Synergistic Binding of SARS-CoV-2 to ACE2 and Gangliosides in Native Lipid Membranes.

Viruses utilize cell surface glycans and plasma membrane receptors to attain an adequate attachment strength for initiating cellular entry. We show that SARS-CoV-2 particles bind to endogenous ACE2 receptors and added sialylated gangliosides in near-native membranes. This was explored using supported membrane bilayers (SMBs) that were formed using plasma membrane vesicles having endogenous ACE2 and GD1a gangliosides reconstituted in lipid vesicles. The virus binding rate to the SMBs is influenced by GD1a and inhibition of the ganglioside reduces the extent of virus binding to the membrane receptors. Using combinations of inhibition assays, we confirm that added GD1a in lipid membranes increases the availability of the endogenous ACE2 receptor and results in the synergistic binding of SARS-CoV-2 to the membrane receptors in SMBs.

Disruption Mechanisms of Enveloped Viruses by Ionic and Nonionic Surfactants.

The world has witnessed multiple pandemics and endemics caused by enveloped viruses in the past century. To name a few, the ongoing COVID-19 pandemic and other pandemics/endemics caused by coronaviruses, influenza viruses, HIV-1, etc. The external and topical applications of surfactants have been effective in limiting the spread of viruses. While it is well-known that surfactants inactivate virus particles (virions), the mechanism of action of surfactants against enveloped virions has not yet been established. In this work, we have evaluated the surfactant-induced disruption mechanism of a cocktail of enveloped viruses containing particles of mumps, measles, and rubella viruses. We applied the total internal reflection fluorescence microscopy technique to trace the temporal changes in the fluorescence signal from single virions upon the addition of a surfactant solution. We report that surfactants solubilize either the viral lipid membrane, proteins, or both. Ionic surfactants, depending on their charge and interaction type with the viral lipids and proteins, can cause bursting or perforation of the viral envelope, whereas a nonionic surfactant can cause either symmetric expansion or perforation of the viral envelope depending on the surfactant concentration.

Preventing amyloid-ß oligomerization and aggregation with berberine: Investigating the mechanism of action through computational methods.

Neurological disorders (NDs) have become a major cause of both cognitive and physical disabilities worldwide. In NDs, misfolded proteins tend to adopt a ß-sheet-rich fibrillar structure called amyloid. Amyloid beta (Aß) plays a crucial role in the nervous system. The misfolding and aggregation of Aß are primary factors in the progression of Alzheimer's disease (AD). Inhibiting the oligomerization and aggregation of Aß is considered as an effective strategy against NDs. While it is known that berberine analogs exhibit anti-Aß aggregation properties, the precise mechanism of action remains unclear. In this study, we have employed computational approaches to unravel the possible mechanism by which berberine combats Aß aggregation. The introduction of berberine was observed to delay the equilibrium of Aß16-21 oligomerization. Initially, within the first 10 ns of simulation, ß-sheets content was 12.89 % and gradually increased to 22.19 % within the first 20 ns. This upward trend continued, reaching 32.80 %. However, berberine substantially reduced the formation of ß-sheets to 1.36 %. These findings decipher the potency of berberine against Aß16-21 oligomerization, a crucial step for ß-sheet formation. Additionally, a remarkable decrease in total number of hydrogen bonds was found in the presence of berberine. Berberine also led to a slight reduction in the flexibility of Aß16-21, which may be due to the formation of a more stable structures. This study offers valuable insights at the mechanistic level, which could prove beneficial in the development of new drugs to combat NDs.

Multi-targeting of virulence factors of P. aeruginosa by ß-lactam antibiotics to combat antimicrobial resistance.

