Role of lipids in HIV-1 pathogenesis. Implications in viral infectivity and development of antiretroviral drugs.

El sumario, el resumen y el capítulo 5 están sujetos a confidencialidad por el autor. 283 p.En esta tesis doctoral se ha estudiado el papel de los lípidos en distintos pasos del ciclo infectivo del VIH-1, y su potencial uso para el desarrollo de agentes antiretrovirales. Los resultados de este trabajo demuestran que la proteína gp41 del VIH-1 interacciona con colesterol tanto en la membrana plasmática como en la membrana viral, y que las secuencias LLP de la cola citoplásmatica de la proteína están involucradas en esta interacción.También se ha estudiado el mecanismo de acción de compuestos antirretrovirales sintéticos similares a lípidos naturales, conocidos como lipidomiméticos. Se ha descubierto que éstos llevan a cabo su acción antirretroviral inhibiendo el paso de entrada al alterar la estructura de la membrana viral y el estado de empaquetamiento de los lípidos. En esta tesis se desarrolló también un sistema de nanopartículas con gangliósidos que pueden ser específicamente focalizadas a células dendríticas maduras y transferidas a células latentemente infectadas mediante la ruta gangliósido/Siglec-1 para el transporte de agentes reactivadores de latencia, con el fin de purgar los reservorios virales que establecen una infección latente

Raman Spectroscopy as a Tool to Study the Pathophysiology of Brain Diseases

The Raman phenomenon is based on the spontaneous inelastic scattering of light, which depends on the molecular characteristics of the dispersant. Therefore, Raman spectroscopy and imaging allow us to obtain direct information, in a label-free manner, from the chemical composition of the sample. Since it is well established that the development of many brain diseases is associated with biochemical alterations of the affected tissue, Raman spectroscopy and imaging have emerged as promising tools for the diagnosis of ailments. A combination of Raman spectroscopy and/or imaging with tagged molecules could also help in drug delivery and tracing for treatment of brain diseases. In this review, we first describe the basics of the Raman phenomenon and spectroscopy. Then, we delve into the Raman spectroscopy and imaging modes and the Raman-compatible tags. Finally, we center on the application of Raman in the study, diagnosis, and treatment of brain diseases, by focusing on traumatic brain injury and ischemia, neurodegenerative disorders, and brain cancer.The APC was funded by grant PID2020-117405GB100, funded by MCIN/AEI/10.13039/501100011033 and, as appropriate, by “ERDF A way of making Europe” by the “European Union” or by the “European Union NextGenerationEU/PRTR”; by the Basque Government, grant numbers ELKARTEK22/86 and IT1625-22; and by Fundación Ramón Areces, grant number CIVP20S11276

Novel Methodology for the Detection of Enveloped Viruses

Presented at Viruses 2020—Novel Concepts in Virology, Barcelona, Spain, 5–7 February 2020 (abstract)Viral infections in humans cause a huge burden in worldwide healthcare that has increased due to the emergence of new pathogenic viruses, such as in the recent Ebola virus (EBOV) outbreaks. Viral particles in body fluids are often at very low levels, making diagnosis difficult. In order to address this problem, we have developed a new detection platform to isolate and detect different enveloped viruses. We have recently identified that sialic acid-binding Ig‑like lectin 1 (Siglec-1/CD169) is one cellular receptor used by EBOV and HIV-1 to enter myeloid cells, key target cells for infection and pathogenesis. For viral uptake, the V-set domain of this myeloid cell receptor recognizes the gangliosides of viral membranes that were dragged during viral budding from the plasma membrane of infected cells. We took advantage of this specific interaction between Siglec‑1 and viral gangliosides to develop a new detection methodology. We have generated a recombinant protein that contains the V-set domain of Siglec-1 fused to the human IgG Fc domain for anchoring in latex beads. These coated beads allow the isolation of viral particles and their measurement by flow cytometry. We have tested its efficacy to detect HIV-1 and EBOV and its specificity by using anti-Siglec‑1 antibodies that prevent the interaction and serve as a negative control. To test the capacity of our method, we used synthetic liposomes to assess the effect of ganglioside concentration in membranes as well as the size of viral particles. This methodology would facilitate the diagnosis of infections by concentrating viral particles in a fast and direct method. At a time when global human mobility facilitates the dissemination of infectious agents, our approach represents a rapid and effective method to maximize the identification of both known and emerging enveloped viruses as part of public health viral surveillance strategies

