Hydroxypropyl-ß-Cyclodextrin Depletes Membrane Cholesterol and Inhibits SARS-CoV-2 Entry into HEK293T-ACEhi Cells.

Vaccination has drastically decreased mortality due to coronavirus disease 19 (COVID-19), but not the rate of acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Alternative strategies such as inhibition of virus entry by interference with angiotensin-I-converting enzyme 2 (ACE2) receptors could be warranted. Cyclodextrins (CDs) are cyclic oligosaccharides that are able to deplete cholesterol from membrane lipid rafts, causing ACE2 receptors to relocate to areas devoid of lipid rafts. To explore the possibility of reducing SARS-CoV-2 entry, we tested hydroxypropyl-ß-cyclodextrin (HPßCD) in a HEK293T-ACE2hi cell line stably overexpressing human ACE2 and Spike-pseudotyped SARS-CoV-2 lentiviral particles. We showed that HPßCD is not toxic to the cells at concentrations up to 5 mM, and that this concentration had no significant effect on cell cycle parameters in any experimental condition tested. Exposure of HEK293T-ACEhi cells to concentrations of HPßCD starting from 2.5 mM to 10 mM showed a concentration-dependent reduction of approximately 50% of the membrane cholesterol content. In addition, incubation of HEK293T-ACEhi cells with HIV-S-CoV-2 pseudotyped particles in the presence of increasing concentrations of HPßCD (from 0.1 to 10 mM) displayed a concentration-dependent effect on SARS-CoV-2 entry efficiency. Significant effects were detected at concentrations at least one order of magnitude lower than the lowest concentration showing toxic effects. These data indicate that HPßCD is a candidate for use as a SARS-CoV-2 prophylactic agent.

Thermoplasmonic Vesicle Fusion Reveals Membrane Phase Segregation of Influenza Spike Proteins.

Many cellular processes involve the lateral organization of integral and peripheral membrane proteins into nanoscale domains. Despite the biological significance, the mechanisms that facilitate membrane protein clustering into nanoscale lipid domains remain enigmatic. In cells, the analysis of membrane protein phase affinity is complicated by the size and temporal nature of ordered and disordered lipid domains. To overcome these limitations, we developed a method for delivering membrane proteins from transfected cells into phase-separated model membranes that combines optical trapping with thermoplasmonic-mediated membrane fusion and confocal imaging. Using this approach, we observed clear phase partitioning into the liquid disordered phase following the transfer of GFP-tagged influenza hemagglutinin and neuraminidase from transfected cell membranes to giant unilamellar vesicles. The generic platform presented here allows investigation of the phase affinity of any plasma membrane protein which can be labeled or tagged with a fluorescent marker.

Altered lipid profile in patients with COVID-19 infection

Background: In this study, we aimed to investigate the pathological alterations of LDL-cholesterol, HDL-cholesterol, total cholesterol and triglycerides in COVID-19 patients during the acute phase of infection, and after recovery. Method(s): A retrospective study was performed to examine serum levels of LDL-cholesterol, HDL-cholesterol, total cholesterol and triglycerides on 55 COVID-19 patients who were hospitalized in our center between February and April 2020. The lipid profile and the hematological parameters were analyzed in the same group of patients before (Group before) and after clinical management (Group after). The laboratory tests results were compared between these two groups, as well as with a group of healthy subjects (Healthy controls), matched for age and sex and selected among the blood donors. Result(s): LDL-cholesterol, HDL-cholesterol, total cholesterol levels were significantly lower in COVID-19 patients (Group before) as compared with normal subjects (P<0.0001). Comparing healthy controls and the group after, statistically significant differences were observed for all parameters except for total cholesterol (P=0.9006). Total cholesterol, HDL-cholesterol, LDL-cholesterol and triglyceride were found to be significantly higher after recovery than during the acute phase of infection (P<0.0001). C-reactive protein levels were found to be inversely correlated with those of LDL-cholesterol (rs =-0573, P<0.0001), total cholesterol (r=-0.732, P<0.0001), and HDL-cholesterol (r=-0.700, P<0.0001). Conclusion(s): The results of our study seemingly attest that lipids, especially cholesterol, may play an important role in viral replication, internalization and immune activation in patients with COVID-19 infection. Moreover, lipid abnormalities observed during and after this infection could be used for assessing indirectly the response to clinical treatment.Copyright © Journal of Laboratory and Precision Medicine. All rights reserved.