Antimicrobial resistance poses a significant challenge to public health, especially in developing countries, due to a substantial rise in bacterial resistance. This situation has become so concerning that we are now at risk of losing the effectiveness of antibiotics altogether. Recent research has firmly established that bacteria engage in a process called quorum sensing (QS). QS regulates various functions, including nutrient scavenging, immune response suppression, increased virulence, biofilm formation and mobility. Pseudomonas aeruginosa, an opportunistic bacterial pathogen, plays a significant role in various medical conditions such as chronic wounds, corneal infections, burn wounds and cystic fibrosis. While antibiotics are effective in killing bacteria, only a few antibiotics, particularly those from the ß-lactam group, have been studied for their impact on the quorum sensing of P. aeruginosa. Given the lack of concentrated efforts in this area, we have investigated the role of ß-lactam antibiotics on various potential targets of P. aeruginosa. Based on their toxicological profiles and the average binding energy obtained through molecular docking, azlocillin and moxalactam have emerged as lead antibiotics. The binding energy for the docking of azlocillin and moxalactam with LasA was determined to be -8.2 and -8.6 kcal/mol, respectively. Molecular simulation analysis has confirmed the stable interaction of both these ligands with all three target proteins (LasI, LasA and PqsR) under physiological conditions. The results of this research underscore the effectiveness of azlocillin and moxalactam. These two antibiotics may be repurposed to target the quorum sensing of P. aeruginosa.Communicated by Ramaswamy H. Sarma.

Particle-based phasor-FLIM-FRET resolves protein-protein interactions inside single viral particles.

Fluorescence lifetime imaging microscopy (FLIM) is a popular modality to create additional contrast in fluorescence images. By carefully analyzing pixel-based nanosecond lifetime patterns, FLIM allows studying complex molecular populations. At the single-molecule or single-particle level, however, image series often suffer from low signal intensities per pixel, rendering it difficult to quantitatively disentangle different lifetime species, such as during Förster resonance energy transfer (FRET) analysis in the presence of a significant donor-only fraction. In this article we investigate whether an object localization strategy and the phasor approach to FLIM have beneficial effects when carrying out FRET analyses of single particles. Using simulations, we first showed that an average of ∼300 photons, spread over the different pixels encompassing single fluorescing particles and without background, is enough to determine a correct phasor signature (SD < 5% for a 4-ns lifetime). For immobilized single- or double-labeled dsDNA molecules, we next validated that particle-based phasor-FLIM-FRET readily allows estimating fluorescence lifetimes and FRET from single molecules. Thirdly, we applied particle-based phasor-FLIM-FRET to investigate protein-protein interactions in subdiffraction HIV-1 viral particles. To do this, we first quantitatively compared the fluorescence brightness, lifetime, and photostability of different popular fluorescent protein-based FRET probes when genetically fused to the HIV-1 integrase enzyme in viral particles, and conclude that eGFP, mTurquoise2, and mScarlet perform best. Finally, for viral particles coexpressing FRET-donor/acceptor-labeled IN, we determined the absolute FRET efficiency of IN oligomers. Available in a convenient open-source graphical user interface, we believe that particle-based phasor-FLIM-FRET is a promising tool to provide detailed insights in samples suffering from low overall signal intensities.

Kinetics of Ganglioside-Rich Supported Lipid Bilayer Formation with Tracer Vesicle Fluorescence Imaging.

Gangliosides, forming a class of lipids complemented by sugar chains, influence the lateral distribution of membrane proteins or membrane-binding proteins, act as receptors for viruses and bacterial toxins, and mediate several types of cellular signaling. Gangliosides incorporated into supported lipid bilayers (SLBs) have been widely applied as a model system to examine these biological processes. In this work, we explored how ganglioside composition affects the kinetics of SLB formation using the vesicle rupturing method on a solid surface. We imaged the attachment of vesicles and the subsequent SLB formation using the time-lapse total internal reflection fluorescence microscopy technique. In the early phase, the ganglioside type and concentration influence the adsorption kinetics of vesicles and their residence/lifetime on the surface before rupturing. Our data confirm that a simultaneous rupturing of neighboring surface-adsorbed vesicles forms microscopic lipid patches on the surface and it is triggered by a critical coverage of the vesicles independent of their composition. In the SLB growth phase, lipid patches merge, forming a continuous SLB. The propagation of patch edges catalyzes the process and depends on the ganglioside type. Our pH-dependent experiments confirm that the polar/charged head groups of the gangliosides have a critical role in these steps and phases of SLB formation kinetics.

