Engineering small extracellular vesicles with multivalent DNA probes for precise tumor targeting and enhanced synergistic therapy.

Small extracellular vesicles (sEVs) have gained wide attention as efficient carriers for disease treatment. However, the proclivity of sEVs to be ingested by source cells is insufficient to accurately target specific sites, posing a challenge in realizing controlled targeting treatment. Here, we developed an engineered sEV nanocarrier capable of precise tumor targeting and enhanced synergistic therapy. Multivalent DNA probes, comprising abundant AS1411 aptamers and telomerase primers, were innovatively modified on the sEV membrane (M-D-sEV) for precise tumor targeting. To achieve synergistic therapy, gold nanorod-cerium oxide nanostructures (Au NRs-CeO2) and manganese dioxide nanosheets-doxorubicin (MnO2 NSs-DOX) were encapsulated into liposomes (Lip-Mat). Then M-D-sEV and Lip-Mat were fused together through membrane fusion to obtain nanocarriers. Owing to the multivalence of the probes, the surface of the nanocarriers was loaded with numerous aptamers, which greatly enhances their targeting ability and promotes the accumulation of drugs. When nanocarriers were ingested by tumor cells, telomerase and multivalent DNA probes triggered their aggregation, enhancing the therapeutic effect. Furthermore, under laser irradiation, Au NRs-CeO2 converted light into hyperthermia, thereby inducing the destruction of nanocarriers membrane. This process initiated a series of reactions involving glutathione and H2O2 consumption, as well as DOX release, ultimately achieving synergistic tumor therapy. In vitro and in vivo studies demonstrated the remarkable targeting ability of multivalent DNA probes and excellent therapeutic effect of this strategy. The engineered strategy of sEVs provide a promising approach for precise tumor therapy and hold great potential for the development of efficient, safe, and personalized drug delivery systems.

Conformational dynamics of SARS-CoV-2 Omicron spike trimers during fusion activation at single molecule resolution.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron entry involves spike (S) glycoprotein-mediated fusion of viral and late endosomal membranes. Here, using single-molecule Förster resonance energy transfer (sm-FRET) imaging and biochemical measurements, we directly visualized conformational changes of individual spike trimers on the surface of SARS-CoV-2 Omicron pseudovirions during fusion activation. We observed that the S2 domain of the Omicron spike is a dynamic fusion machine. S2 reversibly interchanges between the pre-fusion conformation and two previously undescribed intermediate conformations. Acidic pH shifts the conformational equilibrium of S2 toward an intermediate conformation and promotes the membrane hemi-fusion reaction. Moreover, we captured conformational reversibility in the S2 domain, which suggests that spike can protect itself from pre-triggering. Furthermore, we determined that Ca2+ directly promotes the S2 conformational change from an intermediate conformation to post-fusion conformation. In the presence of a target membrane, low pH and Ca2+ stimulate the irreversible transition to S2 post-fusion state and promote membrane fusion.

Lipid remodeling in acrosome exocytosis: unraveling key players in the human sperm.

It has long been thought that exocytosis was driven exclusively by well-studied fusion proteins. Some decades ago, the role of lipids became evident and escalated interest in the field. Our laboratory chose a particular cell to face this issue: the human sperm. What makes this cell special? Sperm, as terminal cells, are characterized by their scarcity of organelles and the complete absence of transcriptional and translational activities. They are specialized for a singular membrane fusion occurrence: the exocytosis of the acrosome. This unique trait makes them invaluable for the study of exocytosis in isolation. We will discuss the lipids' role in human sperm acrosome exocytosis from various perspectives, with a primary emphasis on our contributions to the field. Sperm cells have a unique lipid composition, very rare and not observed in many cell types, comprising a high content of plasmalogens, long-chain, and very-long-chain polyunsaturated fatty acids that are particular constituents of some sphingolipids. This review endeavors to unravel the impact of membrane lipid composition on the proper functioning of the exocytic pathway in human sperm and how this lipid dynamic influences its fertilizing capability. Evidence from our and other laboratories allowed unveiling the role and importance of multiple lipids that drive exocytosis. This review highlights the role of cholesterol, diacylglycerol, and particular phospholipids like phosphatidic acid, phosphatidylinositol 4,5-bisphosphate, and sphingolipids in driving sperm acrosome exocytosis. Furthermore, we provide a comprehensive overview of the factors and enzymes that regulate lipid turnover during the exocytic course. A more thorough grasp of the role played by lipids transferred from sperm can provide insights into certain causes of male infertility. It may lead to enhancements in diagnosing infertility and techniques like assisted reproductive technology (ART).

