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The emergence of SARS-CoV-2 has heightened the need to evaluate the detection of enveloped viruses in the environment, particularly in wastewater, within the context of wastewater-based epidemiology. The studies published over the past 80 years focused primarily on non-enveloped viruses due to their ability to survive longer in environmental matrices such as wastewater or sludge compared to enveloped viruses. However, different enveloped viruses survive in the environment for different lengths of time. Therefore, it is crucial to be prepared to assess the potential infectious risk that may arise from future emerging enveloped viruses. This will require appropriate tools, notably suitable viral concentration methods that do not compromise virus infectivity. This review has a dual purpose: first, to gather all the available literature on the survival of infectious enveloped viruses, specifically at different pH and temperature conditions, and in contact with detergents; second, to select suitable concentration methods for evaluating the infectivity of these viruses in wastewater and sludge. The methodology used in this data collection review followed the systematic approach outlined in the PRISMA (Preferred Reporting Items for Systematic Review and Meta-Analysis) guidelines. Concentration methods cited in the data gathered are more tailored towards detecting the enveloped viruses' genome. There is a lack of suitable methods for detecting infectious enveloped viruses in wastewater and sludge. Ultrafiltration, ultracentrifugation, and polyethylene glycol precipitation methods, under specific/defined conditions, appear to be relevant approaches. Further studies are necessary to validate reliable concentration methods for detecting infectious enveloped viruses. The choice of culture system is also crucial for detection sensitivity. The data also show that the survival of infectious enveloped viruses, though lower than that of non-enveloped ones, may enable environmental transmission. Experimental data on a wide range of enveloped viruses is required due to the variability in virus persistence in the environment.

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Monkeypox (mpox) is an infectious disease caused by the mpox virus and can potentially lead to fatal outcomes. It resembles infections caused by viruses from other families, challenging identification. The pathogenesis, transmission, and clinical manifestations of mpox and other Orthopoxvirus species are similar due to their closely related genetic material. This review provides a comprehensive discussion of the roles of various proteins, including extracellular enveloped virus (EEV), intracellular mature virus (IMV), and profilin-like proteins of mpox. It also highlights recent diagnostic techniques based on these proteins to detect this infection rapidly.

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Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic, or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity to exploit innovative solutions against these hazards. Recently, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs was evaluated against the non-enveloped RNA viruses, hepatitis A virus (HAV) and murine norovirus (MNV), a surrogate for human norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. The results showed that almost all peptides displayed virucidal effects, with about 90% of infectivity reduction in HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles causing their damage or hindering the recognition of cellular receptors. Hence, RiLK1 could represent a versatile antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

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Growing concerns regarding the emergence of highly transmissible viral diseases highlight the urgent need to expand the repertoire of antiviral therapeutics. For this reason, new strategies for neutralizing and inhibiting these viruses are necessary. A promising approach involves targeting the glycans present on the surfaces of enveloped viruses. Lectins, known for their ability to recognize specific carbohydrate molecules, offer the potential for glycan-targeted antiviral strategies. Indeed, numerous studies have reported the antiviral effects of various lectins of both endogenous and exogenous origins. However, many lectins in their natural forms, are not suitable for use as antiviral therapeutics due to toxicity, other unfavorable pharmacological effects, and/or unreliable manufacturing sources. Therefore, improvements are crucial for employing lectins as effective antiviral therapeutics. A novel approach to enhance lectins' suitability as pharmaceuticals could be the generation of recombinant lectin-Fc fusion proteins, termed "lectibodies." In this review, we discuss the scientific rationale behind lectin-based antiviral strategies and explore how lectibodies could facilitate the development of new antiviral therapeutics. We will also share our perspective on the potential of these molecules to transcend their potential use as antiviral agents.