SARS-CoV-2 Binding to Terminal Sialic Acid of Gangliosides Embedded in Lipid Membranes.

Multiple recent reports indicate that the S protein of SARS-CoV-2 specifically interacts with membrane receptors and attachment factors other than ACE2. They likely have an active role in cellular attachment and entry of the virus. In this article, we examined the binding of SARS-CoV-2 particles to gangliosides embedded in supported lipid bilayers (SLBs), mimicking the cell membrane-like environment. We show that the virus specifically binds to sialylated (sialic acid (SIA)) gangliosides, i.e., GD1a, GM3, and GM1, as determined from the acquired single-particle fluorescence images using a time-lapse total internal reflection fluorescence (TIRF) microscope. The data of virus binding events, the apparent binding rate constant, and the maximum virus coverage on the ganglioside-rich SLBs show that the virus particles have a higher binding affinity toward the GD1a and GM3 compared to the GM1 ganglioside. Enzymatic hydrolysis of the SIA-Gal bond of the gangliosides confirms that the SIA sugar unit of GD1a and GM3 is essential for virus attachment to the SLBs and even the cell surface sialic acid is critical for the cellular attachment of the virus. The structural difference between GM3/GD1a and GM1 is the presence of SIA at the main or branched chain. We conclude that the number of SIA per ganglioside can weakly influence the initial binding rate of SARS-CoV-2 particles, whereas the terminal or more exposed SIA is critical for the virus binding to the gangliosides in SLBs.

Membrane attachment and fusion of HIV-1, influenza A, and SARS-CoV-2: resolving the mechanisms with biophysical methods.

Attachment to and fusion with cell membranes are two major steps in the replication cycle of many human viruses. We focus on these steps for three enveloped viruses, i.e., HIV-1, IAVs, and SARS-CoV-2. Viral spike proteins drive the membrane attachment and fusion of these viruses. Dynamic interactions between the spike proteins and membrane receptors trigger their specific attachment to the plasma membrane of host cells. A single virion on cell membranes can engage in binding with multiple receptors of the same or different types. Such dynamic and multivalent binding of these viruses result in an optimal attachment strength which in turn leads to their cellular entry and membrane fusion. The latter process is driven by conformational changes of the spike proteins which are also class I fusion proteins, providing the energetics of membrane tethering, bending, and fusion. These viruses exploit cellular and membrane factors in regulating the conformation changes and membrane processes. Herein, we describe the major structural and functional features of spike proteins of the enveloped viruses including highlights on their structural dynamics. The review delves into some of the case studies in the literature discussing the findings on multivalent binding, membrane hemifusion, and fusion of these viruses. The focus is on applications of biophysical tools with an emphasis on single-particle methods for evaluating mechanisms of these processes at the molecular and cellular levels.

Degradation of Xenobiotic Pollutants: An Environmentally Sustainable Approach.

The ability of microorganisms to detoxify xenobiotic compounds allows them to thrive in a toxic environment using carbon, phosphorus, sulfur, and nitrogen from the available sources. Biotransformation is the most effective and useful metabolic process to degrade xenobiotic compounds. Microorganisms have an exceptional ability due to particular genes, enzymes, and degradative mechanisms. Microorganisms such as bacteria and fungi have unique properties that enable them to partially or completely metabolize the xenobiotic substances in various ecosystems.There are many cutting-edge approaches available to understand the molecular mechanism of degradative processes and pathways to decontaminate or change the core structure of xenobiotics in nature. These methods examine microorganisms, their metabolic machinery, novel proteins, and catabolic genes. This article addresses recent advances and current trends to characterize the catabolic genes, enzymes and the techniques involved in combating the threat of xenobiotic compounds using an eco-friendly approach.

