Biology subjects

Favard, C.

Publications and source records attributed to Favard, C..

14 recordsLinked to original sources

bioRxiv2026

HIV-1 Assembly is Assisted by IRSp53 Through an Interaction that Extends Gag Conformation

Budding of the immature virion is an essential part of the HIV-1 life cycle. However, understanding and inhibiting this process has been challenging, as both simulations and theoretical studies suggest that the energy required to bend the membrane is too high for the complete assembly of a spherical viral particle at the plasma membrane without the assistance of cellular host factors. Recent results suggest that the I-BAR protein IRSp53 may be one such host factor, but the molecular mechanism by which the Gag-IRSp53 interaction promotes HIV-1 assembly and budding remains poorly understood. We employ molecular dynamics simulations, starting from AlphaFold models, to show that the SH3 domain of IRSp53 interacts directly with the MACANTD domain of Gag and we experimentally validate this interaction. Our simulations show that the specificity of the interaction with IRSp53 arises from its ability to extend the MACANTD Gag conformation in a manner similar to RNA binding, which is known to activate Gag assembly. We then computationally design HIV-1 Gag mutants that, as confirmed by simulations, do not undergo conformational changes upon interaction with theIRSp53 SH3 domain. Experimentally, these Gag mutants are tested and shown to accumulate in cells. In addition, for these mutants we observe large quantities of Gag assembling at the plasma membrane but exhibiting large budding defects. These results validate the proposed mechanism by which the Gag-IRSp53 interaction promotes completion of HIV-1 budding and suggest new routes for inhibiting viral assembly or budding.

molecular biology↗

bioRxiv2026

SARS-CoV-2 nucleocapsid protein engages with viral RNA and ERGIC lipids to drive viral core assembly

Severe acute respiratory syndrome coronavirus 2 assembles at the ER-Golgi intermediate compartment (ERGIC), yet the molecular basis of nucleocapsid (N) protein interactions with host membranes remains unclear. Using in vitro reconstituted lipid membranes and viral RNA- N complexes, we show that full-length N binds phosphatidylinositol (PI)- and phosphatidylserine (PS)-containing membranes and induces lipid clustering, an effect amplified by viral RNA and ERGIC-like membrane lipid composition. The isolated N-terminal domain lacks this activity, whereas the C-terminal domain retains membrane-associated multimerization. Although, lipid bilayers promote co-clustering of N and PI lipids, facilitating ribonucleoprotein (RNP) assembly, even on simple membranes. Importantly, ERGIC- mimicking membranes enhances this co-clustering further stabilizing RNPs of dimensions matching the viral core. In cells, viral RNA enhances N clustering without altering particle production. These findings reveal cooperative interactions between N, viral RNA, and ERGIC lipids as key drivers of lipid-dependent viral core formation, providing a mechanistic framework for the early steps of viral assembly.

biophysics↗

bioRxiv2025

Conserved architecture of a functional lncRNA-protein interaction in the DNA damage response pathway

LncRNAs are typically poorly conserved at the sequence level, which complicates the use of sequence-based approaches to identify conserved functions. The stress-associated lincRNA-p21 is a paradigm for a sequence-divergent lncRNA. In mice, the interaction between lincRNA-p21 and the hnRNP K protein activates the Cdkn1a gene in cis, encoding the cell-cycle regulator p21, a key effector of the cellular stress response to DNA damage. However, the molecular details of this interaction, and its conservation in other species, remained unknown. To address this, we applied an integrated structural and functional approach, combining in silico, in vitro, ex cellulo, and in cellulo assays. We identified a structural element in mouse and human lincRNA-p21 containing a tetranucleotide motif conserved across more than 100 mammals. This element directly mediates weak binding to the hnRNP K protein in both species, which may enable rapid and reversible modulation of Cdkn1a and timely resolution of the DNA damage response. Conserved hnRNP K binding in humans suggests that the mechanism of Cdkn1a activation may also occur in this species. Our findings establish a shared RNA-protein contact between two syntenic but sequence-divergent transcripts, offering a general strategy for identifying functional RNA elements in other lncRNAs with limited sequence conservation.

molecular biology↗

bioRxiv2023

Boosting the toolbox for live imaging of translation

Live imaging of translation based on tag recognition by a single chain antibody is a powerful technique to assess translation regulation in living cells. However, especially in a multicellular organism, this approach is challenging and requires optimization in terms of expression level and detection sensitivity of the system. Here, we improved existing fluorescent tools and developed new ones to image and quantify nascent translation in the living Drosophila embryo and in mammalian cells. We tested and characterized five different Green Fluorescent Protein variants fused to the single chain fragment variable (scFv) and uncover photobleaching, aggregation and intensity disparities. Using different strengths of germline and somatic drivers, we determined that the availability of the scFv is critical in order to detect translation throughout development. We introduced a new translation imaging method based on a nanobody/tag system named ALFA-array, allowing the sensitive and simultaneous detection of the translation of several distinct mRNA species. Finally, we developed a largely improved RNA imaging system based on an MCP-tdStaygold fusion.

developmental biology↗

bioRxiv2022

HIV-1 diverts actin debranching mechanismsfor particle assembly and release in CD4 Tlymphocytes.

