Fusion Pore Research Articles

Maximal Fusion Capacity and Efficient Replenishment of the Dense Core Vesicle Pool in Hippocampal Neurons.

Neuropeptides and neurotrophins, stored in dense core vesicles (DCVs), are together the largest currently known group of chemical signals in the brain. Exocytosis of DCVs requires high-frequency or patterned stimulation, but the determinants to reach maximal fusion capacity and for efficient replenishment of released DCVs are unknown. Here, we systematically studied fusion of DCV with single vesicle resolution on different stimulation patterns in mammalian CNS neurons. We show that tetanic stimulation trains of 50-Hz action potential (AP) bursts maximized DCV fusion, with significantly fewer fusion event during later bursts of the train. This difference was omitted by introduction of interburst intervals but did not increase total DCV fusion. Interburst intervals as short as 5 s were sufficient to restore the fusion capacity. Theta burst stimulation (TBS) triggered less DCV fusion than tetanic stimulation, but a similar fusion efficiency per AP. Prepulse stimulation did not alter this. However, low-frequency stimulation (4 Hz) intermitted with fast ripple stimulation (200 APs at 200 Hz) produced substantial DCV fusion, albeit not as much as tetanic stimulation. Finally, individual fusion events had longer durations with more intense stimulation. These data indicate that trains of 50-Hz AP stimulation patterns triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.SIGNIFICANCE STATEMENT Neuropeptides and neurotrophins modulate multiple regulatory functions of human body like reproduction, food intake or mood. They are packed into dense core vesicles (DCVs) that undergo calcium and action potential (AP) fusion with the plasma membrane. In order to study the fusion of DCVs in vitro, techniques like perfusion with buffer containing high concentration of potassium or electric field stimulation are needed to trigger the exocytosis of DCVs. Here, we studied the relationship between DCVs fusion properties and different electric field stimulation patterns. We used six different stimulation patterns and showed that trains of 50-Hz action potential bursts triggered DCV exocytosis most efficiently and more intense stimulation promotes longer DCV fusion pore openings.

Mechanisms of SNARE proteins in membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of small conserved eukaryotic proteins that mediate membrane fusion between organelles and with the plasma membrane. SNAREs are directly or indirectly anchored to membranes. Prior to fusion, complementary SNAREs assemble between membranes with the aid of accessory proteins that provide a scaffold to initiate SNARE zippering, pulling the membranes together and mediating fusion. Recent advances have enabled the construction of detailed models describing bilayer transitions and energy barriers along the fusion pathway and have elucidated the structures of SNAREs complexed in various states with regulatory proteins. In this Review, we discuss how these advances are yielding an increasingly detailed picture of the SNARE-mediated fusion pathway, leading from first contact between the membranes via metastable non-bilayer intermediates towards the opening and expansion of a fusion pore. We describe how SNARE proteins assemble into complexes, how this assembly is regulated by accessory proteins and how SNARE complexes overcome the free energy barriers that prevent spontaneous membrane fusion.

Host heparan sulfate promotes ACE2 super-cluster assembly and enhances SARS-CoV-2-associated syncytium formation

SARS-CoV-2 infection causes spike-dependent fusion of infected cells with ACE2 positive neighboring cells, generating multi-nuclear syncytia that are often associated with severe COVID. To better elucidate the mechanism of spike-induced syncytium formation, we combine chemical genetics with 4D confocal imaging to establish the cell surface heparan sulfate (HS) as a critical stimulator for spike-induced cell-cell fusion. We show that HS binds spike and promotes spike-induced ACE2 clustering, forming synapse-like cell-cell contacts that facilitate fusion pore formation between ACE2-expresing and spike-transfected human cells. Chemical or genetic inhibition of HS mitigates ACE2 clustering, and thus, syncytium formation, whereas in a cell-free system comprising purified HS and lipid-anchored ACE2, HS stimulates ACE2 clustering directly in the presence of spike. Furthermore, HS-stimulated syncytium formation and receptor clustering require a conserved ACE2 linker distal from the spike-binding site. Importantly, the cell fusion-boosting function of HS can be targeted by an investigational HS-binding drug, which reduces syncytium formation in vitro and viral infection in mice. Thus, HS, as a host factor exploited by SARS-CoV-2 to facilitate receptor clustering and a stimulator of infection-associated syncytium formation, may be a promising therapeutic target for severe COVID.