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV-1 Env Clustering

HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751-854 in the cytoplasmic tail (CT751-854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751-854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.The authors are grateful to Barbara Müller, N. Landau, and Tom Hope for providing the plasmids pCHIV and pCAGGS NL4-3 Env, pMM310, and peGFP-Vpr, respectively. Proteomic analysis was performed by the SGIKER service of the University of the Basque Country. The authors would like to thank Advanced Light Microscopy Unit at the Centre for Genomic Regulation (CRG), Barcelona, Spain for the access to Leica STED microscope. The following reagents were obtained through the NIH AIDS Reagent Program (Division of NIAID, NIH): Anti-HIV-1 gp41 Hybridome (Chessie 8) (Cat# 526) from Dr. George Lewis; Antiviral bicyclam JM-2987 (hydrobromide salt of AMD-3100) from NIAID, DAIDS (cat# 8128). This project was supported by the Basque Government (grant number IT1264-19 to M.L. and F.-X.C.) and the Spanish Ministry of Science, Innovation, and Universities (BFU-2015-68981-P). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.-G. was supported by a FI predoctoral fellowship from the Basque Government and currently by Fundación Biofísica Bizkaia. A.A. was supported by Fundación Biofísica Bizkaia. S.O. was supported by an IKASIKER fellowship from the Basque Government. J.C. was supported by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 793830. H.G.-K. was supported by a grant from the Deutsche Forschungsgemeischaft within TRR86

Identification of a New Cholesterol-Binding Site within the IFN-gamma Receptor that is Required for Signal Transduction

[EN] The cytokine interferon-gamma (IFN-gamma) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN-gamma exerts it signaling action by binding to a specific cell surface receptor, the IFN-gamma receptor (IFN-gamma R), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN-gamma R signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN-gamma R2 chains into plasma membrane lipid nanodomains, orchestrating IFN-gamma R oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol-binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN-gamma R transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol-IFN gamma R2 interaction may represent a potential therapeutic strategy for various IFN-gamma-dependent diseases.This work was supported by grants from the Spanish Ministry of Science, Innovation, and Universities (BFU-2015-68981-P and PID2020-117405GB-I00) and the Basque Government (IT1264-19, IT1625-22) to F.-X.C. and M.L. F.-X.C. acknowledge the generous support of Fundacion Ramon Areces (grant CIVP20S11276). O.T. was supported by a Basque Government grant (IT1270-19) I.R.-B., O.M., J.A.N.-G., and D.C. were supported by the Fundacion Biofisica Bizkaia. The Lamaze laboratory was supported from Agence Nationale de la Recherche grants ANR-11-LABX-0038, ANR-10-IDEX-0001-02, and ANR NanoGammaR-15-CE11-0025-01. The Bernardino de la Serna Lab acknowledges support from Belinda and Bill Gates Foundation and BBSRC (INV-016631 and BB/V019791/1, respectively). This work was supported in part by the Fundacion Biofisica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. The authors thank J. M. Gonzalez Manas for helpful comments on the manuscript. The authors thank the technical and human support provided by the analytical and high-resolution microscopy facility (SGIker) of UPV/EHU and European funding (ERDF and ESF)

Super-Resolution Microscopy Using a Bioorthogonal-Based Cholesterol Probe Provides Unprecedented Capabilities for Imaging Nanoscale Lipid Heterogeneity in Living Cells

Despite more than 20 years of work since the lipid raft concept was proposed, the existence of these nanostructures remains highly controversial due to the lack of noninvasive methods to investigate their native nanorganization in living unperturbed cells. There is an unmet need for probes for direct imaging of nanoscale membrane dynamics with high spatial and temporal resolution in living cells. In this paper, a bioorthogonal-based cholesterol probe (chol-N-3) is developed that, combined with nanoscopy, becomes a new powerful method for direct visualization and characterization of lipid raft at unprecedented resolution in living cells. The chol-N-3 probe mimics cholesterol in synthetic and cellular membranes without perturbation. When combined with live-cell super-resolution microscopy, chol-N-3 demonstrates the existence of cholesterol-rich nanodomains of <50 nm at the plasma membrane of resting living cells. Using this tool, the lipid membrane structure of such subdiffraction limit domains is identified, and the nanoscale spatiotemporal organization of cholesterol in the plasma membrane of living cells reveals multiple cholesterol diffusion modes at different spatial localizations. Finally, imaging across thick organ samples outlines the potential of this new method to address essential biological questions that were previously beyond reach.M.L., O.T., and J.A.N.-G. contributed equally to this work. This work was supported by grants from the Spanish Ministry of Science Innovation and Universities, (Grant No. BFU-2015-68981-P) and the Basque Government (Grant No. IT1264-19) to F.-X.C. and M.L.. The authors thank J. M. Gonzalez Manas and Sergio Perez Acebron for helful comments on the manuscript. The authors thank the technical and human support provided by the analytical and high-resolution microscopy facility (SGIker) of UPV/EHU and European funding (ERDF and ESF). J.B.d.l.S. acknowledges funding from the Bill and Melinda Gates Foundation and the BBSRC (Grant Nos. INV-016631 and BB/V019791/1, respectively). This work was supported in part by the Fundacion Biofisica Bizkaia (FBB) and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.-G. was supported by a FI predoctoral fellowship from the Basque Government and currently by FBB. Documen