Single-Cell Imaging Shows That the Transcriptional State of the HIV-1 Provirus and Its Reactivation Potential Depend on the Integration Site.

Current antiretroviral treatment fails to cure HIV-1 infection since latent provirus resides in long-lived cellular reservoirs, rebounding whenever therapy is discontinued. The molecular mechanisms underlying HIV-1 latency are complex where the possible link between integration and transcription is poorly understood. HIV-1 integration is targeted toward active chromatin by the direct interaction with a host protein, lens epithelium-derived growth factor (LEDGF/p75). LEDGINs are small-molecule inhibitors of the LEDGF/p75-integrase (IN) interaction that effectively inhibit and retarget HIV-1 integration out of preferred integration sites, resulting in residual provirus that is more latent. Here, we describe a single-cell branched DNA imaging method for simultaneous detection of viral DNA and RNA. We investigated how treatment with LEDGINs affects the location, transcription, and reactivation of HIV-1 in both cell lines and primary cells. This approach demonstrated that LEDGIN-mediated retargeting hampered the baseline transcriptional state and the transcriptional reactivation of the provirus, evidenced by the reduction in viral RNA expression per residual copy. Moreover, treatment of primary cells with LEDGINs induced an enrichment of provirus in deep latency. These results corroborate the impact of integration site selection for the HIV-1 transcriptional state and support block-and-lock functional cure strategies in which the latent reservoir is permanently silenced after retargeting. IMPORTANCE A longstanding question exists on the impact of the HIV-1 integration site on viral gene expression. This unsolved question has significant implications for the search toward an HIV-1 cure, as eradication strategies set up to reactivate and eliminate HIV-1 depend on the site where the provirus is integrated. The main determinant for integration site selection is the interaction of the HIV-1 integrase (IN) and the host chromatin targeting factor, LEDGF/p75. LEDGINs are small-molecule inhibitors of the LEDGF/p75-IN interaction that inhibit and retarget HIV-1 integration out of preferred integration sites. Using both LEDGINs and branched DNA (bDNA) imaging, we now investigated, in much detail, the impact of integration site selection on the three-dimensional location of the provirus, HIV-1 transcription, and reactivation. Our results provide evidence for a "block-and-lock" functional cure strategy that aims to permanently silence HIV-1 by LEDGIN-mediated retargeting to sites that are less susceptible to reactivation after treatment interruption.

Dissimilar Deformation of Fluid- and Gel-Phase Liposomes upon Multivalent Interaction with Cell Membrane Mimics Revealed Using Dual-Wavelength Surface Plasmon Resonance.

The mechanical properties of biological nanoparticles play a crucial role in their interaction with the cellular membrane, in particular for cellular uptake. This has significant implications for the design of pharmaceutical carrier particles. In this context, liposomes have become increasingly popular, among other reasons due to their customizability and easily varied physicochemical properties. With currently available methods, it is, however, not trivial to characterize the mechanical properties of nanoscopic liposomes especially with respect to the level of deformation induced upon their ligand-receptor-mediated interaction with laterally fluid cellular membranes. Here, we utilize the sensitivity of dual-wavelength surface plasmon resonance to probe the size and shape of bound liposomes (∼100 nm in diameter) as a means to quantify receptor-induced deformation during their interaction with a supported cell membrane mimic. By comparing biotinylated liposomes in gel and fluid phases, we demonstrate that fluid-phase liposomes are more prone to deformation than their gel-phase counterparts upon binding to the cell membrane mimic and that, as expected, the degree of deformation depends on the number of ligand-receptor pairs that are engaged in the multivalent binding.

Physicochemical tools for studying virus interactions with targeted cell membranes in a molecular and spatiotemporally resolved context.