Fusion pore dynamics of large secretory vesicles define a distinct mechanism of exocytosis.

Exocrine cells utilize large secretory vesicles (LSVs) up to 10 μm in diameter. LSVs fuse with the apical surface, often recruiting actomyosin to extrude their content through dynamic fusion pores. The molecular mechanism regulating pore dynamics remains largely uncharacterized. We observe that the fusion pores of LSVs in the Drosophila larval salivary glands expand, stabilize, and constrict. Arp2/3 is essential for pore expansion and stabilization, while myosin II is essential for pore constriction. We identify several Bin-Amphiphysin-Rvs (BAR) homology domain proteins that regulate fusion pore expansion and stabilization. We show that the I-BAR protein Missing-in-Metastasis (MIM) localizes to the fusion site and is essential for pore expansion and stabilization. The MIM I-BAR domain is essential but not sufficient for localization and function. We conclude that MIM acts in concert with actin, myosin II, and additional BAR-domain proteins to control fusion pore dynamics, mediating a distinct mode of exocytosis, which facilitates actomyosin-dependent content release that maintains apical membrane homeostasis during secretion.

Elucidating the role of pulse shaping on defects formation in aluminum alloys fabricated by powder bed fusion

The Powder Bed Fusion (PBF) additive manufacturing technique, although widely studied and implemented in the industry, still presents issues of porosity and lack of fusion even when optimal processing parameters are used. The benefits and drawbacks of PBF utilizing pulsed lasers are both consequences of the transient temperature field induced by laser-matter interaction. During laser processing with nanosecond pulse durations, intense heating and cooling rates are induced, setting the melt pool dimensions, which are directly related to the occurrence of lack of fusion, and ablation of the processed material, leading to recoil pressure that induces the keyhole effect, increasing the occurrence of porosity. Consequently, controlling the transient temperature field is a major challenge in incorporating pulsed wave laser technology into PBF to minimize porosity and lack of fusion defects. In this study, to reduce porosity and lack of fusion, a smooth heating rate able to sinter powder grains is designed to facilitate heat flow through the heat affected zone, followed by a sharper heating rate that generates a fully molten region while reducing ablation at the same time. This work represents the first attempt to control the transient temperature field, by shaping a nanosecond laser pulse in the time domain, thereby affecting the formation of defects. The effects of the pulse shape on build quality quantified by lack of fusion and high-energy spherical pores were investigated using X-ray microtomography, molecular dynamics (MD), X-ray diffraction (XRD), Electron Backscatter Diffraction (EBSD), and finite element simulations. The variation of porosity and lack of fusion within manufactured samples were revealed by the high-resolution 3D X-ray micro computed tomography. The differences in the transient temperature field, resulting residual stresses, and microstructure are attributed to the differences in the energy delivery rates within the time domain shaped laser pulses. Laser pulse shaping is an additional parameter that plays an important role in optimizing the PBF as-processed part's production opening a new research niche.

Turbocharging synaptic transmission.

Evidence from biochemistry, genetics, and electron microscopy strongly supports the idea that a ring of Synaptotagmin is central to the clamping and release of synaptic vesicles (SVs) for synchronous neurotransmission. Recent direct measurements in cell-free systems suggest there are 12 SNAREpins in each ready-release vesicle, consisting of six peripheral and six central SNAREpins. The six central SNAREpins are directly bound to the Synaptotagmin ring, are directly released by Ca++ , and they initially open the fusion pore. The six peripheral SNAREpins are indirectly bound to the ring, each linked to a central SNAREpin by a bridging molecule of Complexin. We suggest that the primary role of peripheral SNAREpins is to provide additional force to 'turbocharge' neurotransmitter release, explaining how it can occur much faster than other forms of membrane fusion. The SV protein Synaptophysin forms hexamers that bear two copies of the v-SNARE VAMP at each vertex, one likely assembling into a peripheral SNAREpin and the other into a central SNAREpin.

