Membrane-enveloped Viruses Research Articles

Unraveling How Antimicrobial Lipid Mixtures Disrupt Virus-Mimicking Lipid Vesicles: A QCM-D Study.

Single-chain lipid amphiphiles such as fatty acids and monoglycerides are promising antimicrobial alternatives to replace industrial surfactants for membrane-enveloped pathogen inhibition. Biomimetic lipid membrane platforms in combination with label-free biosensing techniques offer a promising route to compare the membrane-disruptive properties of different fatty acids and monoglycerides individually and within mixtures. Until recently, most related studies have utilized planar model membrane platforms, and there is an outstanding need to investigate how antimicrobial lipid mixtures disrupt curved model membrane platforms such as intact vesicle adlayers that are within the size range of membrane-enveloped virus particles. This need is especially evident because certain surfactants that completely disrupt planar/low-curvature membranes are appreciably less active against high-curvature membranes. Herein, we conducted quartz crystal microbalance-dissipation (QCM-D) measurements to investigate the membrane-disruptive properties of glycerol monolaurate (GML) monoglyceride and lauric acid (LA) fatty acid mixtures to rupture high-curvature, ~75 nm diameter lipid vesicle adlayers. We identified that the vesicle rupture activity of GML/LA mixtures mainly occurred above the respective critical micelle concentration (CMC) of each mixture, and that 25/75 mol% GML/LA micelles exhibited the greatest degree of vesicle rupture activity with ~100% efficiency that exceeded the rupture activity of other tested mixtures, individual compounds, and past reported values with industrial surfactants. Importantly, 25/75 GML/LA micelles outperformed 50/50 GML/LA micelles, which were previously reported to have the greatest membrane-disruptive activity towards planar model membranes. We discuss the mechanistic principles behind how antimicrobial lipid engineering can influence membrane-disruptive activity in terms of optimizing the balance between competitive membrane remodeling processes and inducing anisotropic vs. isotropic spontaneous curvature in lipid membrane systems.

Glycerol Monolaurate Inhibits Wild-Type African Swine Fever Virus Infection in Porcine Macrophages.

Naturally abundant antimicrobial lipids, such as fatty acids and monoglycerides, that disrupt membrane-enveloped viruses are promising mitigants to inhibit African swine fever virus (ASFV). Among mitigant candidates in this class, glycerol monolaurate (GML) has demonstrated particularly high antiviral activity against laboratory-adapted ASFV strains. However, there is an outstanding need to further determine the effects of GML on wild-type ASFV strains, which can have different virulence levels and sensitivities to membrane-disrupting compounds as compared to laboratory-adapted strains. Herein, we investigated the antiviral effects of GML on a highly virulent strain of a wild-type ASFV isolate (Armenia/07) in an in vitro porcine macrophage model. GML treatment caused a concentration-dependent reduction in viral infectivity, and there was a sharp transition between inactive and active GML concentrations. Low GML concentrations had negligible effect on viral infectivity, whereas sufficiently high GML concentrations caused a >99% decrease in viral infectivity. The concentration onset of antiviral activity matched the critical micelle concentration (CMC) value of GML, reinforcing that GML micelles play a critical role in enabling anti-ASFV activity. These findings validate that GML can potently inhibit wild-type ASFV infection of porcine macrophages and support a biophysical explanation to guide antimicrobial lipid performance optimization for pathogen mitigation applications.

Lipid acyl chain protrusion induced by the influenza virus hemagglutinin fusion peptide detected by NMR paramagnetic relaxation enhancement

