Biology subjects

Padilla-Parra, S.

Publications and source records attributed to Padilla-Parra, S..

15 recordsLinked to original sources

bioRxiv2026

Gag proteolysis drives HIV-1 envelope conformational closure through the gp41 cytoplasmic tail

HIV-1 must present a fusion-competent envelope glycoprotein (Env) to enter host cells while concealing the very conformation targeted by broadly neutralising antibodies (bnAbs). How the virus resolves this conflict has remained unclear. Using a minimally invasive nanobody-labelling strategy targeting Env's V4 loop, we combine single-virus FRET, fluorescence lifetime cross-correlation spectroscopy, and DNA-PAINT super-resolution microscopy to resolve Env conformation on individual, intact virions from the primary isolate JR-FL. We show that immature virions predominantly display an open Env conformation, while Gag maturation drives acquisition of the tightly closed, fusion-competent State 1 that harbours the principal bnAb epitope. Truncating the Env cytoplasmic tail abolishes this regulation, identifying the gp41 tail as the conduit linking Gag lattice maturation to Env's conformational fate. We further show that bnAb b12 preferentially binds open Env on immature virions, supporting a decoy mechanism that shields mature virions from immune recognition, with direct implications for HIV-1 vaccine design.

biophysics↗

bioRxiv2026

Structural analysis reveals high-order Nipah Virus Fusion Protein assemblies and interactions with neutralising nanobodies

Nipah virus (NiV) is a highly pathogenic emerging henipavirus for which there are no licensed vaccines or therapeutics currently available. Viral entry is mediated by the coordinated action of the attachment (NiVG) and fusion (NiVF) glycoproteins yet the organisation of NiVF on viral particles and its influence on antibody neutralisation is not completely understood. Using cryo-electron tomography of NiV pseudovirions and virus-like particles (VLPs), we directly observe inter-F contacts on viral membranes. These interactions occur frequently but are distributed stochastically, forming variably sized assemblies rather than ordered arrays, consistent with transient or low-affinity associations. We have generated single domain antibodies (termed nanobodies) to the NiVF glycoprotein that bind to an epitope at the interface between neighbouring NiVF trimers and therefore occlude the inter-NiVF contacts. We show that these nanobodies inhibit VLP cell fusion and neutralise NiV pseudoviruses in vitro, which suggests that the interaction between NiVF trimers is important for NiV virus entry. The identification of the inter-NiVF contacts on viral particles adds a previously unrecognised structural feature to the organisation of the NiV entry machinery which could be targeted to prevent viral infection.

molecular biology↗

bioRxiv2026

MX2 Mediates Collapse of the HIV-1 Capsid

The HIV-1 capsid core encapsulates the viral genome and mediates its delivery into the host cells nucleus. It is composed of multiple copies of the Capsid (CA, p24Gag) protein, assembled into hexamers and pentamers to create a lattice that forms a fullerene-like cone. Myxovirus resistance 2 (MX2) is an HIV-1 restriction factor that binds to the capsid core and blocks nuclear import of the viral genome. Here, we define a minimal region of MX2 required for HIV-1 restriction and produce a corresponding functional recombinant protein. We have used cryo-electron microscopy to determine the structure of this MX2 fragment bound to the tri-hexamer interface of the capsid lattice, revealing a large, buried interface combining electrostatic and hydrophobic interactions. This structure, together with assays that measure capsid core destabilisation, shows that MX2 binding induces conformational rearrangements in the capsid lattice that culminate in a loss of integrity. These results support a model whereby MX2 exerts its antiviral activity by disrupting the capsid lattice, inducing premature fragmentation and preventing HIV-1 nuclear import. By revealing the structural basis for MX2-mediated restriction, this work also provides the framework for the development of anti-HIV molecules that mimic MX2 restriction.