Optical tomography and machine learning for in-situ defects detection in laser powder bed fusion: A self-organizing map and U-Net based approach

The inherently stochastic nature of the laser powder bed fusion (LPBF) process presents a significant challenge in developing dependable and efficient defect detection algorithms that can flexibly adjust to a variety of machine configurations and process parameters. The present study proposes a machine learning-based approach that employs the optical tomography (OT) data acquired during LPBF for identifying defects, specifically lack of fusion and keyhole porosity. This novel approach includes a machine learning framework consisting of a self-organizing map (SOM) and a custom U-Net model, to predict porosity effectively and automatically across different process parameter choices, making it robust and computationally efficient. The proposed approach was validated with various sets of experiments, including analyzing the influence of process parameter changes, as well as intentionally and randomly mimicking these defects. These defects predicted by the developed model are validated using computed tomography (CT)-scanning to evaluate the algorithm’s performance. The proposed approach effectively predicted porosity caused by lack of fusion or keyhole for different process parameter choices by comparing the similarity based on Euclidean distance between the normalized porosity curves for CT scan and the developed model using Dynamic Time Wrapping (DTW) technique, leading to an average distance score of 0.243 for randomized lack of fusion pores and an average score of 0.6 for randomized keyhole pores. These results demonstrate the effectiveness of the proposed approach in predicting defects during the LPBF process, and its potential to improve in-situ monitoring and quality assurance.

Three membrane fusion pore families determine the pathway to pore dilation

During exocytosis secretory vesicles fuse with a target membrane and release neurotransmitters, hormones, or other bioactive molecules through a membrane fusion pore. The initially small pore may subsequently dilate for full contents release, as commonly observed in amperometric traces. The size, shape, and evolution of the pore is critical to the course of contents release, but exact fusion pore solutions accounting for membrane tension and bending energy constraints have not been available. Here, we obtained exact solutions for fusion pores between two membranes. We find three families: a narrow pore, a wide pore, and an intermediate tether-like pore. For high tensions these are close to the catenoidal and tether solutions recently reported for freely hinged membrane boundaries. We suggest membrane fusion initially generates a stable narrow pore, and the dilation pathway is a transition to the stable wide pore family. The unstable intermediate pore is the transition state that sets the energy barrier for this dilation pathway. Pore dilation is mechanosensitive, as the energy barrier is lowered by increased membrane tension. Finally, we study fusion pores in nanodiscs, powerful systems for the study of individual pores. We show that nanodiscs stabilize fusion pores by locking them into the narrow pore family.

HIV Infection: Shaping the Complex, Dynamic, and Interconnected Network of the Cytoskeleton.

HIV-1 has evolved a plethora of strategies to overcome the cytoskeletal barrier (i.e., actin and intermediate filaments (AFs and IFs) and microtubules (MTs)) to achieve the viral cycle. HIV-1 modifies cytoskeletal organization and dynamics by acting on associated adaptors and molecular motors to productively fuse, enter, and infect cells and then traffic to the cell surface, where virions assemble and are released to spread infection. The HIV-1 envelope (Env) initiates the cycle by binding to and signaling through its main cell surface receptors (CD4/CCR5/CXCR4) to shape the cytoskeleton for fusion pore formation, which permits viral core entry. Then, the HIV-1 capsid is transported to the nucleus associated with cytoskeleton tracks under the control of specific adaptors/molecular motors, as well as HIV-1 accessory proteins. Furthermore, HIV-1 drives the late stages of the viral cycle by regulating cytoskeleton dynamics to assure viral Pr55Gag expression and transport to the cell surface, where it assembles and buds to mature infectious virions. In this review, we therefore analyze how HIV-1 generates a cell-permissive state to infection by regulating the cytoskeleton and associated factors. Likewise, we discuss the relevance of this knowledge to understand HIV-1 infection and pathogenesis in patients and to develop therapeutic strategies to battle HIV-1.