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The coagulation process has a high potential as a treatment method that can handle pathogenic viruses including emerging enveloped viruses in drinking water treatment process which can lower infection risk through drinking water consumption. In this study, a surrogate enveloped virus, bacteriophage Փ6, and surrogate non-enveloped viruses, including bacteriophage MS-2, T4, ՓX174, were used to evaluate removal efficiencies and mechanisms by the conventional coagulation process with alum, poly­aluminum chloride, and ferric chloride at pH 5, 7, and 9 in turbid water. Also, treatability of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a recent virus of global concern by coagulation was evaluated as SARS-CoV-2 can presence in drinking water sources. It was observed that an increase in the coagulant dose enhanced the removal efficiency of turbidity and viruses, and the condition that provided the highest removal efficiency of enveloped and non-enveloped viruses was 50 mg/L of coagulants at pH 5. In addition, the coagulation process was more effective for enveloped virus removal than for the non-enveloped viruses, and it demonstrated reduction of SARS-CoV-2 Omicron BA.2 over 0.83-log with alum. According to culture- and molecular-based assays (qPCR and CDDP-qPCR), the virus removal mechanisms were floc adsorption and coagulant inactivation. Through inactivation with coagulants, coagulants caused capsid destruction, followed by genome damage in non-enveloped viruses; however, damage to a lipid envelope is suggested to contribute to a great extend for enveloped virus inactivation. We demonstrated that conventional coagulation is a promising method for controlling emerging and re-emerging viruses in drinking water.

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Non-enveloped viruses employ unique entry mechanisms to breach and infect host cells. Understanding these mechanisms is crucial for developing antiviral strategies. Prevailing perspective suggests that non-enveloped viruses release membrane lytic peptides to breach host membranes. However, the precise involvement of the viral capsid in this entry remains elusive. Our study presents direct observations elucidating the dynamically distinctive steps through which metastable reovirus capsids disrupt host lipid membranes as they uncoat into partially hydrophobic intermediate particles. Using both live cells and model membrane systems, our key finding is that reovirus capsids actively deform and permeabilize lipid membranes in a cholesterol-dependent process. Unlike membrane lytic peptides, these metastable viral capsids induce more extensive membrane perturbations, including budding, bridging between adjacent membranes, and complete rupture. Notably, cholesterol enhances subviral particle adsorption, resulting in the formation of pores equivalent to the capsid size. This cholesterol dependence is attributed to the lipid condensing effect, particularly prominent at intermediate cholesterol level. Furthermore, our results reveal a positive correlation between membrane disruption extent and efficiency of viral variants in establishing infection. This study unveils the crucial role of capsid-lipid interaction in non-enveloped virus entry, providing new insights into how cholesterol homeostasis influences virus infection dynamics.

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HYPOTHESIS: Enveloped viruses are pivotal in causing various illnesses, including influenza and COVID-19. The antimicrobial peptide LL-37, a critical part of the human innate immune system, exhibits potential as an antiviral agent capable of thwarting these viral threats. Its mode of action involves versatile and non-specific interactions that culminate in dismantling the viral envelope, ultimately rendering the viruses inert. However, the exact mechanism of action is not yet understood. EXPERIMENTS: Here, the mechanism of LL-37 triggered changes in the structure and function of an enveloped virus is investigated. The bacteriophage "Phi6" is used as a surrogate for pathogenic enveloped viruses. Small angle X-ray and neutron scattering combined with light scattering techniques demonstrate that LL-37 actively integrates into the virus's lipid envelope. FINDINGS: LL-37 addition to Phi6 leads to curvature modification in the lipid bilayer, ultimately separating the envelope from the nucleocapsid. Additional biological assays confirm the loss of virus infectivity in the presence of LL-37, which coincides with the structural transformations. The results provide a fundamental understanding of the structure-activity relationship related to enveloped viruses. The knowledge of peptide-virus interactions can guide the design of future peptide-based antiviral drugs and therapies.

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Cryo-electron tomography (cryo-ET) has emerged as a powerful tool in structural biology to study viruses and is undergoing a resolution revolution. Enveloped viruses comprise several RNA and DNA pleomorphic viruses that are pathogens of clinical importance to humans and animals. Considerable efforts in cryogenic correlative light and electron microscopy (cryo-CLEM), cryogenic focused ion beam milling (cryo-FIB), and integrative structural techniques are helping to identify virus structures within cells leading to a rise of in situ discoveries shedding light on how viruses interact with their hosts during different stages of infection. This chapter reviews recent advances in the application of cryo-ET in imaging enveloped viruses and the structural and mechanistic insights revealed studying the viral infection cycle within their eukaryotic cellular hosts, with particular attention to viral entry, replication, assembly, and egress during infection.

