane phase state, phase separation, charge, and the effects of extensive fusion on membrane organization and may give insights in the regulation of the interactions between cells and liposomes that are used in drug delivery systems.

Ulf Diederichsen (1963–2021)

“For he was our own! … Fruitful both in counsel and deed; this we experienced and appreciated.” With the death of Ulf Diederichsen on November 11, 2021, after a short but severe illness, we lost a great scientist in chemical biology and good friend. He was a generous and extremely gifted man whose strength was to tackle essential biological problems at a molecular level with simple model systems and to draw insightful conclusions realizing a hallmark of chemical biology by addressing biological challenges with chemical approaches derived from simple principles. Ulf Diederichsen was born on October 7, 1963, in Munich and grew up in Göttingen, enjoying excellent scholar education at the Max-Planck Gymnasium. He moved to Freiburg im Breisgau in 1983 to study Chemistry at the Albert-Ludwigs-University. For his doctorate he moved to Zurich in 1988 to work with Prof. Eschenmoser at the ETH on the question why Nature chose pentose—but not hexose nucleic acids. He received the Dr. rer. nat. in 1993 on the Base pairing of hypoxanthine in HOMO DNA oligonucleotides and the question of the pairing behavior of glucopyranosyl oligonucleotides. He then joined the group of Prof. Dennis P. Curran at the University of Pennsylvania (Pittsburgh/USA) as postdoctoral researcher (1993–1994) investigating radical chemistry. Back to Europe he started his habilitation on “Linear nucleic acid analogues with peptidic backbones” under the mentorship of Prof. Horst Kessler at the Technical University of Munich, which was finished in 1999. He then joined the University of Würzburg as Professor of Organic Chemistry, and finally, in 2001, the University of Göttingen. He served as Dean (2005 to 2007) and Vice President for Research (2015 to 2021). In addition, he was Guest Professor at the Ludwig-Maximilians-University in Munich (1998–1999) and Goering Visiting Professor at the University of Wisconsin, USA (2000). He was elected member of the Academy of Sciences in Göttingen in 2012 and became its president in 2020. His scientific interest was mainly centered on the non-covalent interaction of peptide helices with respect to the incorporation, organization, and dynamics in lipid membranes. He successfully employed spin-labeled peptides for distance measurements. Similarly, he addressed the membrane attachment and fusion processes of SNARE proteins with model systems (conjugates of transmembrane peptides with coiled coil peptides or peptide nucleic acids). He was interested in functionalized β-peptides as artificial matrices for aggregation and modification of model membranes. The excellent results of this outstanding research were summarized in largely over 100 publications in highly renowned international journals. Besides his exceptional scientific achievements and admirable mentorship for a large number of doctoral students and postdocs, he deserves high recognition for his excellent research management particularly as Vice President of the University of Göttingen. He also served as a highly esteemed reviewer in various panels, including the German Research Foundation. The large experience collected in this important activity, particularly also for the German peptide community, he adapted in a very successful manner also in his editorial engagement as Deputy Editor of the Journal of Peptide Science since 2012, where he was supposed to take over the full responsibility as the Editor-in-Chief from 2022. Those of us who knew him are saddened by his sudden passing away and mourn the great loss of a dear friend and colleague. He will always be remembered as an inspiring teacher and mentor, as an enthusiastic scientist, but first and foremost as a warm and sincere human being, as a “feiner Kerl.”

Studying membrane fusion using supported lipid bilayers on superparamagnetic beads

The fusion between two lipid membranes is a ubiquitous mechanism in cell traffic and pathogens invasion. Yet it is not well understood how two distinct bilayers overcome the energy barriers towards fusion and reorganize themselves to form a unique continuous bilayer. The magnitudes and numbers of these energy barriers are themselves an open question. To tackle these issues, we developed a new tool that allows to control the forces applied between two supported lipid bilayers (SLBs) deposited on superparamagnetic beads. By applying a magnetic field, the beads self-organize along field lines in chains of beads and compress the two membranes on the contact zone. Using the diffusion of fluorescently labelled lipids from one bilayer to the other allows us to identify fusion of the bilayers in contact. We applied increasing forces on SLBs and increased the occurrence of fusion. This experimental system allows the simultaneous study of tens of facing bilayers in a single experiment and mitigates the stochasticity of the fusion process. It is thus a powerful tool to test the various parameters involved in the membrane fusion process.

Human Claudin-Derived Peptides Block the Membrane Fusion Process of Zika Virus and Are Broad Flavivirus Inhibitors.