Processing defect, microstructure evolution and mechanical properties of laser powder bed fusion Al-12Si alloys

The laser power and scan speed are significant processing parameters influencing the defects and microstructures due to affecting the thermal state within the melt pool during laser powder bed fusion (LPBF). To this end, these process parameter combinations under a constant laser energy density are correlated with defects (balling phenomenon and pores) and microstructures (melt pool arrangement and cell morphology). Setting the parameter combination (laser power and scan speed are 200 W and 910 mm/s, respectively) yields large balling droplets (average size of 150 μm) due to the relatively high melt viscosity resulting from the low melt pool temperature. In this case, a limited surface tension difference acting on a liquid surface cannot provide sufficient driving forces against viscous drags to promote the efficient spreading of the molten alloy. Cavity regions are formed between the large balling droplets and the previous layer, forming lack-of -fusion pores. By increasing the laser power and scan speed gradually, samples with relative densities greater than 99.5% are produced. This phenomenon can be attributed to the increases in laser power and scan speed, which improves the spreading ability of the molten alloy. With increasing laser power and scan speed, the cell size decreases (from 0.51 μm to 0.42 μm) because of the increase in the cooling rate. The sample (laser power and scan speed are 350 W and 1590 mm/s, respectively) has the highest performance (310 MPa yield strength, 470 MPa ultimate tensile strength, and 6.6% fracture strain) due to its highest relative density (greater than 99.5%) and fine microstructure (average cell size of 0.45 μm). All four samples exhibit anisotropic mechanical properties resulting from the combination of the melt pool boundary and lack-of-fusion pores.

Ca2+ and cAMP open differentially dilating synaptic fusion pores.

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. Here, we used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then show that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP).

α-Synuclein is required for sperm exocytosis at a post-fusion stage.

The sperm acrosome is a large dense-core granule whose contents are secreted by regulated exocytosis at fertilization through the opening of numerous fusion pores between the acrosomal and plasma membranes. In other cells, the nascent pore generated when the membrane surrounding a secretory vesicle fuses with the plasma membrane may have different fates. In sperm, pore dilation leads to the vesiculation and release of these membranes, together with the granule contents. α-Synuclein is a small cytosolic protein claimed to exhibit different roles in exocytic pathways in neurons and neuroendocrine cells. Here, we scrutinized its function in human sperm. Western blot revealed the presence of α-synuclein and indirect immunofluorescence its localization to the acrosomal domain of human sperm. Despite its small size, the protein was retained following permeabilization of the plasma membrane with streptolysin O. α-Synuclein was required for acrosomal release, as demonstrated by the inability of an inducer to elicit exocytosis when permeabilized human sperm were loaded with inhibitory antibodies to human α-synuclein. The antibodies halted calcium-induced secretion when introduced after the acrosome docked to the cell membrane. Two functional assays, fluorescence and transmission electron microscopies revealed that the stabilization of open fusion pores was responsible for the secretion blockage. Interestingly, synaptobrevin was insensitive to neurotoxin cleavage at this point, an indication of its engagement in cis SNARE complexes. The very existence of such complexes during AE reflects a new paradigm. Recombinant α-synuclein rescued the inhibitory effects of the anti-α-synuclein antibodies and of a chimeric Rab3A-22A protein that also inhibits AE after fusion pore opening. We applied restrained molecular dynamics simulations to compare the energy cost of expanding a nascent fusion pore between two model membranes and found it higher in the absence than in the presence of α-synuclein. Hence, our results suggest that α-synuclein is essential for expanding fusion pores.