The glycoprotein spikes of membrane-enveloped viruses include a subunit that catalyzes fusion (joining) of the viral and target cell membranes. For influenza virus, this is subunit 2 of hemagglutinin which has a ∼ 20-residue N-terminal fusion peptide (Fp) region that binds target membrane. An outstanding question is whether there are associated membrane changes important for fusion. Several computational studies have found increased “protrusion” of lipid acyl chains near Fp, i.e. one or more chain carbons are closer to the aqueous region than the headgroup phosphorus. Protrusion may accelerate initial joining of outer leaflets of the two membranes into a stalk intermediate. In this study, higher protrusion probability in membrane with vs. without Fp is convincingly detected by larger Mn2+-associated increases in chain 13C NMR transverse relaxation rates (Γ2's). Data analysis provides a ratio Γ2,neighbor/Γ2,distant for lipids neighboring vs. more distant from the Fp. The calculated ratio depends on the number of Fp-neighboring lipids and the experimentally-derived range of 4 to 24 matches the range of increased protrusion probabilities from different simulations. For samples either with or without Fp, the Γ2 values are well-fitted by an exponential decay as the 13C site moves closer to the chain terminus. The decays correlate with free-energy of protrusion proportional to the number of protruded -CH2 groups, with free energy per -CH2 of ∼0.25 kBT. The NMR data support one major fusion role of the Fp to be much greater protrusion of lipid chains, with highest protrusion probability for chain regions closest to the headgroups.

Curvature-sensing peptide inhibits tumour-derived exosomes for enhanced cancer immunotherapy.

Tumour-derived exosomes (T-EXOs) impede immune checkpoint blockade therapies, motivating pharmacological efforts to inhibit them. Inspired by how antiviral curvature-sensing peptides disrupt membrane-enveloped virus particles in the exosome size range, we devised a broadly useful strategy that repurposes an engineered antiviral peptide to disrupt membrane-enveloped T-EXOs for synergistic cancer immunotherapy. The membrane-targeting peptide inhibits T-EXOs from various cancer types and exhibits pH-enhanced membrane disruption relevant to the tumour microenvironment. The combination of T-EXO-disrupting peptide and programmed cell death protein-1 antibody-based immune checkpoint blockade therapy improves treatment outcomes in tumour-bearing mice. Peptide-mediated disruption of T-EXOs not only reduces levels of circulating exosomal programmed death-ligand 1, but also restores CD8+ T cell effector function, prevents premetastatic niche formation and reshapes the tumour microenvironment in vivo. Our findings demonstrate that peptide-induced T-EXO depletion can enhance cancer immunotherapy and support the potential of peptide engineering for exosome-targeting applications.

Lipid Membrane Interface Viewpoint: From Viral Entry to Antiviral and Vaccine Development.

Membrane-enveloped viruses are responsible for most viral pandemics in history, and more effort is needed to advance broadly applicable countermeasures to mitigate the impact of future outbreaks. In this Perspective, we discuss how biosensing techniques associated with lipid model membrane platforms are contributing to improving our mechanistic knowledge of membrane fusion and destabilization that is closely linked to viral entry as well as vaccine and antiviral drug development. A key benefit of these platforms is the simplicity of interpreting the results which can be complemented by other techniques to decipher more complicated biological observations and evaluate the biophysical functionalities that can be correlated to biological activities. Then, we introduce exciting application examples of membrane-targeting antivirals that have been refined over time and will continue to improve based on biophysical insights. Two ways to abrogate the function of viral membranes are introduced here: (1) selective disruption of the viral membrane structure and (2) alteration of the membrane component. While both methods are suitable for broadly useful antivirals, the latter also has the potential to produce an inactivated vaccine. Collectively, we emphasize how biosensing tools based on membrane interfacial science can provide valuable information that could be translated into biomedicines and improve their selectivity and performance.

The Interaction of Hypericin with SARS-CoV-2 Reveals a Multimodal Antiviral Activity

Hypericin is a photosensitizing drug that is active against membrane-enveloped viruses and therefore constitutes a promising candidate for the treatment of SARS-CoV-2 infections. The antiviral efficacy of hypericin is largely determined by its affinity toward viral components and by the number of active molecules loaded on single viruses. Here we use an experimental approach to follow the interaction of hypericin with SARS-CoV-2, and we evaluate its antiviral efficacy, both in the dark and upon photoactivation. Binding to viral particles is directly visualized with fluorescence microscopy, and a strong affinity for the viral particles, most likely for the viral envelope, is measured spectroscopically. The loading of a maximum of approximately 30 molecules per viral particle is estimated, despite with marked heterogeneity among particles. Because of this interaction, nanomolar concentrations of photoactivated hypericin substantially reduce virus infectivity on Vero E6 cells, but a partial effect is also observed in dark conditions, suggesting multiple mechanisms of action for this drug.