ABSTRACTZika virus (ZIKV) is a mosquito-borne flavivirus that emerged in the Pacific islands in 2007 and spread to the Americas in 2015. The infection remains asymptomatic in most cases but can be associated with severe neurological disorders. Despite massive efforts, no specific drug or vaccine against ZIKV infection is available to date. Claudins are tight-junction proteins that favor the entry of several flaviviruses, including ZIKV. In this study, we identified two peptides derived from the N-terminal sequences of claudin-7 and claudin-1, named CL7.1 and CL1.1, respectively, that inhibited ZIKV infection in a panel of human cell lines. Using cell-to-cell fusion assays, we demonstrated that these peptides blocked the ZIKV E-mediated membrane fusion. A comparison of the antiviral efficacy of CL1.1 and CL7.1 pointed to the importance of the peptide amphipathicity. Electron microscopic analysis revealed that CL1.1 altered the ultrastructure of the viral particles likely by binding the virus lipid envelope. However, amphipathicity could not fully explain the antiviral activity of CL1.1. In silico docking simulations suggested that CL1.1 may also interact with the E protein, near its stem region. Overall, our data suggested that claudin-derived peptides inhibition may be linked to simultaneous interaction with the E protein and the viral lipid envelope. Finally, we found that CL1.1 also blocked infection by yellow fever and Japanese encephalitis viruses but not by HIV-1 or SARS-CoV-2. Our results provide a basis for the future development of therapeutics against a wide range of endemic and emerging flaviviruses.IMPORTANCE Zika virus (ZIKV) is a flavivirus transmitted by mosquito bites that have spread to the Pacific Islands and the Americas over the past decade. The infection remains asymptomatic in most cases but can cause severe neurological disorders. ZIKV is a major public health threat in areas of endemicity, and there is currently no specific antiviral drug or vaccine available. We identified two antiviral peptides deriving from the N-terminal sequences of claudin-7 and claudin-1 with the latter being the most effective. These peptides block the envelope-mediated membrane fusion. Our data suggested that the inhibition was likely achieved by simultaneously interacting with the viral lipid envelope and the E protein. The peptides also inhibited other flaviviruses. These results could provide the basis for the development of therapies that might target a wide array of flaviviruses from current epidemics and possibly future emergences.

Structural and Mechanistic Evidence for Calcium Interacting Sites in the HIV Transmembrane Protein gp41 Involved in Membrane Fusion.

The HIV envelope protein gp160 comprises two subunits, gp120 and gp41, responsible for receptor binding and membrane fusion during viral entry, respectively. In the course of the membrane fusion process, gp41 undergoes a conformational change, leading to the formation of a six-helix bundle (SHB), which ultimately drives membrane fusion. The gp41 C-terminal and N-terminal heptad repeats (CHR and NHR) interact with one another to form the SHB, and this step can be targeted by peptide inhibitors, which are used in the clinic to mitigate HIV infection. Here, we discover the calcium interaction motifs (CIMs) in the gp41 CHR and NHR regions via NMR spectroscopy. We find that the assembly of the CHR–NHR SHB is facilitated in Ca2+-containing media and impaired in CIM mutants. Of note, the clinically approved, gp41-derived fusion inhibitor T20, which does not contain the CIM motif, exhibits reduced inhibitory efficiency when challenged with calcium. This finding could have important implications for the development of better fusion inhibitors for HIV.

Effect of Slp4-a on Membrane Bending During Prefusion of Vesicles in Blood-Brain Barrier.

Vesicle exocytosis is a promising pathway for brain drug delivery through the blood-brain barrier to treat neurodegenerative diseases. In vesicle exocytosis, the membrane fusion process is initiated by the calcium sensor protein named synaptotagmin-like protein4-a (Slp4-a). Understanding conformational changes of Slp4-a during the prefusion stage of exocytosis will help to develop vesicle-based drug delivery to the brain. In this work, we use molecular dynamics (MD) simulations with a hybrid force field coupling united-atom protein model with MARTINI coarse-grained (CG) solvent to capture the conformational changes of Slp4-a during the prefusion stage. These hybrid coarse-grained simulations are more efficient than all-atom MD simulations and can capture protein interactions and conformational changes. Our simulation results show that the calcium ions play critical roles during the prefusion stage. Only one calcium ion can remain in each calcium-binding pocket of Slp4-a C2 domains. The C2B domain of calcium-unbound Slp4-a remains parallel to the endothelial membrane, while the C2B domain of calcium-bound Slp4-a rotates perpendicular to the endothelial membrane to approach the vesicular membrane. For the calcium-bound case, three Slp4-a proteins can effectively bend lipid membranes at the prefusion stage, which could later trigger lipid stalk between membranes. This work provides a better understanding how C2 domains of Slp4-a operate during vesicle exocytosis from an endothelial cell.