Intrinsic Disorder in α-Synuclein Regulates the Exocytotic Fusion Pore Transition.

Today, it is widely accepted that intrinsic disorder is strongly related to the cell cycle, during mitosis, differentiation, and apoptosis. Of particular interest are hybrid proteins possessing both structured and unstructured domains that are critical in human health and disease, such as α-synuclein. In this work, we describe how α-synuclein interacts with the nascent fusion pore as it evolves toward expansion. We unveil the key role played by its intrinsically disordered region as a thermodynamic regulator of the nucleation-expansion energy barrier. By analyzing a truncated variant of α-synuclein that lacks the disordered region, we find that the landscape of protein interactions with PIP2 and POPS lipids is highly altered, ultimately increasing the energy cost for the fusion pore to transit from nucleation to expansion. We conclude that the intrinsically disordered region in full-length α-synuclein recognizes and allocates pivotal protein:lipid interactions during membrane remodeling in the first stages of the fusion pore.

Acoustic feature based geometric defect identification in wire arc additive manufacturing

ABSTRACT In additive manufacturing of metals, numerous techniques have been employed to sense print defects. Among these, acoustic-based sensing has the advantage of low cost and shows the most potential to identify both external and internal defects as an in-situ monitoring system. Using acoustic signals, researchers have broadly investigated non-machine learning and machine learning-based approaches to identify defects like balling, micro defects, lack of fusion pores, keyhole pores, cracks, and porosity. While most of these works have shown promising results for laser-based AM systems, few have explored how acoustic signals can be used effectively for Wire Arc Additive Manufacturing (WAAM) defect detection. This paper proposes a methodology to construct machine learning (ML)-based models on identifying geometrically defective bead segments using acoustic signals during the WAAM process. Geometrically defective bead segment or geometric defect is a defect that causes voids in the final printed part due to incomplete fusion between two non-uniform overlapping bead segments. Such a defect is currently not explored in the literature. The proposed methodology uses a novel dataset labeling approach to identify good and bad bead segments based on an optimal threshold of the range of mean curvature. Furthermore, the methodology targets defective bead segments based on acoustic feature inputs like Principal Components (PC) or Mel Frequency Cepstral Coefficients (MFCC). To understand the resulting performance of the defect identification models constructed based on the proposed methodology, experiments are performed and tested on a variety of ML models (KNN, SVM, RF, NN, and CNN) based on the Inconel 718 material. The results show that the combinatorics of two acoustic input features and five ML models can be able to identify geometrically defective segments accurately with F1 score that ranges from 80% to 85%.

Manufacturability study in laser powder bed fusion of biomedical Ti alloys for orthopedic implants: an investigation of mechanical properties, process-induced porosity and surface roughness