The objective of this critical review is to provide an overview of how emerging bioanalytical techniques are expanding our understanding of the complex physicochemical nature of virus interactions with host cell surfaces. Herein, selected model viruses representing both non-enveloped (simian virus 40 and human norovirus) and enveloped (influenza A virus, human herpes simplex virus, and human immunodeficiency virus type 1) viruses are highlighted. The technologies covered utilize a wide range of cell membrane mimics, from supported lipid bilayers (SLBs) containing a single purified host membrane component to SLBs derived from the plasma membrane of a target cell, which can be compared with live-cell experiments to better understand the role of individual interaction pairs in virus attachment and entry. These platforms are used to quantify binding strengths, residence times, diffusion characteristics, and binding kinetics down to the single virus particle and single receptor, and even to provide assessments of multivalent interactions. The technologies covered herein are surface plasmon resonance (SPR), quartz crystal microbalance with dissipation (QCM-D), dynamic force spectroscopy (DFS), total internal reflection fluorescence (TIRF) microscopy combined with equilibrium fluctuation analysis (EFA) and single particle tracking (SPT), and finally confocal microscopy using multi-labeling techniques to visualize entry of individual virus particles in live cells. Considering the growing scientific and societal needs for untangling, and interfering with, the complex mechanisms of virus binding and entry, we hope that this review will stimulate the community to implement these emerging tools and strategies in conjunction with more traditional methods. The gained knowledge will not only contribute to a better understanding of the virus biology, but may also facilitate the design of effective inhibitors to block virus entry.

A Systematic Review of Piezoelectric Materials and Energy Harvesters for Industrial Applications.

In the last three decades, smart materials have become popular. The piezoelectric materials have shown key characteristics for engineering applications, such as in sensors and actuators for industrial use. Because of their excellent mechanical-to-electrical and vice versa energy conversion properties, piezoelectric materials with high piezoelectric charge and voltage coefficient have been tested in renewable energy applications. The fundamental component of the energy harvester is the piezoelectric material, which, when subjected to mechanical vibrations or applied stress, induces the displaced ions in the material and results in a net electric charge due to the dipole moment of the unit cell. This phenomenon builds an electric potential across the material. In this review article, a detailed study focused on the piezoelectric energy harvesters (PEH's) is reported. In addition, the fundamental idea about piezoelectric materials, along with their modeling for various applications, are detailed systematically. Then a summary of previous studies based on PEH's other applications is listed, considering the technical aspects and methodologies. A discussion has been provided as a critical review of current challenges in this field. As a result, this review can provide a guideline for the scholars who want to use PEH's for their research.

Impurity-induced nematic-isotropic transition of liquid crystals.

Complex fluids made of liquid crystals (LCs) and small molecules, surfactants, nanoparticles or 1D/2D nanomaterials show novel and interesting features, making them suitable materials for various applications starting from optoelectronics to biosensing. While these additives (impurities) introduce new features in the complex fluids, they may also alter the phase transition behaviour of LCs depending on the physiochemical properties of the added impurity. This article reports on the phase transition of 4-cyano-4'-alkylbiphenyl (nCB) LCs in the presence of an associative impurity, i.e., water and a non-associative impurity, i.e., hexane employing computational methods and experiments. In particular, all-atom (AA) simulations and coarse-grained (CG) models were designed for two complex systems, i.e., 6CB + water and 6CB + hexane and corresponding spectrophotometry experiments were performed using a homologous LC, i.e., 5CB. Results from the simulations and experiments elucidate that the phase transition of LCs depends on the mixing/demixing phenomenon of the impurity in the LC. While associative liquids like water which do not mix with LCs do not influence the nematic-to-isotropic phase transition of LCs, hexane, being a non-associative liquid, mixes well with LCs and induces a sharp impurity-induced nematic-to-isotropic phase transition. Upon application of both AA and CG simulations, we could reach the conclusion that the mixing/demixing phenomenon in an LC + impurity system influences the entropy of the system and hence the observed phase transitions are entropy-driven.

A vaccine combination of lipid nanoparticles and a cholera toxin adjuvant derivative greatly improves lung protection against influenza virus infection.