Human parainfluenza virus 3 field strains undergo extracellular fusion protein cleavage to activate entry.

Human parainfluenza virus 3 (HPIV3) infection is driven by the coordinated action of viral surface glycoproteins hemagglutinin-neuraminidase (HN) and fusion protein (F). Receptor-engaged HN activates F to insert into the target cell membrane and drive virion-cell membrane fusion. For F to mediate entry, its precursor (F0) must first be cleaved by host proteases. F0 cleavage has been thought to be executed during viral glycoprotein transit through the trans-Golgi network by the ubiquitously expressed furin because F0 proteins of laboratory-adapted viruses contain a furin recognition dibasic cleavage motif RXKR around residue 108. Here, we show that the F proteins of field strains have a different cleavage motif from laboratory-adapted strains and are cleaved by unidentified proteases expressed in only a narrow subset of cell types. We demonstrate that extracellular serine protease inhibitors block HPIV3 F0 cleavage for field strains, suggesting F0 cleavage occurs at the cell surface facilitated by transmembrane proteases. Candidate proteases that may process HPIV3 F in vivo were identified by a genome-wide CRISPRa screen in HEK293/dCas9-VP64 + MPH cells. The lung-expressed extracellular serine proteases TMPRSS2 and TMPRSS13 are both sufficient to cleave HPIV3 F and enable infectious virus release by otherwise non-permissive cells. Our findings support an alternative mechanism of F activation in vivo, reliant on extracellular membrane-bound serine proteases expressed in a narrow subset of cells. The proportion of HPIV3 F proteins cleaved and infectious virus release is determined by host cell expression of requisite proteases, allowing just-in-time activation of F and positioning F cleavage as another key regulator of HPIV3 spread. IMPORTANCE: Enveloped viruses cause a wide range of diseases in humans. At the first step of infection, these viruses must fuse their envelope with a cell membrane to initiate infection. This fusion is mediated by viral proteins that require a critical activating cleavage event. It was previously thought that for parainfluenza virus 3, an important cause of respiratory disease and a representative of a group of important pathogens, this cleavage event was mediated by furin in the cell secretory pathways prior to formation of the virions. We show that this is only true for laboratory strain viruses, and that clinical viruses that infect humans utilize extracellular proteases that are only made by a small subset of cells. These results highlight the importance of studying authentic clinical viruses that infect human tissues for understanding natural infection.

All-in-One Fusogenic Nanoreactor for the Rapid Detection of Exosomal MicroRNAs for Breast Cancer Diagnosis.

Molecular-profiling-based cancer diagnosis has significant implications for predicting disease prognosis and selecting targeted therapeutic interventions. The analysis of cancer-derived extracellular vesicles (EVs) provides a noninvasive and sequential method to assess the molecular landscape of cancer. Here, we developed an all-in-one fusogenic nanoreactor (FNR) encapsulating DNA-fueled molecular machines (DMMs) for the rapid and direct detection of EV-associated microRNAs (EV miRNAs) in a single step. This platform was strategically designed to interact selectively with EVs and induce membrane fusion under a specific trigger. After fusion, the DMMs recognized the target miRNA and initiated nonenzymatic signal amplification within a well-defined reaction volume, thus producing an amplified fluorescent signal within 30 min. We used the FNRs to analyze the unique expression levels of three EV miRNAs in various biofluids, including cell culture, urine, and plasma, and obtained an accuracy of 86.7% in the classification of three major breast cancer (BC) cell lines and a diagnostic accuracy of 86.4% in the distinction between patients with cancer and healthy donors. Notably, a linear discriminant analysis revealed that increasing the number of miRNAs from one to three improved the accuracy of BC patient discrimination from 78.8 to 95.4%. Therefore, this all-in-one diagnostic platform performs nondestructive EV processing and signal amplification in one step, providing a straightforward, accurate, and effective individual EV miRNA analysis strategy for personalized BC treatment.