Uptake of severe acute respiratory syndrome coronavirus 2 spike protein mediated by angiotensin converting enzyme 2 and ganglioside in human cerebrovascular cells.

Introduction: There is clinical evidence of neurological manifestations in coronavirus disease-19 (COVID-19). However, it is unclear whether differences in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)/spike protein (SP) uptake by cells of the cerebrovasculature contribute to significant viral uptake to cause these symptoms. Methods: Since the initial step in viral invasion is binding/uptake, we used fluorescently labeled wild type and mutant SARS-CoV-2/SP to study this process. Three cerebrovascular cell types were used (endothelial cells, pericytes, and vascular smooth muscle cells), in vitro. Results: There was differential SARS-CoV-2/SP uptake by these cell types. Endothelial cells had the least uptake, which may limit SARS-CoV-2 uptake into brain from blood. Uptake was time and concentration dependent, and mediated by angiotensin converting enzyme 2 receptor (ACE2), and ganglioside (mono-sialotetrahexasylganglioside, GM1) that is predominantly expressed in the central nervous system and the cerebrovasculature. SARS-CoV-2/SPs with mutation sites, N501Y, E484K, and D614G, as seen in variants of interest, were also differentially taken up by these cell types. There was greater uptake compared to that of the wild type SARS-CoV-2/SP, but neutralization with anti-ACE2 or anti-GM1 antibodies was less effective. Conclusion: The data suggested that in addition to ACE2, gangliosides are also an important entry point of SARS-CoV-2/SP into these cells. Since SARS-CoV-2/SP binding/uptake is the initial step in the viral penetration into cells, a longer exposure and higher titer are required for significant uptake into the normal brain. Gangliosides, including GM1, could be an additional potential SARS-CoV-2 and therapeutic target at the cerebrovasculature.

Convergent Evolution Dynamics of SARS-CoV-2 and HIV Surface Envelope Glycoproteins Driven by Host Cell Surface Receptors and Lipid Rafts: Lessons for the Future.

Although very different, in terms of their genomic organization, their enzymatic proteins, and their structural proteins, HIV and SARS-CoV-2 have an extraordinary evolutionary potential in common. Faced with various selection pressures that may be generated by treatments or immune responses, these RNA viruses demonstrate very high adaptive capacities, which result in the continuous emergence of variants and quasi-species. In this retrospective analysis of viral proteins, ensuring the adhesion of these viruses to the plasma membrane of host cells, we highlight many common points that suggest the convergent mechanisms of evolution. HIV and SARS-CoV-2 first recognize a lipid raft microdomain that acts as a landing strip for viral particles on the host cell surface. In the case of mucosal cells, which are the primary targets of both viruses, these microdomains are enriched in anionic glycolipids (gangliosides) forming a global electronegative field. Both viruses use lipid rafts to surf on the cell surface in search of a protein receptor able to trigger the fusion process. This implies that viral envelope proteins are both geometrically and electrically compatible to the biomolecules they select to invade host cells. In the present study, we identify the surface electrostatic potential as a critical parameter controlling the convergent evolution dynamics of HIV-1 and SARS-CoV-2 surface envelope proteins, and we discuss the impact of this parameter on the phenotypic properties of both viruses. The virological data accumulated since the emergence of HIV in the early 1980s should help us to face present and future virus pandemics.

Electrostatic Surface Potential as a Key Parameter in Virus Transmission and Evolution: How to Manage Future Virus Pandemics in the Post-COVID-19 Era.

