Nagma Parveen

We report the rational design and synthesis of nitrogen-doped sialic acid-derived carbon dots (SiaMPDA CDs) with excitation-independent green emission and high quantum yield and demonstrate their application as efficient Förster resonance energy transfer (FRET) donors for the selective detection of influenza A virus (IAV) subtypes. The surface functionalization of the CDs with sialic acid moieties enables specific molecular recognition of the viral hemagglutinin (HA) protein via multivalent hydrogen-bonding interactions. In combination with Rhodamine B (RhB) as an acceptor, the SiaMPDA–RhB pair exhibits efficient FRET under native conditions. Upon interaction with IAV (H3N2 and H1N1), the FRET system displays anomalous spectral behavior characterized by a simultaneous increase in both donor and acceptor emission intensities without any spectral shift. Mechanistic investigations suggest the formation of a ternary IAV–SiaMPDA–RhB complex, wherein long-range charge transfer coupled with FRET governs the observed fluorescence response. Control experiments with proteins, membrane vesicles, and liposomes confirm that the signal specificity arises from targeted interactions between the sialic acid functionalities of the CDs and the viral surface proteins. The sensing platform enables selective and sensitive detection of IAV subtypes in the picomolar range, comparable to existing carbon dot-based detection systems. These findings establish N-doped sialic acid carbon dots as promising nanoscale probes for virus detection and provide insights into charge-transfer-coupled FRET mechanisms in complex bionano interfaces.

We investigated the binding of influenza A viruses (IAVs) to gangliosides containing either α,2–3- or α,2–8-linked sialic acid residues, or a combination of both. The respective gangliosides were reconstituted into phase-separated giant unilamellar vesicles to mimic the cellular membrane environment. Dual-color fluorescence imaging of influenza particles interacting with these vesicles revealed that the order of virus binding density and the lateral mobility of bound particles does not correlate with (i) the intrinsic affinity of viral hemagglutinin (HA) for specific sialic acid conformations or (ii) the number of sialic acid residues present in the gangliosides. Instead, we observe that the extent of viral attachment closely follows the surface charge of the ganglioside-rich vesicles. Because influenza particles carry a net negative charge, they experience electrostatic repulsion when approaching negatively charged membranes. Consequently, electrostatic interactions modulate the ability of viruses to establish multivalent contacts with membrane-bound sialic acids. Taken together, our findings demonstrate that electrostatic repulsion, glycosidic linkage, and lipid packing collectively govern influenza binding to phase-separated membranes. These results suggest that the commonly discussed preferential binding of influenza to specific sialic acid linkages and its association with viral species tropism should be reconsidered in the broader context of membrane surface charge and lipid organization.

We report that soluble serine proteases such as trypsin and chymotrypsin trigger the shedding and/or detachment of SARS-CoV-2 particles bound to lipid membranes. Shedding kinetics were evaluated upon fluorescence imaging of the labeled virus particles on native membrane mimics. The temporal change in the fluorescence signal, supported by kinetic data from SPR, confirms the fastest shedding of the Delta variant. B.1.1.8 (near Wuhan) and Omicron exhibit comparable shedding kinetics. Membrane-bound Delta variants become laterally mobile upon protease treatment. The other variants are shed from the immobile state. The mean square displacement and probability distribution of lateral displacements of the particles evaluated upon single-particle tracking reveal Brownian yet non-Gaussian diffusion, arising from the intermittent mobility of the Delta variant. These findings are discussed with a model that describes the "hopping motion" of the Delta variant caused by dynamic membrane binding in the presence of a soluble serine protease.

The low scaling factor confirms that cationic LNPs fuse upon mass transfer. Lag time decreases significantly for LNPs containing DOPE or DSPC + Chol helper lipids, enhancing their gene delivery efficacy compared to conventional DOPC/DSPC systems.

The altered tropism and infection severity of the evolved SARS-CoV-2 variants indicate engagement of attachment factors other than the ACE2 receptor for the cellular attachment and entry of the virus. In this work, we report the binding of Omicron, Delta, and B.1.1.8 (A2a type) variants to gangliosides (GD1a, GM3, GM1) with terminal sialic acid (SA). The binding kinetics of intact virus particles to these ganglioside-embedded lipid membranes reveal that the affinity of Omicron for GD1a (two SA residues) is the highest, and the lowest affinity is that of B.1.1.8 for GM1 (one SA at the branched chain). Our TIRF imaging data confirm that SA and acetylated SA can inhibit the virus attachment to the bilayers but at millimolar concentration. We evaluated tetravalent glycoclusters, i.e., sialo-porphyrin, galactose-porphyrin, and glucose-porphyrin, as multivalent inhibitors of SARS-CoV-2. Our results show that membrane attachment of the variants is blocked by the micromolar concentration of sialo-porphyrin. Even the glycocluster effectively inhibits cellular infection caused by the variants.

We demonstrate rapid membrane mixing between GUVs of pure lipid compositions and membrane vesicles (MVs) isolated from the plasma membrane of Vero cells, resulting in the transfer of native lipids and proteins to the GUVs. The steps involved in the membrane mixing are docking followed by membrane fusion. We show that positively charged lipids of the GUVs are essential for the docking, and the native membrane components of MVs drive the fusion. The interleaflet and lateral asymmetry and a change in the membrane tension upon the membrane mixing trigger membrane invagination. We detected outward and inward invagination sites at the rim of the GUVs within 10-40 min of the membrane mixing. The extent of the invaginations depends on the cholesterol and sphingomyelin (SM) contents in the GUVs. Cholesterol content above a critical concentration disfavors membrane invaginations, and the SM lipid is an essential molecular factor for membrane invagination.

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.

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.

Membrane receptors, attachment factors and various other membrane factors play a critical role in the specific attachment of virus particles to a cell surface. Researchers have been employing a range of model systems starting from receptor‐reconstituted lipid membranes to live cells for quantitative evaluation of virus‐receptor/attachment factor interactions. In this work, we show that giant plasma membrane vesicles (GPMVs) derived from adherent cells are a suitable cell‐mimicking model system to examine the binding of SARS‐CoV‐2 to cellular receptors. We evaluated the binding of cell‐cultured SARS‐CoV‐2 to GPMVs by imaging single virus particles with the wide‐field fluorescence microscopy technique. The GPMVs were derived from three different cell lines, i. e., Vero, HEK293T and A549. We compared the number of bound virus particles per cell, cell‐attached GPMV, and cell‐free GPMV of three different cell lines. We explain the observed trend in the data from the expression level of endogenous ACE2 protein and also, conclude that the protein is functional in GPMVs for virus and antibody binding.