Molecular Basis of VCPIP1 and P97/VCP Interaction Reveals Its Functions in Post-Mitotic Golgi Reassembly.

The VCPIP1-P97/VCP (Valosin-Containing Protein) complex is required for post-mitotic Golgi cisternae reassembly and maintenance in interphase. However, the organization and mechanism of this complex in regulating Golgi membrane fusion is still elusive. Here, the cryo-electron microscopy (cryo-EM) structures of the human VCPIP1-P97/VCP complex are presented. These studies reveal that three independent VCPIP1 molecules sit over the C-terminal substrate exit tunnel formed by P97/VCP homo-hexamer, resulting in an unusual C3 to C6 symmetric barrel architecture. The UFD1 (unknown function domain 1) from VCPIP1, but not the N-terminal OTU domain and the C-terminal UBL domain, docks to the two adjacent D2 domains of P97/VCP, allosterically causing the cofactors binding domain-NTDs (N-terminal domains) of P97/VCP in a "UP" and D1 domain in an ATPase competent conformation. Conversely, VCPIP1 bound P97/VCP hexamer favors the binding of P47, and thus the intact SNARE complex, promoting Golgi membrane fusion. These studies not only reveal the unexpected organization of humanVCPIP1-P97/VCP complex, but also provide new insights into the mechanism of VCPIP1-P97/VCP mediated Golgi apparatus reassembly, which is a fundamental cellular event for protein and lipid processing.

Extracellular vesicles-hitchhiking boosts the deep penetration of drugs to amplify anti-tumor efficacy.

Developing drug delivery systems capable of achieving deep tumor penetration is a challenging task, yet there is a significant demand for such systems in cancer treatment. Hitchhiking on tumor-derived extracellular vesicles (EVs) represents a promising strategy for enhancing drug penetration into tumors. However, the limited drug assembly on EVs restricts its further application. Here, we present a novel approach to efficiently attach antitumor drugs to EVs using an engineered cell membrane-based vector. This vector includes the AS1411 aptamer for tumor-specific targeting, the vesicular stomatitis virus glycoprotein (VSV-G) for tumor cell membrane fusion, and a photosensitizer as the therapeutic agent while ensuring optimal drug encapsulation and stability. Upon injection, photosensitizers are firstly transferred to the tumor cell membrane and subsequently piggybacked onto EVs with the inherent secretion process. By hitchhiking with EVs, photosensitizers can be transferred layer by layer deep into the solid tumors. The results suggest that this EVs-hitchhiking strategy enables photosensitizers to penetrate deeply into tumor tissue, thereby enhancing the efficacy of phototherapy. This study offers broad application prospects for delivering drugs deeply into tumor tissues.

Mitochondrial membrane potential and oxidative stress interact to regulate Oma1-dependent processing of Opa1 and mitochondrial dynamics.

Mitochondrial form and function are regulated by the opposing forces of mitochondrial dynamics: fission and fusion. Mitochondrial dynamics are highly active and consequential during neuronal ischemia/reperfusion (I/R) injury. Mitochondrial fusion is executed at the mitochondrial inner membrane by Opa1. The balance of long (L-Opa1) and proteolytically cleaved short (S-Opa1) isoforms is critical for efficient fusion. Oma1 is the predominant stress-responsive protease for Opa1 processing. In neuronal cell models, we assessed Oma1 and Opa1 regulation during mitochondrial stress. In an immortalized mouse hippocampal neuron line (HT22), Oma1 was sensitive to mitochondrial membrane potential depolarization (rotenone, FCCP) and hyperpolarization (oligomycin). Further, oxidative stress was sufficient to increase Oma1 activity and necessary for depolarization-induced proteolysis. We generated Oma1 knockout (KO) HT22 cells that displayed normal mitochondrial morphology and fusion capabilities. FCCP-induced mitochondrial fragmentation was exacerbated in Oma1 KO cells. However, Oma1 KO cells were better equipped to perform restorative fusion after fragmentation, presumably due to preserved L-Opa1. We extended our investigations to a combinatorial stress of neuronal oxygen-glucose deprivation and reoxygenation (OGD/R), where we found that Opa1 processing and Oma1 activation were initiated during OGD in an ROS-dependent manner. These findings highlight a novel dependence of Oma1 on oxidative stress in response to depolarization. Further, we demonstrate contrasting fission/fusion roles for Oma1 in the acute response and recovery stages of mitochondrial stress. Collectively, our results add intersectionality and nuance to the previously proposed models of Oma1 activity.