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The endosomal sorting complex required for transport (ESCRT) is an essential molecular machinery in eukaryotic cells that facilitates the invagination of endosomal membranes, leading to the formation of multivesicular bodies (MVBs). It participates in various cellular processes, including lipid bilayer remodeling, cytoplasmic separation, autophagy, membrane fission and re-modeling, plasma membrane repair, as well as the invasion, budding, and release of certain enveloped viruses. The ESCRT complex consists of five complexes, ESCRT-0 to ESCRT-III and VPS4, along with several accessory proteins. ESCRT-0 to ESCRT-II form soluble complexes that shuttle between the cytoplasm and membranes, mainly responsible for recruiting and transporting membrane proteins and viral particles, as well as recruiting ESCRT-III for membrane neck scission. ESCRT-III, a soluble monomer, directly participates in vesicle scission and release, while VPS4 hydrolyzes ATP to provide energy for ESCRT-III complex disassembly, enabling recycling. Studies have confirmed the hijacking of ESCRT complexes by enveloped viruses to facilitate their entry, replication, and budding. Recent research has focused on the interaction between various components of the ESCRT complex and different viruses. In this review, we discuss how different viruses hijack specific ESCRT regulatory proteins to impact the viral life cycle, aiming to explore commonalities in the interaction between viruses and the ESCRT system.

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Dengue virus (DENV) is one of the most prevalent arthropod-borne diseases. It may cause dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), while no effective vaccines and drugs are available. Our study demonstrated that conessine exhibits broad antiviral activity against several enveloped viruses, including DENV, vesicular stomatitis virus, and herpes simplex virus. In addition, conessine has no direct destructive effect on the integrity or infectivity of virions. Both pre-treatment and post-treatment with conessine significantly reduce DENV replication. Pre-treatment with conessine disrupts the endocytosis of enveloped viruses, while post-treatment disturbs DENV RNA replication or translation at an early stage. Through screening differentially expressed genes by transcriptome sequencing, we found that conessine may affect cholesterol biosynthesis, metabolism or homeostasis. Finally, we confirmed that conessine inhibits virus replication through up-regulating cholesterol levels. Our work suggests that conessine could be developed as a prophylactic and therapeutic treatment for infectious diseases caused by enveloped viruses.

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Ebola virus (EBOV) causes a hemorrhagic fever with fatality rates up to 90%. The EBOV entry process is complex and incompletely understood. Following attachment to host cells, EBOV is trafficked to late endosomes/lysosomes where its glycoprotein (GP) is processed to a 19-kDa form, which binds to the EBOV intracellular receptor Niemann-Pick type C1. We previously showed that the cathepsin protease inhibitor, E-64d, blocks infection by pseudovirus particles bearing 19-kDa GP, suggesting that further cathepsin action is needed to trigger fusion. This, however, has not been demonstrated directly. Since 19-kDa Ebola GP fusion occurs in late endosomes, we devised a system in which enriched late endosomes are used to prepare supported planar endosomal membranes (SPEMs), and fusion of fluorescent (pseudo)virus particles is monitored by total internal reflection fluorescence microscopy. We validated the system by demonstrating the pH dependencies of influenza virus hemagglutinin (HA)-mediated and Lassa virus (LASV) GP-mediated fusion. Using SPEMs, we showed that fusion mediated by 19-kDa Ebola GP is dependent on low pH, enhanced by Ca2+, and augmented by the addition of cathepsins. Subsequently, we found that E-64d inhibits full fusion, but not lipid mixing, mediated by 19-kDa GP, which we corroborated with the reversible cathepsin inhibitor VBY-825. Hence, we provide both gain- and loss-of-function evidence that further cathepsin action enhances the fusion activity of 19-kDa Ebola GP. In addition to providing new insights into how Ebola GP mediates fusion, the approach we developed employing SPEMs can now be broadly used for studies of virus and toxin entry through endosomes. IMPORTANCE Ebola virus is the causative agent of Ebola virus disease, which is severe and frequently lethal. EBOV gains entry into cells via late endosomes/lysosomes. The events immediately preceding fusion of the viral and endosomal membranes are incompletely understood. In this study, we report a novel in vitro system for studying virus fusion with endosomal membranes. We validated the system by demonstrating the low pH dependencies of influenza and Lassa virus fusion. Moreover, we show that further cathepsin B action enhances the fusion activity of the primed Ebola virus glycoprotein. Finally, this model endosomal membrane system should be useful in studying the mechanisms of bilayer breaching by other enveloped viruses, by non-enveloped viruses, and by acid-activated bacterial toxins.