Virus-cell interactions involve fundamental parameters that need to be considered in strategies implemented to control viral outbreaks. Among these, the surface electrostatic potential can give valuable information to deal with new epidemics. In this article, we describe the role of this key parameter in the hemagglutination of red blood cells and in the co-evolution of synaptic receptors and neurotransmitters. We then establish the functional link between lipid rafts and the electrostatic potential of viruses, with special emphasis on gangliosides, which are sialic-acid-containing, electronegatively charged plasma membrane components. We describe the common features of ganglioside binding domains, which include a wide variety of structures with little sequence homology but that possess key amino acids controlling ganglioside recognition. We analyze the role of the electrostatic potential in the transmission and intra-individual evolution of HIV-1 infections, including gatekeeper and co-receptor switch mechanisms. We show how to organize the epidemic surveillance of influenza viruses by focusing on mutations affecting the hemagglutinin surface potential. We demonstrate that the electrostatic surface potential, by modulating spike-ganglioside interactions, controls the hemagglutination properties of coronaviruses (SARS-CoV-1, MERS-CoV, and SARS-CoV-2) as well as the structural dynamics of SARS-CoV-2 evolution. We relate the broad-spectrum antiviral activity of repositioned molecules to their ability to disrupt virus-raft interactions, challenging the old concept that an antibiotic or anti-parasitic cannot also be an antiviral. We propose a new concept based on the analysis of the electrostatic surface potential to develop, in real time, therapeutic and vaccine strategies adapted to each new viral epidemic.

A Vaccine Strategy Based on the Identification of an Annular Ganglioside Binding Motif in Monkeypox Virus Protein E8L.

The recent outbreak of Monkeypox virus requires the development of a vaccine specifically directed against this virus as quickly as possible. We propose here a new strategy based on a two-step analysis combining (i) the search for binding domains of viral proteins to gangliosides present in lipid rafts of host cells, and (ii) B epitope predictions. Based on previous studies of HIV and SARS-CoV-2 proteins, we show that the Monkeypox virus cell surface-binding protein E8L possesses a ganglioside-binding motif consisting of several subsites forming a ring structure. The binding of the E8L protein to a cluster of gangliosides GM1 mimicking a lipid raft domain is driven by both shape and electrostatic surface potential complementarities. An induced-fit mechanism unmasks selected amino acid side chains of the motif without significantly affecting the secondary structure of the protein. The ganglioside-binding motif overlaps three potential linear B epitopes that are well exposed on the unbound E8L surface that faces the host cell membrane. This situation is ideal for generating neutralizing antibodies. We thus suggest using these three sequences derived from the E8L protein as immunogens in a vaccine formulation (recombinant protein, synthetic peptides or genetically based) specific for Monkeypox virus. This lipid raft/ganglioside-based strategy could be used for developing therapeutic and vaccine responses to future virus outbreaks, in parallel to existing solutions.

Serum Proteomics and Metabolomics Study on Susceptibility to Sars-Cov-2 Infection in Ibd Patients