microbiology↗

bioRxiv2025

Early HIV-1 maturation drives Env clustering and fusion competence

Human immunodeficiency virus type 1 (HIV-1) particles are initially released from the cell in an immature state that cannot cause infection. The proteolytic processing of the Gag structural polyprotein into its individual components triggers a dramatic structural reorganisation that produces fully mature and infectious virions. HIV-1 maturation remodels the Gag lattice to enable membrane fusion, but how early proteolytic steps regulate the envelope glycoprotein (Env) organisation is unknown. Although cryo-electron tomography has defined structural intermediates of maturation, it has not resolved how Env conformers relate to specific early maturation states. Here, we show that partial Gag cleavage, prior to capsid formation, allosterically reorganises the membrane-proximal lattice, promoting Env clustering and fusion-competent conformations. Using native virus preparations combined with cryogenic fluorescence-lifetime imaging correlated to cryo-electron tomography, we map Env conformational signatures onto defined Gag-processing intermediates. This reveals an unrecognised early maturation stage in which incomplete Gag processing primes Env for entry, shifting current models by positioning functional Env activation upstream of core maturation. These results establish that early lattice remodelling is a key determinant of HIV-1 fusion competence and expose a previously inaccessible, intervention-sensitive step with implications for therapeutic and vaccine strategies targeting maturation-entry coupling.

microbiology↗

bioRxiv2025

Alpha-BET: Functional labeling of envelope glycoproteins with single domain antibodies for in-virus single molecule imaging

We present Alpha-BET, a structure-guided strategy leveraging AlphaFold to identify optimal ALFA-tag insertion sites for minimally disruptive labeling of viral glycoproteins with high-affinity nanobodies. Applied to HIV-1 Env, SARS-CoV-2 S, and NiV G, Alpha-BET preserves structural integrity and function. For HIV-1 Env, we demonstrate super-resolution DNA-PAINT MINFLUX 3D imaging enabled by tag insertion, showcasing its power for visualizing native trimers in single virions and potential for broader applications in virus research.

biophysics↗

bioRxiv2023

Early steps of multi-receptor viral interactions dissected by high-density, multi-color single molecule mapping in living cells

Direct visualization of the early steps of multi-receptor viral interactions at the singlemolecule level has been largely impeded by the technical challenges associated to imaging individual multi-molecular systems at relevant spatial (nanometer) and temporal (millisecond) scales. Here, we present a four-color, high-density single-molecule spatiotemporal mapping methodology to capture real-time interactions between individual viruses and three different viral (co-)receptors on the membrane of living immune cells derived from donors. Together with quantitative tools, our approach revealed the existence of a coordinated spatiotemporal diffusion of the three different (co-)receptors prior to viral-engagement. By varying the temporal-windows of cumulated single-molecule localizations, we discovered that such a concerted diffusion impacts on the residence time of viruses on the host membrane and potential viral infectivity. Overall, our methodology opens a new door to the systematic analysis of the initial steps of viralhost interactions and paves the way to the investigation of other multi-molecular systems at the single-molecule level.

biophysics↗

bioRxiv2022

Live Cell Multicolour Lifetime Imaging Using Genetically Encodable Fluorophores

Nine fluorescent proteins (FPs) simultaneous imaging has been demonstrated in a single acquisition using fluorescence lifetime imaging microscopy (FLIM) combined with pulsed interleaved excitation (PIE) for three laser lines. We also show how to unmix spectrally similar FPs in a pixel-by-pixel manner with an analytical non-fitting solution.

biophysics↗

bioRxiv2020

Glycolysis downregulation is a hallmark of HIV-1 latency and sensitizes infected cells to oxidative stress

HIV-1 infects lymphoid and myeloid cells, which can harbor a latent proviral reservoir responsible for maintaining lifelong infection. Glycolytic metabolism has been identified as a determinant of susceptibility to HIV-1 infection, but its role in the development and maintenance of HIV-1 latency has not been elucidated. By combining transcriptomic, proteomic and metabolomic analysis, we here show that transition to latent HIV-1 infection downregulates glycolysis, while viral reactivation by conventional stimuli reverts this effect. Decreased glycolytic output in latently infected cells is associated with downregulation of NAD+/NADH. Consequently, infected cells rely on the parallel pentose phosphate pathway and its main product, the antioxidant NADPH, fueling antioxidant pathways maintaining HIV-1 latency. Of note, blocking NADPH downstream effectors, thioredoxin and glutathione, favors HIV-1 reactivation from latency in lymphoid and myeloid cellular models. This provides a "shock and kill effect" decreasing proviral DNA in cells from people-living-with-HIV/AIDS. Overall, our data show that downmodulation of glycolysis is a metabolic signature of HIV-1 latency that can be exploited to target latently infected cells with eradication strategies.