PurposeSince the biomedical implants with an improved compressive strength, near bone elastic modulus, controlled porosity, and sufficient surface roughness, can assist in long term implantation. Therefore, the fine process tuning plays its crucial role to develop optimal settings to achieve these desired properties. This paper aims to find applications for fine process tuning in laser powder bed fusion of biomedical Ti alloys for load-bearing implants.Design/methodology/approachIn this work, the parametric porosity simulations were initially performed to simulate the process-induced porosity for selective laser-melted Ti6Al4V as per full factorial design. Continually, the experiments were performed to validate the simulation results and perform multiresponse optimization to fine-tune the processing parameters. Three levels of each control variable, namely, laser power – Pl (180, 190, 200) W, scanning speed – Vs (1500, 1600, 1700) mm/s and scan orientation – ϴ{1(0,0), 2(0,67°), 3(0,90°)} were used to investigate the processing performance. The measured properties from this study include compressive yield strength, elastic modulus, process-induced porosity and surface roughness. Finally, confirmatory experiments and comparisons with the already published works were also performed to validate the research results.FindingsThe results of porosity parametric simulation and experiments in selective laser melting of Ti6Al4V were found close to each other with overall porosity (less than 10%). The fine process tuning was resulted in optimal settings [Pl (200 W), Vs (1500 mm/s), ϴ (0,90°)], [Pl (200 W), Vs (1500 mm/s), ϴ (0,67°)], [Pl (200 W), Vs (1500 mm/s), ϴ (0,0)] and [Pl (200 W), Vs (1500 mm/s), ϴ (0,0)] with higher compressive strength (672.78 MPa), near cortical bone elastic modulus (12.932 GPa), process-induced porosity (0.751%) and minimum surface roughness (2.72 µm). The morphology of the selective laser melted (SLMed) surface indicated that the lack of fusion pores was prominent because of low laser energy density among the laser and powder bed. Confirmatory experimentation revealed that an overall percent improvement of around 15% was found between predicted and the experimental values.Originality/valueSince no significant works are available on the collaborative optimization and fine process tuning in laser powder bed fusion of biomedical Ti alloys for different load bearing implants. Therefore, this work involves the comprehensive investigation and multi-objective optimization to determine optimal parametric settings for better mechanical and physical properties. Another novel aspect is the parametric porosity simulation using Ansys Additive to assist in process parameters and their levels selection. As a result, selective laser melted Ti alloys at optimal settings may help in examining the possibility for manufacturing metallic implants for load-bearing applications.

Potential assessment in laser powder bed fusion of bionic porous Ti scaffolds concerning compressive behavior, porosity, and surface roughness

The fabrication of cellular structures with biocompatible materials for implants has increased in recent years because of human aging and traffic road accidents. Laser powder bed fusion or Selective laser melting (SLM) can fabricate various load-bearing implants with mass customization in design and manufacturing. This work involves a manufacturability study in selective laser melting of biomedical Ti alloy implant plug for Osteoarthritis patients. The investigation involved the control variables like laser power (175,185,195) watt, scanning speed (1100,1250,1400) mm/s, and hatch spacing (0.065,0.0725,0.08) mm alongwith output responses such as Compressive yield strength, Elastic modulus, porosity, and surface roughness. The experiments were performed using Box Behnken design in Response surface modelling for collaborative optimization. The results were explicitly explained with an in-depth process physics supported with Characterization involving Scanning electron microscopy, Electro-dispersive spectroscopy, X-ray diffraction, micro-computed tomography, optical profilometry analyses, and process schematics. The optimal setting with laser power (195 W), scanning speed (1100 mm/s), and hatch spacing (0.068 mm) yielded high compressive yield strength (22.41 MPa), near trabecular bone Elastic modulus (0.891GPa), controlled porosity formation (29.32 %), and optimal surface roughness (3.647 μm). Topography resulted in a prominent lack of fusion pores because of low energy density levels, whereas the optimal settings produced around 15 % improvement from design of experiments data.

High curvature promotes fusion of lipid membranes: Predictions from continuum elastic theory

The fusion of lipid membranes progresses through a series of hemifusion intermediates with two significant energy barriers related to the formation of stalk and fusion pore, respectively. These energy barriers determine the speed and success rate of many critical biological processes, including the fusion of highly curved membranes, for example synaptic vesicles and enveloped viruses. Here we use continuum elastic theory of lipid monolayers to determine the relationship between membrane shape and energy barriers to fusion. We find that the stalk formation energy decreases with curvature by up to 31 kBT in a 20-nm-radius vesicle compared with planar membranes and by up to 8 kBT in the fusion of highly curved, long, tubular membranes. In contrast, the fusion pore formation energy barrier shows a more complicated behavior. Immediately after stalk expansion to the hemifusion diaphragm, the fusion pore formation energy barrier is low (15–25 kBT) due to lipid stretching in the distal monolayers and increased tension in highly curved vesicles. Therefore, the opening of the fusion pore is faster. However, these stresses relax over time due to lipid flip-flop from the proximal monolayer, resulting in a larger hemifusion diaphragm and a higher fusion pore formation energy barrier, up to 35 kBT. Therefore, if the fusion pore fails to open before significant lipid flip-flop takes place, the reaction proceeds to an extended hemifusion diaphragm state, which is a dead-end configuration in the fusion process and can be used to prevent viral infections. In contrast, in the fusion of long tubular compartments, the surface tension does not accumulate due to the formation of the diaphragm, and the energy barrier for pore expansion increases with curvature by up to 11 kBT. This suggests that inhibition of polymorphic virus infection could particularly target this feature of the second barrier.