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New vaccine delivery technologies, such as mRNA, have played a critical role in the rapid and efficient control of SARS-CoV-2, helping to end the COVID-19 pandemic. Enveloped virus-like particles (eVLPs) are often more immunogenic than protein subunit immunogens and could be an effective vaccine platform. Here, we investigated whether the genetic delivery of eVLPs could achieve strong immune responses in mice as previously reported with the immunization of in vitro purified eVLPs. We utilized Newcastle disease virus-like particles (NDVLPs) to display SARS-CoV-2 prefusion-stabilized spikes from the WA-1 or Beta variant (S-2P or S-2Pᵦ, respectively) and evaluated neutralizing murine immune responses achieved by a single-gene-transcript DNA construct for the WA-1 or Beta variant (which we named S-2P-NDVLP-1T and S-2Pᵦ-NDVLP-1T, respectively), by multiple-gene-transcript DNA constructs for the Beta variant (S-2Pᵦ-NDVLP-3T), and by a protein subunit-DNA construct for the WA-1 or Beta variant (S-2P-TM or S-2Pᵦ-TM, respectively). The genetic delivery of S-2P-NDVLP-1T or S-2Pᵦ-NDVLP-1T yielded modest neutralizing responses after a single immunization and high neutralizing responses after a second immunization, comparable to previously reported results in mice immunized with in vitro purified S-2P-NDVLPs. Notably, genetic delivery of S-2Pᵦ-NDVLP-3T yielded significantly higher neutralizing responses in mice after a second immunization than S-2Pᵦ-NDVLP-1T or S-2Pᵦ-TM. Genetic delivery also elicited high spike-specific T-cell responses. Collectively, these results indicate that genetic delivery can provide an effective means to immunize eVLPs and that a multiple-gene transcript eVLP platform may be especially efficacious and inform the design of improved vaccines.

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In a framework aimed at the recovery and enhancement of medicinal plants endemic to the territory of the Cilento and Vallo di Diano National Park, Lavandula austroapennina N.G. Passal., Tundis and Upson has aroused interest. An insight into the chemical composition of the corolla, calyx, leaf, stem, and root organs was carried out following ultrasound-assisted maceration in n-hexane. The obtained lipophilic extracts were explored using ultra-high-performance chromatography coupled to high-resolution mass spectrometry (UHPLC-ESI-QqTOF-MS/MS). The extracts from the different organs varied in their relative content of fatty acids, ursanes, and oleanane-type triterpenes. In particular, the oleanolic acid content appeared to increase in the order of corolla < leaf < stem. An MTT assay was performed to verify the possible cytotoxicity of the organ extracts of L. austroapennina at a concentration ranging from 12.5 to 400 µg/mL on the Vero CCL-81 cell line. Antiviral activity against herpes simplex virus type 1 (HSV-1), alpha human coronavirus 229E (HCoV-229E), and poliovirus type 1 (PV-1) was evaluated via a plaque reduction assay in the same cellular model. All the extracts did not show cytotoxic effects after 2 and 24 h exposure times, and the antiviral efficacy was particularly important for the stem extract, capable of completely inhibiting the tested viruses at low doses. The antiviral activity in a non-enveloped virus PV-1 allowed the assertion that the extracts from the organs of L. austroapennina, and especially the stem extract, interfered directly with the viral envelope. This study underlines how much knowledge of a territory's medicinal plant heritage is a harbinger of promising discoveries in the health field.