Lipid Selectivity of Membrane Action of the Fragments of Fusion Peptides of Marburg and Ebola Viruses.

The life cycle of Ebola and Marburg viruses includes a step of the virion envelope fusion with the cell membrane. Here, we analyzed whether the fusion of liposome membranes under the action of fragments of fusion peptides of Ebola and Marburg viruses depends on the composition of lipid vesicles. A fluorescence assay and electron microscopy were used to quantify the fusogenic activity of the virus fusion peptides and to identify the lipid determinants affecting membrane merging. Differential scanning calorimetry of lipid phase transitions revealed alterations in the physical properties of the lipid matrix produced by virus fusion peptides. Additionally, we found that plant polyphenols, quercetin, and myricetin inhibited vesicle fusion induced by the Marburg virus fusion peptide.

Identification of SLC25A46 interaction interfaces with mitochondrial membrane fusogens Opa1 and Mfn2.

Mitochondrial fusion requires the sequential merger of four bilayers to two. The outer-membrane solute carrier family 25 member (SLC25A46) interacts with both the outer and inner membrane dynamin family GTPases mitofusin 1/2 and optic atrophy 1 (Opa1). While SLC25A46 levels are known to affect mitochondrial morphology, how SLC25A46 interacts with mitofusin 1/2 and Opa1 to regulate membrane fusion is not understood. In this study, we use crosslinking mass spectrometry and AlphaFold 2 modeling to identify interfaces mediating an SLC25A46 interaction with Opa1 and Mfn2. We reveal that the bundle signaling element of Opa1 interacts with SLC25A46, and present evidence of an Mfn2 interaction involving the SLC25A46 cytosolic face. We validate these newly identified interaction interfaces and show that they play a role in mitochondrial network maintenance.

Harringtonine metabolites: 5'-de-O-methylharringtonine and cephalotaxine, targeting spike protein and TMPRSS2 to double block membrane fusion of SARS-CoV-2 and its variants.

Membrane fusion is the main pathway for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) to invade host cells. Harringtonine (HT), derived from cephalotaxus fortunei Hook. f., has been recognized as an effective antagonist of SARS-CoV-2. It can directly block the active binding of spike (S) protein to host angiotensin converting enzyme 2 (ACE2), as well as hinder the enzymolysis of transmembrane serine proteases 2 (TMPRSS2). This study examined the potential of HT metabolites, 5'-de-O-methylharringtonine and cephalotaxine, as the membrane fusion inhibitors for SARS-CoV-2. 5'-De-O-methylharringtonine was synthesized and subsequently characterized by high resolution mass spectrometry and nuclear magnetic resonance to be structurally consistent, with a purity of 92.677% determined by reverse phase high performance liquid chromatography. Both 5'-de-O-methylharringtonine and cephalotaxine can specifically bind to SARS-CoV-2 S protein and TMPRSS2 using cell membrane chromatography. They can form hydrogen bonds with key sites that correlated highly with the enhanced binding affinity of SARS-CoV-2 and its variants to ACE2 or nafamostat to TMPRSS2. Moreover, 5'-de-O-methylharringtonine and cephalotaxine can inhibit pseudotyped virus entry and membrane fusion in a dose-dependent manner, with enhanced effectiveness upon elevated expression of TMPRSS2. Importantly, they displayed low cytotoxic effects on human normal cell lines. Our study suggested that 5'-de-O-methylharringtonine and cephalotaxine were of low toxicity and safety for humans as potential antagonists of SARS-CoV-2 and its variants, which deserve further validation in a biosafety level 3 facility.

Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation.

Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus utilizes glycoprotein B (gB) to fuse the viral and host cell membranes upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion state whereby the central trimeric coiled-coils, formed by domain (Dom)III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking or one disulfide bond-breaking mutation in the DIII α3 to study different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations, gB_Y494P and gB_I495P, were lethal for the virus in either fibroblasts or epithelial cells. The third substitution, gB_G493P, on the other hand, displayed a delayed replication and spread, which was more pronounced in epithelial cells, hinting at an impaired fusion. Interestingly, the disulfide bond-breaker mutation, gB_C507S, performed strikingly differently in the two cell types - lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency, the spread morphology, and the cell-cell fusogenicity suggest a dysregulated fusion process, which could be reverted by second-site mutations mapping predominantly to gB DomV. Our findings underline the functional importance of a stable DomIII core for a well-regulated DomV rearrangement during fusion.IMPORTANCEHuman cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course; however, it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. The virus uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and glycoprotein B (gB). The fusion protein gB undergoes fundamental rearrangements from a metastable pre-fusion to a stable post-fusion conformation. Here, we characterized the viral behavior after the introduction of four single-point mutations in the gB central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the development of a structurally and functionally altered gB, which can further serve as a tool for drug and vaccine development.

Lys716 in the transmembrane domain of yeast mitofusin Fzo1 modulates anchoring and fusion.

Outer mitochondrial membrane fusion, a vital cellular process, is mediated by mitofusins. However, the underlying molecular mechanism remains elusive. We have performed extensive multiscale molecular dynamics simulations to predict a model of the transmembrane (TM) domain of the yeast mitofusin Fzo1. Coarse-grained simulations of the two TM domain helices, TM1 and TM2, reveal a stable interface, which is controlled by the charge status of residue Lys716. Atomistic replica-exchange simulations further tune our model, which is confirmed by a remarkable agreement with an independent AlphaFold2 (AF2) prediction of Fzo1 in complex with its fusion partner Ugo1. Furthermore, the presence of the TM domain destabilizes the membrane, even more if Lys716 is charged, which can be an asset for initiating fusion. The functional role of Lys716 was confirmed with yeast experiments, which show that mutating Lys716 to a hydrophobic residue prevents mitochondrial fusion.

[Preparation of hybrid exosomes based on bovine milk exosomes and liposomes and pharmacodynamic study of sinomenine loaded exosomes in treatment of rheumatoid arthritis].

This study investigates the therapeutic effect of hybrid exosomes loaded with sinomenine(SIN) obtained by membrane fusion of milk exosomes with liposomes in collagen-induced arthritis(CIA) rats. Exosomes were isolated from fresh bovine milk by sucrose density gradient centrifugation, while liposomes were prepared using the emulsion solvent evaporation-low temperature curing method. Hybrid exosomes were characterized after membrane fusion through co-incubation: The morphology was detected by transmission electron microscopy, the particle size and potential by nanoparticle size potentiostat, and the expressions of surface characteristic proteins CD63 and TSG101 before and after fusion by Western blot(WB). The drug loading capacity and encapsulation rate of sinomenine were measured after the loading of sinomenine on exosomes by ultrasonic method. The CIA rat model was induced by collagen antibody. The efficacy experiment consisted of the control group, model group, SIN group, SIN-liposome group, SIN-milk exosome group, SIN-hybrid exosome group and positive drug(dexamethasone) group. The changes in body mass of rats during administration were recorded. Besides, the foot swelling, immune organ index, arthritis index, microcirculation index, synovial histopathology, and serum inflammatory factor levels detected by enzyme-linked immunosorbent assay were observed for pharmacodynamical study. Under transmission electron microscopy, both hybrid exosomes and milk exosomes showed saucer-like appearance. After co-incubation, the exosome particle size increased from(97.92±3.42)nm to(132.70±4.07)nm, and the Zeta potential changed from(-2.01±0.33)mV to(-17.90±2.13)mV. WB assay showed that CD63 and TSG101 proteins were normally expressed in milk exosomes and hybrid exosomes. The encapsulation rate of milk exosomes was 31.64%±2.48%, with a drug loading of 2.35%±0.52%, while the hybrid exosomes exhibited an encapsulation rate of 48.21%±3.12% and drug loading of 3.17%±0.36%, as determined by the microplate reader. Pharmacodynamic results showed that compared with the model group, the general condition, swelling degree of foot, arthritis index and immune organ index of all drug administration groups were significantly improved(P<0.05, P<0.01); microvascular comprehensive score and vascular resistance were significantly decreased(P<0.05, P<0.01); serum levels of TNF-α, IL-1ß and IL-6 inflammatory factors were significantly decreased(P<0.01); and the lesions of synovial tissue were improved to some extent. Meanwhile, compared with the SIN group, SIN-liposome group and SIN-milk exosome group, the SIN-hybrid exosome group had a more stable and durable drug effect. The hybrid exosomes obtained by co-incubation of milk-derived exosomes with liposomes successfully improved the drug carrying capacity of exosomes and biocompatibility of liposomes. The hybrid exosomes loaded with sinomenine have good efficacy on CIA model rats, and can effectively solve the problems of TCM such as sinomenine, which have good efficacy but short biological half-life. The study provides new insights for the development of TCM and the treatment of diseases such as rheumatoid arthritis.