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development

Super-Resolution Microscopy to Study Interorganelle Contact Sites

Interorganelle membrane contact sites (MCS) are areas of close vicinity between the membranes of two organelles that are maintained by protein tethers. Recently, a significant research effort has been made to study MCS, as they are implicated in a wide range of biological functions, such as organelle biogenesis and division, apoptosis, autophagy, and ion and phospholipid homeostasis. Their composition, characteristics, and dynamics can be studied by different techniques, but in recent years super-resolution fluorescence microscopy (SRFM) has emerged as a powerful tool for studying MCS. In this review, we first explore the main characteristics and biological functions of MCS and summarize the different approaches for studying them. Then, we center on SRFM techniques that have been used to study MCS. For each of the approaches, we summarize their working principle, discuss their advantages and limitations, and explore the main discoveries they have uncovered in the field of MCS

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV‐1 Env Clustering

HIV‐1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751–854 in the cytoplasmic tail (CT751–854). Super‐resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751–854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.The authors are grateful to Barbara Müller, N. Landau, and Tom Hope for providing the plasmids pCHIV and pCAGGS NL4‐3 Env, pMM310, and peGFP‐Vpr, respectively. Proteomic analysis was performed by the SGIKER service of the University of the Basque Country. The authors would like to thank Advanced Light Microscopy Unit at the Centre for Genomic Regulation (CRG), Barcelona, Spain for the access to Leica STED microscope. The following reagents were obtained through the NIH AIDS Reagent Program (Division of NIAID, NIH): Anti‐HIV‐1 gp41 Hybridome (Chessie 8) (Cat# 526) from Dr. George Lewis; Antiviral bicyclam JM‐2987 (hydrobromide salt of AMD‐3100) from NIAID, DAIDS (cat# 8128). This project was supported by the Basque Government (grant number IT1264‐19 to M.L. and F.‐X.C.) and the Spanish Ministry of Science, Innovation, and Universities (BFU‐2015‐68981‐P). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.‐G. was supported by a FI predoctoral fellowship from the Basque Government and currently by Fundación Biofísica Bizkaia. A.A. was supported by Fundación Biofísica Bizkaia. S.O. was supported by an IKASIKER fellowship from the Basque Government. J.C. was supported by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No. 793830. H.G.‐K. was supported by a grant from the Deutsche Forschungsgemeischaft within TRR86.Peer reviewe

Actin-regulated Siglec-1 nanoclustering influences HIV-1 capture and virus-containing compartment formation in dendritic cells

The immunoglobulin-like lectin receptor CD169 (Siglec-1) mediates the capture of HIV-1 by activated dendritic cells (DCs) through binding to sialylated ligands. These interactions result in a more efficient virus capture as compared to resting DCs, although the underlying mechanisms are poorly understood. Using a combination of super-resolution microscopy, single-particle tracking and biochemical perturbations we studied the nanoscale organization of Siglec-1 on activated DCs and its impact on viral capture and its trafficking to a single viral-containing compartment. We found that activation of DCs leads to Siglec-1 basal nanoclustering at specific plasma membrane regions where receptor diffusion is constrained by Rho-ROCK activation and formin-dependent actin polymerization. Using liposomes with varying ganglioside concentrations, we further demonstrate that Siglec-1 nanoclustering enhances the receptor avidity to limiting concentrations of gangliosides carrying sialic ligands. Binding to either HIV-1 particles or ganglioside-bearing liposomes lead to enhanced Siglec-1 nanoclustering and global actin rearrangements characterized by a drop in RhoA activity, facilitating the final accumulation of viral particles in a single sac-like compartment. Overall, our work provides new insights on the role of the actin machinery of activated DCs in regulating the formation of basal Siglec-1 nanoclustering, being decisive for the capture and actin-dependent trafficking of HIV-1 into the virus-containing compartment