Multivalency-Induced Shape Deformation of Nanoscale Lipid Vesicles: Size-Dependent Membrane Bending Effects.

The size of membrane-enveloped virus particles, exosomes, and lipid vesicles strongly impacts functional properties in biological and applied contexts. Multivalent ligand-receptor interactions involving nanoparticle shape deformation are critical to such functions, yet the corresponding effect of nanoparticle size remains largely elusive. Herein, using an indirect nanoplasmonic sensing approach, we investigated how the nanoscale size properties of ligand-modified lipid vesicles affect real-time binding interactions, especially vesicle deformation processes, with a receptor-modified, cell membrane-mimicking platform. Together with theoretical analyses, our findings reveal a pronounced, size-dependent transition in the membrane bending properties of nanoscale lipid vesicles between 60 and 180 nm in diameter. For smaller vesicles, a large membrane bending energy enhanced vesicle stiffness while the osmotic pressure energy was the dominant modulating factor for larger, less stiff vesicles. These findings advance our fundamental understanding of how nanoparticle size affects multivalency-induced nanoparticle shape deformation and can provide guidance for the design of biomimetic nanoparticles with tailored nanomechanical properties.

Characterization of Sendai virus binding to supported lipid bilayers at the single-virus level

Sendai virus (murine respirovirus) is a negative-sense, membrane-enveloped RNA virus belonging to the Paramyxoviridae family, which also includes measles and parainfluenza viruses. Despite causing respiratory disease in rodents, Sendai virus is non-pathogenic in humans and therefore has recently gained attention as a virus vector candidate for gene therapy and vaccine development. To infect host cells, Sendai virus attaches to sialic-acid-containing glycolipid receptors on the surface of host cells via the hemagglutinin-neuraminidase (HN) protein.

Supported Lipid Bilayer Platform for Characterizing the Membrane-Disruptive Behaviors of Triton X-100 and Potential Detergent Replacements.

Triton X-100 (TX-100) is a widely used detergent to prevent viral contamination of manufactured biologicals and biopharmaceuticals, and acts by disrupting membrane-enveloped virus particles. However, environmental concerns about ecotoxic byproducts are leading to TX-100 phase out and there is an outstanding need to identify functionally equivalent detergents that can potentially replace TX-100. To date, a few detergent candidates have been identified based on viral inactivation studies, while direct mechanistic comparison of TX-100 and potential replacements from a biophysical interaction perspective is warranted. Herein, we employed a supported lipid bilayer (SLB) platform to comparatively evaluate the membrane-disruptive properties of TX-100 and a potential replacement, Simulsol SL 11W (SL-11W), and identified key mechanistic differences in terms of how the two detergents interact with phospholipid membranes. Quartz crystal microbalance-dissipation (QCM-D) measurements revealed that TX-100 was more potent and induced rapid, irreversible, and complete membrane solubilization, whereas SL-11W caused more gradual, reversible membrane budding and did not induce extensive membrane solubilization. The results further demonstrated that TX-100 and SL-11W both exhibit concentration-dependent interaction behaviors and were only active at or above their respective critical micelle concentration (CMC) values. Collectively, our findings demonstrate that TX-100 and SL-11W have distinct membrane-disruptive effects in terms of potency, mechanism of action, and interaction kinetics, and the SLB platform approach can support the development of biophysical assays to efficiently test potential TX-100 replacements.

Antiviral peptide engineering for targeting membrane-enveloped viruses: Recent progress and future directions

Membrane-enveloped viruses are a major cause of global health challenges, including recent epidemics and pandemics. This mini-review covers the latest efforts to develop membrane-targeting antiviral peptides that inhibit enveloped viruses by 1) preventing virus-cell fusion or 2) disrupting the viral membrane envelope. The corresponding mechanisms of antiviral activity are discussed along with peptide engineering strategies to modulate membrane-peptide interactions in terms of potency and selectivity. Application examples are presented demonstrating how membrane-targeting antiviral peptides are useful therapeutics and prophylactics in animal models, while a stronger emphasis on biophysical concepts is proposed to refine mechanistic understanding and support potential clinical translation.