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During entry, non-enveloped viruses penetrate a host membrane to cause infection, although how this is accomplished remains enigmatic. Polyomaviruses (PyVs) are non-enveloped DNA viruses that penetrate the endoplasmic reticulum (ER) membrane to reach the cytosol en route to the nucleus for infection. To penetrate the ER membrane, the prototype PyV simian virus 40 (SV40) induces formation of ER-escape sites, called foci, composed of repeating units of multi-tubular ER junctions where the virus is thought to exit. How SV40 triggers formation of the ER-foci harboring these multi-tubular ER junctions is unclear. Here, we show that the ER morphogenic atlastin 2 (ATL2) and ATL3 membrane proteins play critical roles in SV40 infection. Mechanistically, ATL3 mobilizes to the ER-foci where it deploys its GTPase-dependent membrane fusion activity to promote formation of multi-tubular ER junctions within the ER-foci. ATL3 also engages an SV40-containing membrane penetration complex. By contrast, ATL2 does not reorganize to the ER-foci. Instead, it supports the reticular ER morphology critical for the integrity of the ATL3-dependent membrane complex. Our findings illuminate how two host factors play distinct roles in the formation of an essential membrane penetration site for a non-enveloped virus. IMPORTANCE Membrane penetration by non-enveloped viruses, a critical infection step, remains enigmatic. The non-enveloped PyV simian virus 40 (SV40) penetrates the endoplasmic reticulum (ER) membrane to reach the cytosol en route for infection. During ER-to-cytosol membrane penetration, SV40 triggers formation of ER-associated structures (called ER-foci) that function as the membrane penetration sites. Here, we discover a role of the ATL ER membrane proteins-known to shape the ER morphology-during SV40-induced ER-foci formation. These findings illuminate how a non-enveloped virus hijacks host components to construct a membrane penetration structure.

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The development of durable new antiviral therapies is challenging, as viruses can evolve rapidly to establish resistance and attenuate therapeutic efficacy. New compounds that selectively target conserved viral features are attractive therapeutic candidates, particularly for combating newly emergent viral threats. The innate immune system features a sustained capability to combat pathogens through production of antimicrobial peptides (AMPs); however, these AMPs have shortcomings that can preclude clinical use. The essential functional features of AMPs have been recapitulated by peptidomimetic oligomers, yielding effective antibacterial and antifungal agents. Here, we show that a family of AMP mimetics, called peptoids, exhibit direct antiviral activity against an array of enveloped viruses, including the key human pathogens Zika, Rift Valley fever, and chikungunya viruses. These data suggest that the activities of peptoids include engagement and disruption of viral membrane constituents. To investigate how these peptoids target lipid membranes, we used liposome leakage assays to measure membrane disruption. We found that liposomes containing phosphatidylserine (PS) were markedly sensitive to peptoid treatment; in contrast, liposomes formed exclusively with phosphatidylcholine (PC) showed no sensitivity. In addition, chikungunya virus containing elevated envelope PS was more susceptible to peptoid-mediated inactivation. These results indicate that peptoids mimicking the physicochemical characteristics of AMPs act through a membrane-specific mechanism, most likely through preferential interactions with PS. We provide the first evidence for the engagement of distinct viral envelope lipid constituents, establishing an avenue for specificity that may enable the development of a new family of therapeutics capable of averting the rapid development of resistance.

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Viruses can be involved in respiratory disorders in horses, with limited therapeutic options. Citrate-complexed silver nanoparticles (C-AgNP) have shown bactericidal properties after in vitro nebulization. The aim of the present study was to assess the virucidal activity of C-AgNP after in vitro instillation or nebulization on equine herpesvirus-1 (EHV-1) and murine norovirus (MNV), the latter used as surrogate for small non-enveloped viruses. Both viruses were instilled or nebulized with C-AgNP of increasing concentrations, and titres were determined via TCID50 method. We demonstrated efficient inactivation of enveloped EHV-1 following instillation and nebulization of C-AgNP (infectivity losses of ≥ three orders of magnitude). While tenacious MNV was inactivated via 2000 ppm C-AgNP instillation, nebulized C-AgNP did not lead to reduction in MNV titres. Nebulization of C-AgNP may represent a novel virucidal therapeutic approach in horses. Further investigations are needed to assess its safety and effective concentrations for in vivo use.