Enveloped viruses assemble and bud from the host cell membranes. Possible roles of cortical actin in these processes have often been a source of controversy. Here, we assessed the involvement of the Arp2/3 mediated branched actin in HIV-1 assembly at the membrane of infected CD4 T lymphocytes. Our results show that actin debranching not only increases HIV-1 release but also the number of individual HIV-1 assembly clusters present at the cell plasma membrane unravelling new mechanisms. Indeed, we showed that, in infected T lymphocytes, HIV-1 Gag prefers areas deficient in F-actin for assembly. In vitro, we could reproduce and quantify this mechanism using model systems. Finally, we found that the actin debranching factor, Arpin, an Arp2/3 inhibitor, is recruited by Gag at the cell membrane to promote virus assembly. Altogether, our data show that HIV-1 favors local actin debranching for assembly and release by subverting the host factor Arpin.

microbiology↗

bioRxiv2022

Quantification of membrane binding and diffusion using Fluorescence Correlation Spectroscopy diffusion laws

Many transient processes in cells arise from the binding of cytosolic proteins to membranes. Quantifying this membrane binding and its associated diffusion in the living cell is therefore of primary importance. Dynamic photonic microscopies, e.g. single/multiple particle tracking, fluorescence recovery after photobleaching and fluorescence correlation spectroscopy (FCS) enable noninvasive measurement of molecular mobility in living cells and their plasma membranes. However, FCS with a single beam waist is of limited applicability with complex, non Brownian, motions. Recently, the development of FCS diffusion laws methods has given access to the characterization of these complex motions, although none of them is applicable to the membrane binding case at the moment. In this study, we combined computer simulations and FCS experiments to propose an FCS diffusion law for membrane binding. First, we generated computer simulations of spot-variation FCS (svFCS) measurements for a membrane binding process combined to 2D and 3D diffusion at the membrane and in the bulk/cytosol, respectively. Then, using these simulations as a learning set, we derived an empirical diffusion law with three free parameters: the apparent binding constant KDapp, the diffusion coefficient on the membrane D2D and the diffusion coefficient in the bulk/cytosol, D3D. Finally, we monitored, using svFCS, the dynamics of retroviral Gag proteins and associated mutants during their binding to supported lipid bilayers of different lipid composition or at plasma membranes of living cells and we quantified KDapp and D2D in these conditions using our empirical diffusion law. Based on these experiments and numerical simulations, we conclude that this new approach enables correct estimation of membrane partitioning and membrane diffusion properties (KDapp and D2D) for peripheral membrane molecules.

biophysics↗

bioRxiv2022

Optimized production and fluorescent labelling of SARS-CoV-2 Virus-Like-Particles to study virus assembly and entry.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemia that conducted in 6 million deaths worldwide so far. SARS-CoV-2 particles are mainly composed of the 4 main structural proteins M, N, E and S to form 100nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the virus RNA ratio allowing SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and S. Furthermore, monochrome, dual-color fluorescent or photoconvertible VLPs were produced. Thanks to live fluorescence and super-resolution microscopy, we quantified VLPs size and concentration. It shows a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a minimum concentration of 10e12 VLP/ml. SARS-CoV-2 VLPs could tolerate the integration of fluorescent N and M tagged proteins without impairing particle assembly. In this condition, we were able to establish incorporation of the mature Spike in fluorescent VLPs. The Spike functionality was then shown by monitoring fluorescent MNES-VLPs docking and endocytosis in human pulmonary cells expressing the receptor hACE2. This work provides new insights on the use of non-fluorescent and fluorescent VLPs to study and visualize the SARS-CoV-2 viral life cycle in a safe environment (BSL-2 instead of BSL-3). Moreover, optimized SARS-CoV-2 VLP production can be further adapted to vaccine design strategies.

microbiology↗

bioRxiv2021

The control of transcriptional memory by stable mitotic bookmarking

To maintain cellular identities during development, gene expression profiles must be faithfully propagated through cell generations. The reestablishment of gene expression patterns upon mitotic exit is thought to be mediated, in part, by mitotic bookmarking by transcription factors (TF). However, the mechanisms and functions of TF mitotic bookmarking during early embryogenesis remain poorly understood. In this study, taking advantage of the naturally synchronized mitoses of Drosophila early embryos, we provide evidence that the pioneer-like transcription factor GAF acts as stable mitotic bookmarker during zygotic genome activation. We report that GAF remains associated to a large fraction of its interphase targets including at cis-regulatory sequences of key developmental genes, with both active and repressive chromatin signatures. GAF mitotic targets are globally accessible during mitosis and are bookmarked via histone acetylation (H4K8ac). By monitoring the kinetics of transcriptional activation in living embryos, we provide evidence that GAF binding establishes competence for rapid activation upon mitotic exit.

developmental biology↗

bioRxiv2021

Full assembly of HIV-1 particles requires assistance of the membrane curvature factor IRSp53.

During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. Partial gene editing of IRSp53 induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein localizes preferentially to IRSp53 I-BAR domain induced curved membranes on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

biophysics↗

bioRxiv2019

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly.