Introduction: Since 2020 COVID-19 pandemic has spread throughout the world and became an ongoing global health crisis due to SARS-CoV-2 virus. Elderly and pre-existing disorders including hypertension, heart problems, diabetes, cancer, autoimmune diseases and IBD are found associated with an increased risk of COVID-19. Although COVID-19 leads to mild flu-like symptoms in the majority of patients, the disease may cause severe complications and death. To date, a few clinical studies suggested that IBD and/or immunomodulation may reduce the susceptibility to COVID-19;however, the mechanisms through which this is happening is largely unknown. Aims & Methods: Aim of this study is to investigate the effects of IBD and different therapies on the risk of SARS-CoV-2 infection and COVID-19 severity through serum proteomics and metabolomics. Between April 2020 and April 2022, 238 IBD patients (N=145 Crohn disease, N=93 Ulcerative colitis) and 45 healthy controls (HC) of the North Italy area were enrolled and serum samples were collected. To evaluate the exposure to SARSCoV- 2, both clinical data were collected and seroprevalence of anti-SARSCoV- 2 Ab were analyzed by means of multiplex technology, the BioPlex 2200 Sars-Cov-2 IgG Panel (biorad, Italy). Serum samples underwent untargeted metabolomics analysis and the frequency of a serum metabolomics signature associated with protection were evaluated in IBD compared to HC and also between anti-TNF and Vedolizumab biological therapies for IBD patients. Result(s): The seroprevalence of anti-SARS-CoV-2 Ab in IBD cohort (22/238) indicates an overall lower incidence of COVID-19 in comparison with the general population of Lombardy. Our data indicated that IBD patients in treated with biologic drugs as anti-TNF (10,5%) and Vedolizumab (7,5%) have a lower incidence than IBD patients treated with conventional therapies (28,0%). Accordingly, we observed that serum metabolomics signature associated with protection was more frequent in IBD patients treated with anti-TNF (N=50, 70%), and with Vedolizumab (N=57, 85%) than healthy controls (N=45, 50%). The metabolomic protective profile is characterized by the presence of fat-soluble Tocopherols family members and Cholecalciferol and also of omega-3 and omega-6 polyunsaturated fatty acid. Conclusion(s): Our study indicates that IBD population treated with biologics has an overall lower risk to contract SARS-CoV-2 infection and a serum proteomic/metabolomic protection profile. The increased presence in IBD patients of radical scavengers such as tocopherols which are incorporated into cell membranes and protect against oxidative damage and anti-inflammatory and immunomodulating fatty acids suggest a better response to SARS-CoV-2 infection. Also increased levels of omega;-3 interfere with the entry of the virus by modulating the Lipid Rafts where ACE2 and TMPRSS2 are mainly expressed and PUFAs inhibit the attachment of SARS-CoV-2 virions to the human ACE2 receptor by interacting directly with the RBD sequence. Mechanistically understanding how this protection profile exerts its effects on COVID-19 severity might shed light on potential targets to increase resistance in higher risk subgroups of patients.

Studies in the antiviral molecular mechanisms of 25-hydroxycholesterol: Disturbing cholesterol homeostasis and post-translational modification of proteins.

Efficient antiviral drug discovery has been a pressing issue of global public health concern since the outbreak of coronavirus disease 2019. In recent years, numerous in vitro and in vivo studies have shown that 25-hydroxycholesterol (25HC), a reactive oxysterol catalyzed by cholesterol-25-hydroxylase, exerts broad-spectrum antiviral activity with high efficiency and low toxicity. 25HC restricts viral internalization and disturbs the maturity of viral proteins using multiple mechanisms. First, 25HC reduces lipid rafts and cholesterol in the cytomembrane by inhibiting sterol-regulatory element binding proteins-2, stimulating liver X receptor, and activating Acyl-coenzyme A: cholesterol acyl-transferase. Second, 25HC impairs endosomal pathways by restricting the function of oxysterol-binding protein or Niemann-pick protein C1, causing the virus to fail to release nucleic acid. Third, 25HC disturbs the prenylation of viral proteins by suppressing the sterol-regulatory element binding protein pathway and glycosylation by increasing the sensitivity of glycans to endoglycosidase. This paper reviews previous studies on the antiviral activity of 25HC in order to fully understand its role in innate immunity and how it may contribute to the development of urgently needed broad-spectrum antiviral drugs.