A single amino acid substitution in the Borna disease virus glycoprotein enhances the infectivity titer of vesicular stomatitis virus pseudotyped virus by altering membrane fusion activity.

Borna disease virus 1 (BoDV-1) causes acute fatal encephalitis in mammals, including humans. Despite its importance, research on BoDV-1 cell entry has been hindered by low infectious viral particle production in cells and the lack of cytopathic effects, which are typically useful for screening. To address these issues, we developed a method to efficiently produce vesicular stomatitis virus (VSV) pseudotyped with glycoprotein (G) of members of the genus Orthobornavirus, including BoDV-1. We discovered that optimal G expression is required to obtain a high infectivity titer of the VSV pseudotyped virus. Remarkably, the infectivity of the VSV pseudotyped virus with G from the BoDV-1 strain huP2br was significantly higher than that of the VSV pseudotyped virus with G from the He/80 strain. Mutational analysis demonstrated that the methionine at BoDV-1-G residue 307 increases the infectivity titer of VSV pseudotyped with BoDV-1-G (VSV-BoDV-1-G). A cell‒cell fusion assay indicated that this residue plays a pivotal role in membrane fusion, thus suggesting that high membrane fusion activity and a broad pH range for membrane fusion are crucial for achieving a high infectivity titer of VSV-BoDV-1-G. This finding may be extended to increase the infectivity titer of VSV pseudotyped virus with other orthobornavirus G. Our study also contributes to identifying functional domains of BoDV-1-G and provides insight into G-mediated cell entry.

Low pinocytic brain endothelial cells primarily utilize membrane fusion to internalize extracellular vesicles.

Extracellular vesicles (EVs) are an emerging class of drug carriers and are primarily reported to be internalized into recipient cells via a combination of endocytic routes such as clathrin-mediated, caveolae-mediated and macropinocytosis pathways. In this work, (1) we investigated potential effects of homotypic vs. heterotypic interactions by studying the cellular uptake of homologous EVs (EV donor cells and recipient cells of the same type) vs. heterologous EVs (EV donor cells and recipient cells of different types) and (2) determined the route of EV internalization into low pinocytic/hard-to-deliver cell models such as brain endothelial cells (BECs) and phagocytic cell model as macrophages. Homotypic interactions led to a greater extent of uptake into the recipient BECs compared to heterotypic interactions. However, we did not see a complete reduction in EV uptake into recipient BECs when endocytic pathways were blocked using pharmacological inhibitors and our findings from a R18-based fusion assay suggest that EVs primarily use membrane fusion to enter low-pinocytic recipient BECs instead of relying on endocytosis. Lipophilic PKH67 dye-labeled EVs but not intravesicular esterase-activated calcein ester-labeled EVs severely reduced particle uptake into BECs while phagocytic macrophages internalized both types of EV-labeled particles to comparable extents. Our results also highlight the importance of carefully choosing labeling dye chemistry to study EV uptake, especially in the case of low pinocytic cells such as BECs.

Allosteric mechanism of membrane fusion activation in a herpesvirus.