immunology↗

bioRxiv2020

Structure and Dynamics of HIV-1 Env Trimers on Native Virions Engaged in Living T Cells

The HIV-1 envelope glycoprotein (Env) mediates viral entry into the host cell. Although the highly dynamic nature of Env intramolecular conformations has been shown with single molecule spectroscopy in vitro, the bona fide Env intra- and intermolecular mechanics when engaged in live T cells remains unknown. We used both, two photon fast fluorescence lifetime imaging detection of single-molecule Forster Resonance Energy Transfer and single molecule photobleaching to reveal transitions between intramolecular and intermolecular conformations that mediate Env clustering. Furthermore, we show that three broad neutralizing anti-Env antibodies directed to different epitopes destabilise Env intramolecular dynamics and their clusters when engaged to living T cells. Importantly, our results show that Env clustering is a common conformation across different HIV-1 Env strains, which depends on efficient virus maturation, and that is disrupted upon binding of Env to CD4 or to neutralizing antibodies. Thus, this study illuminates how different intramolecular conformations and clusters of Env mediate HIV-1 Env-T cell interactions in real time and therefore might control immune evasion.

immunology↗

bioRxiv2020

Homology-guided identification of a conserved motif linking the antiviral functions of IFITM3 to its oligomeric state

The interferon-inducible transmembrane (IFITM) proteins belong to the Dispanin/CD225 family and inhibit diverse virus infections. IFITM3 reduces membrane fusion between cells and virions through a poorly characterized mechanism. We identified a GxxxG motif in many CD225 proteins, including IFITM3 and proline rich transmembrane protein 2 (PRRT2). Mutation of PRRT2, a regulator of neurotransmitter release, at glycine-305 was previously linked to paroxysmal neurological disorders in humans. Here, we show that glycine-305 and the homologous site in IFITM3, glycine-95, drive protein oligomerization from within a GxxxG motif. Mutation of glycine-95 in IFITM3 disrupted its oligomerization and reduced its antiviral activity against Influenza A and HIV-1. The oligomerization-defective variant was used to reveal that IFITM3 promotes membrane rigidity in a glycine-95-dependent manner. Furthermore, a compound which counteracts virus inhibition by IFITM3, amphotericin B, prevented the IFITM3-mediated rigidification of membranes. Overall, these data suggest that IFITM3 oligomers inhibit virus-cell fusion by promoting membrane rigidity.

evolutionary biology↗

bioRxiv2020

Orthogonal fluorescent chemogenetic reporters for multicolor imaging

Fluorescence microscopy is an indispensable tool in biological research, allowing sub-second and sub-micrometer mapping of molecules or processes inside living cells. Moreover, using spectrally separated fluorophores, one can observe multiple targets simultaneously, leading to a deeper understanding of the dynamic molecular interplays that regulate cell function and fate. Chemogenetic systems, which combine a protein tag and a synthetic fluorophore, provide certain advantages over fluorescent proteins since there is no requirement for chromophore maturation. However, the fluorophore promiscuity of chemogenetic systems renders two-color applications challenging. Here, we present the engineering of a set of spectrally orthogonal fluorogen activating tags based on the Fluorescence Activating and absorption Shifting Tag (FAST), that are compatible with two-color, live cell imaging. The resulting tags, greenFAST and redFAST, demonstrate orthogonality not only in their fluorogen recognition capabilities, but also in their one- and two-photon absorption profiles. A two-color cell cycle sensor based on greenFAST and redFAST is capable of detecting very short, early cell cycles in zebrafish development which had previously been difficult to image. Furthermore, this pair of orthogonal tags can be developed into split complementation systems that are capable of detecting multiple protein-protein interactions by live cell fluorescence microscopy.

molecular biology↗

bioRxiv2019

Single-cell glycolytic activity regulates membrane tension 1 and HIV-1 fusion

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 viruses were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane-order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells. As glycolytic activity sets membrane tension levels by altering cellular cholesterol surface levels, our results suggest additional previously unknown benefits of cholesterol-lowering medication in HIV-1 infection.