Identification of a Conserved Intracellular Loop (CIL) Structure That Scaffolds PIP3 to Amplify Oncogenic Signaling during Malignant B-Cell Transformation

Background: Within seconds of antigen-encounter, B-cell receptor (BCR) signaling induces dramatic changes of cell membrane lipid composition, including >40-fold increases of local PIP3-concentrations within lipid rafts. While several structural elements, including pleckstrin homology (PH) domains have been identified as PIP3-binding proteins, the underlying mechanisms that amplify BCR-signaling to assemble large signaling complexes within lipid rafts within 15 to 30 seconds, remained elusive. To understand the mechanistic and biophysical requirements for PIP3 accumulation during normal B-cell activation and acute oncogenic transformation, we identified PIP3-interacting proteins by cell-surface proteomic analyses. Results: In addition to proteins known to bind PIP3 with their PH-domains, we identified the short 133 aa protein IFITM3 (interferon-inducible transmembrane protein 3) as a top-ranking PIP3 scaffold. This was unexpected because IFITM3 was previously identified as endosomal protein that blocks viral infection by stiffening endosomal membranes to firmly contain viral cargo. Previous studies revealed that polymorphisms that lead to the expression of truncated IFITM3 are associated with increased susceptibility to viral infections, including SARS-CoV2. Among known cell membrane lipids, PIP3 has the highest negative charge. Instead of a PH-domain, IFITM3 laterally sequestered PIP3 through electrostatic interactions with two basic lysine residues (K83 and K104) located at the membrane-solution interface. Together with three other basic lysine and arginine residues K83 and K104 form a conserved intracellular loop (CIL), which enable IFITM3 to efficiently capture two PIP3 molecules. Bivalent PIP3-binding of the IFITM3-CIL enables a crosslinking mechanism that results in dramatic amplification of B-cell activation signals and clustering of large signaling complexes within lipid rafts. In normal resting B-cells, Ifitm3 was minimally expressed and mainly localized in endosomes. However, B-cell activation and oncogenic kinases induced phosphorylation at IFITM3-Y20, resulting in translocation of IFITM3 from endosomes and massive accumulation at the cell surface. Ifitm3ˉ /ˉ naïve B-cells developed at normal numbers, however, activation by antigen encounter was compromised. In Ifitm3ˉ /ˉ B-cells, lipid rafts were depleted of PIP3, resulting in defective expression of >60 lipid raft-associated surface receptors and impaired PI3K-signaling. Ifitm3ˉ /ˉ B-cells were unable to undergo affinity maturation and di not contribute to germinal center formation upon immunization. Analyses of gene expression and clinical outcome data from patients in six clinical cohorts for pediatric and adult B-ALL, mantle cell lymphoma, CLL and DLBCL, we consistently identified IFITM3 as a top-ranking predictor of poor clinical outcome. Inducible activation of BCR-ABL1 and NRAS G12D rapidly induced development of B-ALL but failed to transform and initiate B-ALL from Ifitm3ˉ /ˉ B-cell precursors. Conversely, the phospho-mimetic IFITM3-Y20E mutation, mimicking phosphorylation of the IFITM3 N-terminus at Y20 induced constitutive membrane localization of IFITM3, spontaneous aggregation of large oncogenic signaling complexes and readily initiated transformation in a genetic model of pre-malignant B-cells. Conclusions: We conclude that phosphorylation of IFITM3 upon B-cell activation induces a dynamic switch from antiviral effector functions in endosomes to oncogenic signal-amplification at the cell-surface. IFITM3-dependent amplification of PI3K-signaling is critical to enable rapid expansion of activated B-cells. In addition, multiple oncogenes depend on IFITM3 to assemble PIP3-dependent signaling complexes and amplify PI3K-signaling for malignant transformation and initiation of B-lymphoid leukemia and lymphoma. [Formula presented] Disclosures: Weinstock: SecuraBio: Consultancy;ASELL: Consultancy;Bantam: Consultancy;Abcuro: Research Funding;Verastem: Research Funding;Daiichi Sankyo: Consultancy, Research Funding;AstraZeneca: Consultanc ;Travera: Other: Founder/Equity;Ajax: Other: Founder/Equity.

Ganglioside binding domains in proteins: Physiological and pathological mechanisms.