This is a proof-of-principle study demonstrating that the combination of a cholera toxin derived adjuvant, CTA1-DD, and lipid nanoparticles (LNP) can significantly improve the immunogenicity and protective capacity of an intranasal vaccine. We explored the self-adjuvanted universal influenza vaccine candidate, CTA1-3M2e-DD (FPM2e), linked to LNPs. We found that the combined vector greatly enhanced survival against a highly virulent PR8 strain of influenza virus as compared to when mice were immunized with FPM2e alone. The combined vaccine vector enhanced early endosomal processing and peptide presentation in dendritic cells and upregulated co-stimulation. The augmenting effect was CTA1-enzyme dependent. Whereas systemic anti-M2e antibody and CD4+ T-cell responses were comparable to those of the soluble protein, the local respiratory tract IgA and the specific Th1 and Th17 responses were strongly enhanced. Surprisingly, the lung tissue did not exhibit gross pathology upon recovery from infection and M2e-specific lung resident CD4+ T cells were threefold higher than in FPM2e-immunized mice. This study conveys optimism as to the protective ability of a combination vaccine based on LNPs and various forms of the CTA1-DD adjuvant platform, in general, and, more specifically, an important way forward to develop a universal vaccine against influenza.

Competition for Membrane Receptors: Norovirus Detachment via Lectin Attachment.

Virus internalization into the host cells occurs via multivalent interactions, in which a single virus binds to multiple receptors in parallel. Because of analytical and experimental limitations this complex type of interaction is still poorly understood and quantified. Herein, the multivalent interaction of norovirus-like particles (noroVLPs) with H or B type 1 glycosphingolipids (GSLs), embedded in a supported phospholipid bilayer, is investigated by following the competition between noroVLPs and a lectin (from Ralstonia solanacearum) upon binding to these GSLs. Changes in noroVLP and lectin coverage, caused by competition, were monitored for both GSLs and at different GSL concentrations using quartz crystal microbalance with dissipation monitoring. The study yields information about the minimum GSL concentration needed for (i) noroVLPs to achieve firm attachment to the bilayer prior to competition and to (ii) remain firmly attached to the bilayer during competition. We show that these two concentrations are almost identical for the H type 1-noroVLP interaction but differ for B type 1, indicating an accumulation of B type 1 GSLs in the noroVLP-bilayer interaction area. Furthermore, the GSL concentration required for firm attachment is significantly larger for H type 1 than for B type 1, indicating a higher affinity of noroVLP toward B type 1. This finding is supported by extracting the energy of single noroVLP-H type 1 and noroVLP-B type 1 bonds from the competition kinetics, which were estimated to be 5 and 6 kcal/mol, respectively. This demonstrates the potential of utilizing competitive binding kinetics to analyze multivalent interactions, which has remained difficult to quantify using conventional approaches.

Gel Phase 1,2-Distearoyl-sn-glycero-3-phosphocholine-Based Liposomes Are Superior to Fluid Phase Liposomes at Augmenting Both Antigen Presentation on Major Histocompatibility Complex Class II and Costimulatory Molecule Display by Dendritic Cells in Vitro.

Lipid-based nanoparticles have in recent years attracted increasing attention as pharmaceutical carriers. In particular, reports of them having inherent adjuvant properties combined with their ability to protect antigen from degradation make them suitable as vaccine vectors. However, the physicochemical profile of an ideal nanoparticle for vaccine delivery is still poorly defined. Here, we used an in vitro dendritic cell assay to assess the immunogenicity of a variety of liposome formulations as vaccine carriers and adjuvants. Using flow cytometry, we investigated liposome-assisted antigen presentation as well as the expression of relevant costimulatory molecules on the cell surface. Cytokine secretion was further evaluated with an enzyme-linked immunosorbent assay (ELISA). We show that liposomes can successfully enhance antigen presentation and maturation of dendritic cells, as compared to vaccine fusion protein (CTA1-3Eα-DD) administered alone. In particular, the lipid phase state of the membrane was found to greatly influence the vaccine antigen processing by dendritic cells. As compared to their fluid phase counterparts, gel phase liposomes were more efficient at improving antigen presentation. They were also superior at upregulating the costimulatory molecules CD80 and CD86 as well as increasing the release of the cytokines IL-6 and IL-1ß. Taken together, we demonstrate that gel phase liposomes, while nonimmunogenic on their own, significantly enhance the antigen-presenting ability of dendritic cells and appear to be a promising way forward to improve vaccine immunogenicity.