immunology↗

bioRxiv2019

Robin Hood: non-fitting, non-smoothing image detrending for bleaching correction

Recent advances in protein labelling--gene tagging with CRIPSR-Cas9--have made it possible to label proteins of interest endogenously. This represents a major breakthrough in the field of quantitative microscopy, especially when quantifying protein-protein interactions. This is because over-expression of labelled proteins may cause a distortion in localization, function and perhaps artificially force protein-protein interactions due to crowding effects. A microscopy technique that is particularly well suited to detect protein interactions with low photon budgets is number and brightness (N&B). Detrending (removal of global trends in data) is a necessary pre-processing step to N&B calculations, but all current detrending methods perform poorly at low intensities. Here, we present the Robin Hood automatic detrending algorithm which performs well at low intensities, evaluating it with simulated and low photon budget live cell images. RH is available as an ImageJ plugin and as an R package.\n\nSTATEMENT OF SIGNIFICANCEFluorescence microscopy in general and Fluorescence fluctuation methods in particular are very much dependent on detrend algorithms and so far, the user needs to decide an arbitrary number to correct for bleaching when using the box plot or the running average approach. Here, we have developed a tool available to everybody as an ImageJ plugin that is automatic, user-free and able to correct bleached images with very low photon counts.

biophysics↗

bioRxiv2018

The RNA-binding proteins CELF1 and ELAVL1 cooperatively control alternative splicing

ELAVL1 and CELF1 are RNA-binding proteins that are involved in alternative splicing control. To explore their functional relationship, we looked for mRNAs that are differentially spliced following the depletion of CELF1, ELAVL1, or both. We found that these proteins control the usage of their target exons in the same direction. Double depletion has a greater effect than individual depletions, showing that CELF1 and ELAVL1 exert additive control. To confirm these results, we carried out RT-PCR on the alternative cassette exons of several mRNAs, including CD44, WNK1, PHACTR2, MICAL3, SPTBN1, and PPP3CB. Using FRET, we found that CELF1 and ELAVL1 directly interact in cell nuclei. We demonstrated that the combined levels of CELF1 and ELAVL1 are a valuable biomarker in several cancers, even when their individual levels may yield very limited information. CD44 alternative splicing probably accounts in part for the effects of CELF1 and ELAVL1 levels on patient survival. These data point to strong functional interactions between CELF1 and ELAVL1 in the control of mRNA isoform production, resulting in significant impacts on human pathology.

molecular biology↗

bioRxiv2018

Chromatin Condensation Fluctuations Rather Than Steady-State Predict Chromatin Accessibility

Chromatin accessibility to protein factors is critical for genome activities. Dynamic changes in nucleosomal DNA compaction and higher order chromatin structures are expected to allow specific sites to be accessible to regulatory factors and the transcriptional machinery. However, the dynamic properties of chromatin that regulate its accessibility are poorly understood. Here, we took advantage of the microenvironment sensitivity of the fluorescence lifetime of EGFP-H4 histone incorporated in chromatin to map in the nucleus of live cells the dynamics of chromatin condensation and its direct interaction with a tail acetylation recognition domain (the double bromodomain module of human TAFII250, dBD). We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation. Fluctuations are spontaneous, yet their amplitudes are affected by their sub-nuclear localization and by distinct and competing mechanisms dependent on histone acetylation, ATP, and both. Moreover, we show that accessibility of acetylated histone H4 to dBD is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.\n\nSignificanceIn higher eukaryotes, the structure and compaction of chromatin are considered as barriers to genome activities. Epigenetic marks such as post-translational modifications of histones can modify the structure and compaction of chromatin. The accessibility of protein factors to these epigenetic marks is therefore of paramount importance for genome activities. We reveal chromatin condensation fluctuations supported by mechanisms fundamentally distinct from that of condensation itself. We show that accessibility of acetylated histone H4 to double bromodomains is not restricted by chromatin condensation nor predicted by acetylation, rather, it is predicted by chromatin condensation fluctuations.\n\nClassificationBiological Sciences, Cell Biology

cell biology↗