Gangliosides are anionic lipids that form condensed membrane clusters (lipid rafts) and exert major regulatory functions on a wide range of proteins. In this review, we propose a new view of the structural features of gangliosides with special emphasis on emerging properties associated with protein binding modes. We analyze the different possibilities of molecular associations of gangliosides in lipid rafts and the role of cholesterol in this organization. We are particularly interested in amide groups of N-acetylated sugars which make it possible to neutralize the negative charge of the carboxylate group of sialic acids. We refer to this effect as "NH trick" and we demonstrate that it is operative in GM1, GD1a, GD1b and GT1b gangliosides. The NH trick is key to understand the different topologies adopted by gangliosides (chalice-like at the edge of lipid rafts, condensed clusters in central areas) and their impact on protein binding. We define three major types of ganglioside-binding domains (GBDs): α-helical, loop shaped, and large flat surface. We describe the mode of interaction of each GBD with typical reference proteins: synaptotagmin, 5HT1A receptor, cholera and botulinum toxins, HIV-1 surface envelope glycoprotein gp120, SARS-CoV-2 spike protein, cellular prion protein, Alzheimer's ß-amyloid peptide and Parkinson's disease associated α-synuclein. We discuss the common mechanisms and peculiarities of protein binding to gangliosides in the light of physiological and pathological conditions. We anticipate that innovative ganglioside-based therapies will soon show an exponential growth for the treatment of cancer, microbial infections, and neurodegenerative diseases.

Structural dynamics of SARS-CoV-2 variants: A health monitoring strategy for anticipating Covid-19 outbreaks.

OBJECTIVES: the Covid-19 pandemic has been marked by sudden outbreaks of SARS-CoV-2 variants harboring mutations in both the N-terminal (NTD) and receptor binding (RBD) domains of the spike protein. The goal of this study was to predict the transmissibility of SARS-CoV-2 variants from genomic sequence data. METHODS: we used a target-based molecular modeling strategy combined with surface potential analysis of the NTD and RBD. RESULTS: we observed that both domains act synergistically to ensure optimal virus adhesion, which explains why most variants exhibit concomitant mutations in the RBD and in the NTD. Some mutation patterns affect the affinity of the spike protein for ACE-2. However, other patterns increase the electropositive surface of the spike, with determinant effects on the kinetics of virus adhesion to lipid raft gangliosides. Based on this new view of the structural dynamics of SARS-CoV-2 variants, we defined an index of transmissibility (T-index) calculated from kinetic and affinity parameters of coronavirus binding to host cells. The T-index is characteristic of each variant and predictive of its dissemination in animal and human populations. CONCLUSIONS: the T-index can be used as a health monitoring strategy to anticipate future Covid-19 outbreaks due to the emergence of variants of concern.

Leveraging coronavirus binding to gangliosides for innovative vaccine and therapeutic strategies against COVID-19.

Covid-19 is an infectious respiratory disease due to a coronavirus named SARS-CoV-2. A critical step of the infection cycle is the binding of the virus spike S protein to the cellular ACE-2 receptor. This interaction involves a receptor binding domain (RBD) located at the center of the S trimer, whereas the lateral N-terminal domain (NTD) displays a flat ganglioside binding site that enables the virus to bind to lipid rafts of the plasma membrane, where the ACE-2 receptor resides. S protein binding to lipid rafts can be blocked by hydroxychloroquine, which binds to gangliosides, and by azithromycin, which binds to the NTD. Based on these data, we identified the NTD of SARS-CoV-2 as a promising target for both therapeutic and vaccine strategies, a notion later supported by the discovery, in convalescent Covid-19 patients, of a neutralizing antibody (4A8) that selectively binds to the NTD. The 4A8 epitope overlaps the ganglioside binding domain, denying any access of the virus to lipid rafts when the antibody is bound to the S protein. Thus, our data explain why antibody binding to the tip of the NTD results in SARS-CoV-2 neutralization. The high level of conservation of the ganglioside binding domain of SARS-CoV-2 (100% identity in 584 of 600 isolates analyzed worldwide) offers unique opportunities for innovative vaccine/therapeutic strategies.