Lipid Nanoparticle Technology for Delivering Biologically Active Fatty Acids and Monoglycerides.

There is enormous interest in utilizing biologically active fatty acids and monoglycerides to treat phospholipid membrane-related medical diseases, especially with the global health importance of membrane-enveloped viruses and bacteria. However, it is difficult to practically deliver lipophilic fatty acids and monoglycerides for therapeutic applications, which has led to the emergence of lipid nanoparticle platforms that support molecular encapsulation and functional presentation. Herein, we introduce various classes of lipid nanoparticle technology and critically examine the latest progress in utilizing lipid nanoparticles to deliver fatty acids and monoglycerides in order to treat medical diseases related to infectious pathogens, cancer, and inflammation. Particular emphasis is placed on understanding how nanoparticle structure is related to biological function in terms of mechanism, potency, selectivity, and targeting. We also discuss translational opportunities and regulatory needs for utilizing lipid nanoparticles to deliver fatty acids and monoglycerides, including unmet clinical opportunities.

Rapid 2H NMR Transverse Relaxation of Perdeuterated Lipid Acyl Chains of Membrane with Bound Viral Fusion Peptide Supports Large-Amplitude Motions of These Chains That Can Catalyze Membrane Fusion.

An early step in cellular infection by a membrane-enveloped virus like HIV or influenza is joining (fusion) of the viral and cell membranes. Fusion is catalyzed by a viral protein that typically includes an apolar "fusion peptide" (fp) segment that binds the target membrane prior to fusion. In this study, the effects of nonhomologous HIV and influenza fp's on lipid acyl chain motion are probed with 2H NMR transverse relaxation rates (R2's) of a perdeuterated DMPC membrane. Measurements were made between 35 and 0 °C, which brackets the membrane liquid-crystalline-to-gel phase transitions. Samples were made with either HIV "GPfp" at pH 7 or influenza "HAfp" at pH 5 or 7. GPfp induces vesicle fusion at pH 7, and HAfp induces more fusion at pH 5 vs 7. GPfp bound to DMPC adopts an intermolecular antiparallel β sheet structure, whereas HAfp is a monomer helical hairpin. The R2's of the no peptide and HAfp, pH 7, samples increase gradually as temperature is lowered. The R2's of GPfp and HAfp, pH 5, samples have very different temperature dependence, with a ∼10× increase in R2CD2 when temperature is reduced from 25 to 20 °C and smaller but still substantial R2's at 10 and 0 °C. The large R2's with GPfp and HAfp, pH 5, are consistent with large-amplitude motions of lipid acyl chains that can aid fusion catalysis by increasing the population of chains near the aqueous phase, which is the chain location for transition states between membrane fusion intermediates.

Engineered lipid bicelle nanostructures for membrane-disruptive antibacterial applications

Lipid bicelles are cell membrane-mimicking nanostructures that self-assemble from mixtures of long- and short-chain lipidic components and are widely used in various applications related to structural biology, drug delivery, and interfacial science. To date, most research efforts have focused on bicelles as passive structural carriers to host membrane proteins or hydrophobic drugs, while there remains untapped potential to engineer functionally active lipid bicelles that contain biologically important lipidic components for targeted applications. Herein, we developed antibiotic-free, antibacterial bicellar nanostructures composed of a long-chain phospholipid and glycerol monolaurate, which is a monoglyceride that exhibits membrane-disruptive inhibitory activity against various bacteria and membrane-enveloped viruses. Quartz crystal microbalance-dissipation and time-lapse fluorescence microscopy imaging experiments were conducted to identify fusogenic bicellar compositions with optimal levels of pore-like, membrane-disruptive activity that was distinct from the activity of the monoglyceride alone. Cryogenic transmission electron microscopy was performed to characterize the lamellar-phase nanostructure properties of the lead bicelle composition along with in vitro antibacterial assays, which identified that the bicelles inhibited Staphylococcus aureus bacteria via a killing mechanism. Collectively, these findings demonstrate the potential of applying molecular-level engineering strategies to fabricate lipid bicelles with membrane-disruptive properties relevant to anti-infective applications.