Part of anti-covid therapy based on a theoretical model describing mechanics of contact interaction between coronavirus and cell membrane

This article proposes a theoretical model of mechanical feedback in pattern formation on deforming a membrane when coronavirus enters a cell. Coronavirus stiff and flexible spike proteins attach to ACE2 receptors on the cell membrane of the target cell. TMPRSS2 triggers the fusion of the viral and cell membranes with the formation of a fusion pore leading to opening of a capsid surrounded by the coronavirus envelope and viral RNA release into the cell. Based on mechanical feedback analysis of contact interaction and elastic shell theory, a critical value of cell radius, at which a fusion pore is formed and membrane damage occurs locally, is determined. The results revealed that the smaller the cell size, the less likely that the cell will be damaged mechanically when exposed to the virus. One of the ways to reduce the cell size is to decrease intracellular fluid volume through the use of medicines - diuretics. The critical value of cell radius is inversely proportional to the value of binding energy at the time of attachment of the coronavirus to the cell membrane. Further research is required to improve our knowledge of the dependency of binding energy on the shape and sizes of spikelike bumps for various types of coronavirus strains. It may be predicted that when a new coronavirus strain will emerge, it may produce lower binding energy to cell surface and the severity of the disease may decrease. It is necessary to verify the conclusions of the theoretical study by experimental methods.

Molecular Modeling of Viral Type I Fusion Proteins: Inhibitors of Influenza Virus Hemagglutinin and the Spike Protein of Coronavirus.

The fusion of viral and cell membranes is one of the basic processes in the life cycles of viruses. A number of enveloped viruses confer fusion of the viral envelope and the cell membrane using surface viral fusion proteins. Their conformational rearrangements lead to the unification of lipid bilayers of cell membranes and viral envelopes and the formation of fusion pores through which the viral genome enters the cytoplasm of the cell. A deep understanding of all the stages of conformational transitions preceding the fusion of viral and cell membranes is necessary for the development of specific inhibitors of viral reproduction. This review systematizes knowledge about the results of molecular modeling aimed at finding and explaining the mechanisms of antiviral activity of entry inhibitors. The first section of this review describes types of viral fusion proteins and is followed by a comparison of the structural features of class I fusion proteins, namely influenza virus hemagglutinin and the S-protein of the human coronavirus.

IFITM3 blocks influenza virus entry by sorting lipids and stabilizing hemifusion

Interferon-induced transmembrane protein 3 (IFITM3) inhibits the entry of numerous viruses through undefined molecular mechanisms. IFITM3 localizes in the endosomal-lysosomal system and specifically affects virus fusion with target cell membranes. We found that IFITM3 induces local lipid sorting, resulting in an increased concentration of lipids disfavoring viral fusion at the hemifusion site. This increases the energy barrier for fusion pore formation and the hemifusion dwell time, promoting viral degradation in lysosomes. In situ cryo-electron tomography captured IFITM3-mediated arrest of influenza A virus membrane fusion. Observation of hemifusion diaphragms between viral particles and late endosomal membranes confirmed hemifusion stabilization as a molecular mechanism of IFITM3. The presence of the influenza fusion protein hemagglutinin in post-fusion conformation close to hemifusion sites further indicated that IFITM3 does not interfere with the viral fusion machinery. Collectively, these findings show that IFITM3 induces lipid sorting to stabilize hemifusion and prevent virus entry into target cells.