Viscoelastic Properties of Polyelectrolyte Multilayers Swollen with Ionic Liquid Solutions.

Polyelectrolyte multilayers (PEM) obtained by layer-by-layer assembly can be doped with ionic liquid (IL) via the swelling of the films with IL solutions. In order to examine the mechanical properties of IL-containing PEM, we implement a Kelvin-Voigt model to obtain thickness, viscosity and elastic modulus from the frequency and dissipation shifts determined by a dissipative quartz crystal microbalance (QCM-D). We analyze the changes in the modeled thickness and viscoelasticity of PEI(PSS/PADMAC)4PSS and PEI(PSS/PAH)4PSS multilayers upon swelling by increasing the concentration of either 1-Ethyl-3-methylimidazolium chloride or 1-Hexyl-3-methylimidazolium chloride, which are water soluble ILs. The results show that the thickness of the multilayers changes monotonically up to a certain IL concentration, whereas the viscosity and elasticity change in a non-monotonic fashion with an increasing IL concentration. The changes in the modeled parameters can be divided into three concentration regimes of IL, a behavior specific to ILs (organic salts), which does not occur with swelling by simple inorganic salts such as NaCl. The existence of the regimes is attributed to a competition of the hydrophobic interactions of large hydrophobic ions, which enhance the layer stability at a low salt content, with the electrostatic screening, which dominates at a higher salt content and causes a film softening.

Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication.

Viruses are simple agents exhibiting complex reproductive mechanisms. Decades of research have provided crucial basic insights, antiviral medication and moderately successful gene therapy trials. The most infectious viral particle is, however, not always the most abundant one in a population, questioning the utility of classic ensemble-averaging virology. Indeed, viral replication is often not particularly efficient, prone to errors or containing parallel routes. Here, we review different single-molecule sensitive fluorescence methods that we employ routinely to investigate viruses. We provide a brief overview of the microscopy hardware needed and discuss the different methods and their application. In particular, we review how we applied (i) single-molecule Förster resonance energy transfer (smFRET) to probe the subviral human immunodeficiency virus (HIV-1) integrase (IN) quaternary structure; (ii) single particle tracking to study interactions of the simian virus 40 with membranes; (iii) 3D confocal microscopy and smFRET to quantify the HIV-1 pre-integration complex content and quaternary structure; (iv) image correlation spectroscopy to quantify the cytosolic HIV-1 Gag assembly, and finally; (v) super-resolution microscopy to characterize the interaction of HIV-1 with tetherin during assembly. We hope this review is an incentive for setting up and applying similar single-virus imaging studies in daily virology practice.

Membrane Deformation Induces Clustering of Norovirus Bound to Glycosphingolipids in a Supported Cell-Membrane Mimic.

Quartz crystal microbalance with dissipation monitoring and total internal reflection fluorescence microscopy have been used to investigate binding of norovirus-like particles (noroVLPs) to a supported (phospho)lipid bilayer (SLB) containing a few percent of H or B type 1 glycosphingolipid (GSL) receptors. Although neither of these GSLs spontaneously form domains, noroVLPs were observed to form micron-sized clusters containing typically up to about 30 VLP copies, especially for B type 1, which is a higher-affinity receptor. This novel finding is explained by proposing a model implying that VLP-induced membrane deformation promotes VLP clustering, a hypothesis that was further supported by observing that functionalized gold nanoparticles were able to locally induce SLB deformation. Because similar effects are likely possible also at cellular membranes, our findings are interesting beyond a pure biophysicochemical perspective as they shed new light on what may happen during receptor-mediated uptake of viruses as well as nanocarriers in drug delivery.