Single-Virus Investigation of Non-Sialic-Acid-Mediated Sendai Virus Binding to Supported Lipid Bilayers in the Absence of Ganglioside Receptor

Sendai virus is a negative-sense, single-stranded RNA, membrane-enveloped virus of the Paramyxoviridae family that causes respiratory disease in rodents. Because Sendai virus is non-pathogenic in humans, yet can infect human cells in cell culture, it has gained use as a virus vector candidate for gene therapy and vaccination. Previous bulk and cell culture studies indicate Sendai virus infects host cells via sialic-acid-mediated binding (SAMB) by the viral HN attachment protein. This binding then triggers the viral F protein to mediate membrane fusion. Here, we study single-particle Sendai virus binding to glass-supported lipid bilayers (SLBs) using a fluorescence microscopy-based microfluidic assay. Fluorescently labelled virions are observed under the microscope and bound virions are quantified using image processing. We investigate how lipid composition and ganglioside GD1a receptor density in the target membrane influences viral binding. Interestingly, we observe substantial non-sialic-acid-mediated binding (NSAMB) across a variety of conditions, much more than influenza virus strains which also bind the GD1a receptor. Results from monoclonal antibody interference experiments suggest that the viral HN attachment protein is involved in this NSAMB. We examine various hypotheses and experimental approaches to explain this recurring NSAMB, including the effects of electrostatics, hydrophobicity, and different sialic acid terminated glycolipid receptors, such as GD1a and GQ1b.

Stopping Membrane-Enveloped Viruses with Nanotechnology Strategies: Toward Antiviral Drug Development and Pandemic Preparedness.

Membrane-enveloped viruses are a leading cause of viral epidemics, and there is an outstanding need to develop broad-spectrum antiviral strategies to treat and prevent enveloped virus infections. In this review, we critically discuss why the lipid membrane surrounding enveloped virus particles is a promising antiviral target and cover the latest progress in nanotechnology research to design and evaluate membrane-targeting virus inhibition strategies. These efforts span diverse topics such as nanomaterials, self-assembly, biosensors, nanomedicine, drug delivery, and medical devices and have excellent potential to support the development of next-generation antiviral drug candidates and technologies. Application examples in the areas of human medicine and agricultural biosecurity are also presented. Looking forward, research in this direction is poised to strengthen capabilities for virus pandemic preparedness and demonstrates how nanotechnology strategies can help to solve global health challenges related to infectious diseases.

Single Virus Investigation of Sendai Virus Binding and Fusion to Supported Lipid Bilayers

Sendai virus is a membrane-enveloped virus in the Paramyxoviridae family that infects host cells by binding to specific receptors (sialic acid terminated glycolipids or glycoproteins), which initiates membrane fusion. Sendai virus causes disease in rodents, but not in humans, although it can infect human cells in tissue culture. Consequently, it has become a virus vector candidate for gene therapy and vaccination. Here, we present the development of an assay to study the binding and fusion process of Sendai virus using microfluidic flow cells containing target membranes. We visualize single-virus binding and fusion to a glass-supported lipid bilayer (SLB), using fluorescent microscopy and labelling, which we optimized by testing various fluorescent dyes and purification techniques. By manipulating receptor concentrations and lipid compositions, we also investigated the effects of receptor density and the efficiency of gangliosides, GD1a and GQ1b, as specific receptors for Sendai virus binding and fusion.

Gaussian curvature and the budding kinetics of enveloped viruses

The formation of a membrane-enveloped virus starts with the assembly of a curved layer of capsid proteins lining the interior of the plasma membrane (PM) of the host cell. This layer develops into a spherical shell (capsid) enveloped by a lipid-rich membrane. In many cases, the budding process stalls prior to the release of the virus. Recently, Brownian dynamics simulations of a coarse-grained model system reproduced protracted pausing and stalling, which suggests that the origin of pausing/stalling is to be found in the physics of the budding process. Here, we propose that the pausing/stalling observed in the simulations can be understood as a purely kinetic phenomenon associated with the neck geometry. A geometrical potential energy barrier develops during the budding that must be overcome by capsid proteins diffusing along the membrane prior to incorporation into the capsid. The barrier is generated by a conflict between the positive Gauss curvature of the assembling capsid and the negative Gauss curvature of the neck region. A continuum theory description is proposed and is compared with the Brownian simulations of the budding of enveloped viruses.