Herpesviridae infect nearly all humans for life, causing diseases that range from painful to life-threatening1. These viruses penetrate cells by employing a complex apparatus composed of separate receptor-binding, signal-transmitting, and membrane-fusing components2. But how these components coordinate their functions is unknown. Here, we determined the 4.19-angstrom cryoEM reconstruction of the central signal-transmitting component from herpes simplex virus 2, the gH/gL complex, in its elusive pre-activation state. Analysis of the continuum of conformational ensembles observed in cryoEM data revealed a series of structural rearrangements in gH/gL that allosterically transmit the fusion-triggering signal from the receptor-binding glycoprotein gD to the membrane fusogen gB. Furthermore, we identified a structural "switch" element in gH/gL that refolds and flips 180 degrees during the transition from pre-activation to activated form. Conservation of this "switch" in gH/gL homologs suggests that the proposed fusion triggering mechanism may apply to all Herpesviridae and points to a new target for subunit-based vaccines and treatment efforts.

Potential Effects of Hyperglycemia on SARS-CoV-2 Entry Mechanisms in Pancreatic Beta Cells.

The COVID-19 pandemic has revealed a bidirectional relationship between SARS-CoV-2 infection and diabetes mellitus. Existing evidence strongly suggests hyperglycemia as an independent risk factor for severe COVID-19, resulting in increased morbidity and mortality. Conversely, recent studies have reported new-onset diabetes following SARS-CoV-2 infection, hinting at a potential direct viral attack on pancreatic beta cells. In this review, we explore how hyperglycemia, a hallmark of diabetes, might influence SARS-CoV-2 entry and accessory proteins in pancreatic ß-cells. We examine how the virus may enter and manipulate such cells, focusing on the role of the spike protein and its interaction with host receptors. Additionally, we analyze potential effects on endosomal processing and accessory proteins involved in viral infection. Our analysis suggests a complex interplay between hyperglycemia and SARS-CoV-2 in pancreatic ß-cells. Understanding these mechanisms may help unlock urgent therapeutic strategies to mitigate the detrimental effects of COVID-19 in diabetic patients and unveil if the virus itself can trigger diabetes onset.

A look beyond the QR code of SNARE proteins.

Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor (SNARE) proteins catalyze the fusion process of vesicles with target membranes in eukaryotic cells. To do this, they assemble in a zipper-like fashion into stable complexes between the membranes. Structural studies have shown that the complexes consist of four different helices, which we subdivide into Qa-, Qb-, Qc-, and R-helix on the basis of their sequence signatures. Using a combination of biochemistry, modeling and molecular dynamics, we investigated how the four different types are arranged in a complex. We found that there is a matching pattern in the core of the complex that dictates the position of the four fundamental SNARE types in the bundle, resulting in a QabcR complex. In the cell, several different cognate QabcR-SNARE complexes catalyze the different transport steps between the compartments of the endomembrane system. Each of these cognate QabcR complexes is compiled from a repertoire of about 20 SNARE subtypes. Our studies show that exchange within the four types is largely tolerated structurally, although some non-cognate exchanges lead to structural imbalances. This suggests that SNARE complexes have evolved for a catalytic mechanism, a mechanism that leaves little scope for selectivity beyond the QabcR rule.

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

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike is the fusion machine for host cell entry. Still, the mechanism by which spike protein interacts with the target lipid membrane to facilitate membrane fusion during entry is not fully understood. Here, using steady-state membrane fusion and single-molecule fluorescence resonance energy transfer imaging of spike trimers on the surface of SARS-CoV-2 pseudovirion, we directly show that spike protein interacts with phosphatidylserine (PS) lipid in the target membrane for mediating fusion. We observed that the fusion peptide of the spike S2 domain interacts with the PS lipid of the target membrane. Low pH and Ca2+ trigger the spike conformational change and bring fusion peptide in close proximity to the PS lipid of the membrane. The binding of the spike with PS lipid of its viral membrane (cis interaction) impedes the fusion activation. PS on the target membrane promotes spike binding via trans interaction, prevents the cis interaction, and accelerates fusion. Sequestering or absence of PS lipid abrogates the spike-mediated fusion process and restricts SARS-CoV-2 infectivity. We found that PS-dependent interaction for fusion is conserved across all the SARS-CoV-2 spike variants of concern (D614G, Alpha, Beta, Delta, and Omicron). Our study suggests that PS lipid is indispensable for SARS-CoV-2 spike-mediated virus and target membrane fusion for entry, and restricting PS interaction with spike inhibits the SARS-CoV-2 spike-mediated entry. Therefore, PS is an important cofactor and acts as a molecular beacon in the target membrane for SARS-CoV-2 entry. IMPORTANCE: 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.