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Vesicle-encapsulated nonenveloped viruses are a recently recognized alternate form of nonenveloped viruses that can avoid immune detection and potentially increase systemic transmission. Avian orthoreoviruses (ARVs) are the leading cause of various disease conditions among birds and poultry. However, whether ARVs use cellular vesicle trafficking routes for egress and cell-to-cell transmission is still poorly understood. We demonstrated that fusogenic ARV-infected quail cells generated small (~100 nm diameter) extracellular vesicles (EVs) that contained electron-dense material when observed by transmission electron microscope. Cryo-EM tomography indicated that these vesicles did not contain ARV virions or core particles, but the EV fractions of OptiPrep gradients did contain a small percent of the ARV virions released from cells. Western blotting of detergent-treated EVs revealed that soluble virus proteins and the fusogenic p10 FAST protein were contained within the EVs. Notably, virus particles mixed with the EVs were up to 50 times more infectious than virions alone. These results suggest that EVs and perhaps fusogenic FAST-EVs could contribute to ARV virulence.

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Macrophages are critical in the pathogenesis of a diverse group of viral pathogens, both as targets of infection and for eliciting primary defense mechanisms. Our prior in vitro work identified that CD40 signaling in murine peritoneal macrophages protects against several RNA viruses by eliciting IL-12, which stimulates the production of interferon gamma (IFN-γ). Here, we examine the role of CD40 signaling in vivo. We show that CD40 signaling is a critical, but currently poorly appreciated, component of the innate immune response using two distinct infectious agents: mouse-adapted influenza A virus (IAV, PR8) and recombinant VSV encoding the Ebola virus glycoprotein (rVSV-EBOV GP). We find that stimulation of CD40 signaling decreases early IAV titers, whereas loss of CD40 elevated early titers and compromised lung function by day 3 of infection. Protection conferred by CD40 signaling against IAV is dependent on IFN-γ production, consistent with our in vitro studies. Using rVSV-EBOV GP that serves as a low-biocontainment model of filovirus infection, we demonstrate that macrophages are a CD40-expressing population critical for protection within the peritoneum and T-cells are the key source of CD40L (CD154). These experiments reveal the in vivo mechanisms by which CD40 signaling in macrophages regulates the early host responses to RNA virus infection and highlight how CD40 agonists currently under investigation for clinical use may function as a novel class of broad antiviral treatments.

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The surface hydrophobicity of native or engineered non-enveloped viruses and virus-like particles (VLPs) is a key parameter regulating their fate in living and artificial aqueous systems. Its modulation is mainly depending on the structure and environment of particles. Nevertheless, unexplained variations have been reported between structurally similar viruses and with pH. This indicates that some modulating factors of their hydrophobicity remain to be identified. Herein we investigate the potential involvement of RNA cargo in the MS2 phage used as non-enveloped RNA virus model, by examining the SDS-induced electrophoretic mobility shift (SEMS) determined for native MS2 virions and corresponding RNA-free VLPs at various pH. Interestingly, the SEMS of VLPs was larger and more variable from pH 5 to 9 compared to native virions. These observations are discussed in term of RNA-dependent changes in surface hydrophobicity, suggesting that RNA cargo may be a major modulator/regulator of this viral parameter.

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Flaviviruses are enveloped viruses which include human pathogens that are predominantly transmitted by mosquitoes and ticks. Some, such as dengue virus, exhibit the phenomenon of antibody-dependent enhancement (ADE) of disease, making vaccine-based routes of fighting infections problematic. The pH-dependent conformational change of the envelope (E) protein required for fusion between the viral and endosomal membranes is an attractive point of inhibition by antivirals as it has the potential to diminish the effects of ADE. We examined six flaviviruses by employing large-scale molecular dynamics (MD) simulations of raft systems that represent a substantial portion of the flaviviral envelope. We utilised a benzene-mapping approach that led to a discovery of shared hotspots and conserved cryptic sites. A cryptic pocket previously shown to bind a detergent molecule exhibited strain-specific characteristics. An alternative conserved cryptic site at the E protein domain interfaces showed a consistent dynamic behaviour across flaviviruses and contained a conserved cluster of ionisable residues. Constant-pH simulations revealed cluster and domain-interface disruption under low pH conditions. Based on this, we propose a cluster-dependent mechanism that addresses inconsistencies in the histidine-switch hypothesis and highlights the role of cluster protonation in orchestrating the domain dissociation pivotal for the formation of the fusogenic trimer.

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