Giant viruses: spore-like missing links between Rickettsia and mitochondria?

Phyloproteomics indicate common viral origin from ancient cells before archaea, bacteria, and eukaryota split and subsequent size and complexity reductions occurred. Further independent evidence for the cellular origin of viruses is reviewed for the virus order Megavirales, focusing on the family Poxviridae. Megavirales comprises giant viruses, double-stranded DNA viruses whose genomes exceed some bacterial ones and large enough to parasitize large-celled protists (amoeba). Giant viruses, virophages, and mitochondria have homologous DNA and RNA polymerases and share RNA splicing punctuation by stem-loop hairpins. Giant virus factories and amoeban mitochondria colocate, with viral proteins homologous to mitochondrial proteins specifically targeting mitochondrial inner membranes. Mitochondria share asexual budding with many bacterial endospores and membrane-enveloped viruses. These megavirus-mitochondrion similarities are not coincidental: systematic alignment analyses have detected candidate megaviral homologs of each amoeban mitogene (including ribosomal RNAs) distributed across megaviral genomes. These candidate megaviral homologs overall follow mitogene order, and megaviral-mitogenome synteny increases with viral genome reduction. This analysis is repeated within Poxviridae, whose organellar-like morphogenesis is reminiscent of mitochondria. More generally, the results confirm the patterns observed in Megavirales: synteny with amoeban mitogenomes increases with genome reduction. Parsimoniously interpreting this suggests Megavirales and mitochondria have a rickettsia-like common ancestor. Megavirales could be the missing links between bacterial-like cells and mitochondria, implying cellular-to-viral-to-subcellular macroevolution.

Targeting the Achilles Heel of Mosquito-Borne Viruses for Antiviral Therapy.

Mosquito-borne viruses encompass a wide range of pathogens, such as dengue and Zika viruses, that often cocirculate geographically. These viruses affect hundreds of millions of people worldwide, yet no clinically approved therapy is currently available for treating these viral infections. Thus, innovative therapies, especially inhibitors with broad antiviral activities against all these viruses, are urgently needed. While traditional therapeutic strategies mainly focus on inhibiting viral replication in a "one lock, one key" manner (e.g., viral protease and polymerase inhibitors), inhibitors targeting virions have recently emerged as a promising approach to achieve broad antiviral activities. Within this approach, Lipid Envelope Antiviral Disruption (LEAD) molecules were shown to broadly inhibit mosquito-borne viruses and other lipid membrane-enveloped viruses. Several LEAD molecules have been demonstrated to act against viral membranes in vitro, some of which have even shown in vivo efficacy to treat mosquito-borne viral infections. This therapeutic potential is further enhanced by molecular engineering to improve the inhibitors' pharmacological properties, laying the foundation for the LEAD antiviral strategy to be explored for possible treatment of mosquito-borne viral infections.

Structural conservation in a membrane-enveloped filamentous virus infecting a hyperthermophilic acidophile

Different forms of viruses that infect archaea inhabiting extreme environments continue to be discovered at a surprising rate, suggesting that the current sampling of these viruses is sparse. We describe here Sulfolobus filamentous virus 1 (SFV1), a membrane-enveloped virus infecting Sulfolobus shibatae. The virus encodes two major coat proteins which display no apparent sequence similarity with each other or with any other proteins in databases. We have used cryo-electron microscopy at 3.7 Å resolution to show that these two proteins form a nearly symmetrical heterodimer, which wraps around A-form DNA, similar to what has been shown for SIRV2 and AFV1, two other archaeal filamentous viruses. The thin (∼ 20 Å) membrane of SFV1 is mainly archaeol, a lipid species that accounts for only 1% of the host lipids. Our results show how relatively conserved structural features can be maintained across evolution by both proteins and lipids that have diverged considerably.