Membrane destabilization and pore formation induced by the Synechocystis IM30 protein

The inner membrane-associated protein of 30 kDa (IM30) is essential in chloroplasts and cyanobacteria. The spatio-temporal cellular localization of the protein appears to be highly dynamic and triggered by internal as well as external stimuli, mainly light intensity. The soluble fraction of the protein is localized in the cyanobacterial cytoplasm or the chloroplast stroma, respectively. Additionally, the protein attaches to the thylakoid membrane as well as to the chloroplast inner envelope or the cyanobacterial cytoplasmic membrane, respectively, especially under conditions of membrane stress. IM30 is involved in thylakoid membrane biogenesis and/or maintenance, where it either stabilizes membranes and/or triggers membrane-fusion processes. These apparently contradicting functions have to be tightly controlled and separated spatiotemporally in chloroplasts and cyanobacteria. IM30’s fusogenic activity depends on Mg2+ binding to IM30; yet, it still is unclear how Mg2+-loaded IM30 interacts with membranes and promotes membrane fusion. Here, we show that the interaction of Mg2+ with IM30 results in increased binding of IM30 to native, as well as model, membranes. Via atomic force microscopy in liquid, IM30-induced bilayer defects were observed in solid-supported bilayers in the presence of Mg2+. These structures differ dramatically from the membrane-stabilizing carpet structures that were previously observed in the absence of Mg2+. Thus, Mg2+-induced alterations of the IM30 structure switch the IM30 activity from a membrane-stabilizing to a membrane-destabilizing function, a crucial step in membrane fusion.

Visualization of conformational changes and membrane remodeling leading to genome delivery by viral class-II fusion machinery

Chikungunya virus (CHIKV) is a human pathogen that delivers its genome to the host cell cytoplasm through endocytic low pH-activated membrane fusion mediated by class-II fusion proteins. Though structures of prefusion, icosahedral CHIKV are available, structural characterization of virion interaction with membranes has been limited. Here, we have used cryo-electron tomography to visualize CHIKV’s complete membrane fusion pathway, identifying key intermediary glycoprotein conformations coupled to membrane remodeling events. Using sub-tomogram averaging, we elucidate features of the low pH-exposed virion, nucleocapsid and full-length E1-glycoprotein’s post-fusion structure. Contrary to class-I fusion systems, CHIKV achieves membrane apposition by protrusion of extended E1-glycoprotein homotrimers into the target membrane. The fusion process also features a large hemifusion diaphragm that transitions to a wide pore for intact nucleocapsid delivery. Our analyses provide comprehensive ultrastructural insights into the class-II virus fusion system function and direct mechanistic characterization of the fundamental process of protein-mediated membrane fusion.

Interaction of Lassa virus fusion and membrane proximal peptides with late endosomal membranes

Mammarenaviruses include many significant worldwide-widespread human pathogens, among them Lassa virus (LASV), having a dramatic morbidity and mortality rate. They are a potential high-risk menace to the worldwide public health since there are no treatments and there is a high possibility of animal-to-human and human-to-human viral transmission. These viruses enter into the cells by endocytosis fusing its membrane envelope with the late endosomal membrane thanks to the glycoprotein GP2, a membrane fusion protein of class I. This protein contains different domains, among them the N-terminal fusion peptide (NFP), the internal fusion loop (IFL), the membrane proximal external region (MPER) and the transmembrane domain (TMD). All these domains are implicated in the membrane fusion process. In this work, we have used an all-atom molecular dynamics study to know the binding of these protein domains with a complex membrane mimicking the late endosome one. We show that the NFP/IFL domain is capable of spontaneously inserting into the membrane without a significant change of secondary structure, the MPER domain locates at the bilayer interface with an orientation parallel to the membrane surface and tends to interact with other MPER domains, and the TMD domain tilts inside the bilayer. Moreover, they predominantly interact with negatively charged phospholipids. Overall, these membrane-interacting domains would characterise a target that would make possible to find effective antiviral molecules against LASV in particular and Mammarenaviruses in general.

Exploring the pH dependence of the SARS‐CoV‐2 complete fusion domain and the role of its unique structural features

SARS‐CoV‐2 may enter target cells through the process of membrane fusion at either the plasma (~pH 7.4–7.0) or endosomal (~pH 6.5–5.0) membrane in order to deliver its genetic information. The fusion domain (FD) of the spike glycoprotein is responsible for initiating fusion and is thus integral to the viral life cycle. The FD of SARS‐CoV‐2 is unique in that it consists of two structurally distinctive regions referred to as the fusion peptide (FP) and the fusion loop (FL); yet the molecular mechanisms behind how this FD perturbs the membrane to initiate fusion remains unclear. In this study via solution NMR, we witnessed only a slight conformational change in the FD between pH 7.4 and pH 5.0, resulting in a minor elongation of helix 1. However, we found that the FD's ability to mediate membrane fusion has a large and significant pH dependence, with fusion events being more readily induced at low pH. Interestingly, a biphasic relationship between the environmental pH and fusogenicity was discovered, suggesting a preference for the FD to initiate fusion at the late endosomal membrane. Furthermore, the conserved disulfide bond and hydrophobic motif “LLF” were found to be critical for the function of the complete FD, with minimal activity witnessed when either was perturbed. In conclusion, these findings indicate that the SARS‐CoV‐2 FD preferably initiates fusion at a pH similar to the late endosome through a mechanism that heavily relies on the internal disulfide bond of the FL and hydrophobic LLF motif within the FP.