HIV-1 Gag protein self-assembles at the plasma membrane of infected cells for viral particle formation. Gag targets lipids, mainly the phosphatidylinositol (4, 5) bisphosphate, at the inner leaflet of this membrane. Here, we address the question whether Gag is able to trap specifically PI(4,5)P2 or other lipids during HIV-1 assembly in the host CD4+ T lymphocytes. Lipid dynamics within and away from HIV-1 assembly sites was determined using super-resolution STED microscopy coupled with scanning Fluorescence Correlation Spectroscopy in living T cells. Analysis of HIV-1 infected cells revealed that, upon assembly, HIV-1 is able to specifically trap PI(4,5)P2, and cholesterol, but not phosphatidylethanolamine or sphingomyelin. Furthermore, our data show that Gag is the main driving force to restrict PI(4,5)P2 and cholesterol mobility at the cell plasma membrane. This is first direct evidence showing that HIV-1 creates its own specific lipid environment by selectively recruiting PI(4,5)P2 and cholesterol, as a membrane nano-platform for virus assembly.

biophysics↗

bioRxiv2018

Stick-slip dynamics of cell adhesion triggers spontaneous symmetry breaking and directional migration

Directional cell motility during organism and tissue development, homeostasis and disease requires symmetry breaking. This process relies on the ability of single cells to establish a front-rear polarity, and can occur in absence of external cues. The initiation of migration has been attributed to the spontaneous polarization of cytoskeleton components, while the spatiotemporal evolution of cytoskeletal forces arising from continuous mechanical cell-substrate interaction has yet to be resolved. Here, we establish a one-dimensional microfabricated migration assay that mimics complex in vivo fibrillar environment while being compatible with high-resolution force measurements, quantitative microscopy, and optogenetics. Quantification of morphometric and mechanical parameters reveals a generic stick-slip behavior initiated by contractility-dependent stochastic detachment of adhesive contacts at one side of the cell, which is sufficient to drive directional cell motility in absence of pre-established cytoskeleton polarity or morphogen gradients. A theoretical model validates the crucial role of adhesion dynamics during spontaneous symmetry breaking, proposing that the examined phenomenon can emerge independently of a complex self-polarizing system.\n\nOne sentence summaryCells can autonomously break their symmetry through traction force oscillations (mechanical instabilities) that lead to stochastic detachment of adhesion patches on one side of the cell and the subsequent initiation of migration.

biophysics↗

bioRxiv2018

Temporal Control of Transcription by Zelda in living Drosophila embryos

Abstract/introPioneer factors have the exquisite ability to engage their target sites at nucleosomal DNA, which leads to a local remodeling of chromatin and the establishment of a transcriptional competence. However, the direct impact of enhancer priming by pioneer factors on the temporal control of gene expression and on mitotic memory remains elusive. In Drosophila embryos, the maternally deposited activator Zelda (Zld) exhibits key pioneer factor properties and indeed regulates the awakening of the zygotic genome. The analysis of thousands of endogenous Zld bound regions in various genetic contexts, as well as the study of isolated synthetic enhancers with static approaches, led to the proposal that Zld could act as a quantitative developmental timer. Here we employ quantitative live imaging methods and mathematical modeling to directly test the effect of Zld on temporal coordination in gene activation and on mitotic memory. Using an automatic tracking software, we quantified the timing of activation in hundreds of nuclei and their progeny in Drosophila embryos. We demonstrate that increasing the number of Zld binding sites accelerates the kinetics of transcriptional activation regardless of their past transcriptional state. In spite of its known pioneering activities, we show that Zld is not a mitotic bookmarker and is neither necessary nor sufficient to foster mitotic memory. Fluorescent recovery after photo-bleaching and fluorescent correlation spectroscopy experiments reveal that, Zld is highly dynamic and exhibits transient binding to chromatin. We propose that Zld low binding rates could be compensated for by local accumulation of Zld in nuclear microenvironments in vivo, thus allowing rapid and coordinated gene activation.

developmental biology↗

bioRxiv2018

Live single molecule microscopy of HIV-1 assembly in host T cells reveals a spatio-temporal effect of the viral genome.

Monitoring virus assembly dynamic at the nanoscale level in host cells remains a major challenge. Human Immunodeficiency Virus type 1 (HIV-1) components are addressed to the plasma membrane where they assemble to form spherical particles of 100nm in diameter. HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble immature virus. Here, we monitored Gag assembly in host CD4 T lymphocytes using single molecule dynamics microscopy and energy mapping. A workflow allowing long time recordings of single Gag molecule localization, diffusion and effective energy maps was developed for robust quantitative analysis of HIV assembly and budding. Comparison of numerous cell plasma membrane assembling platforms in cells expressing wild type or assembly-defective Gag proteins showed that VLP formation last 15 minutes, with an assembly time of 5 minutes, and that the nucleocapsid domain is mandatory. Importantly, it reveals that the viral genome coordinates spatio-temporally HIV-1 assembly.

biophysics↗

bioRxiv2016

Self assembly of HIV-1 Gag protein on lipid membranes generates PI(4,5)P2/Cholesterolnanoclusters.

The self-assembly of HIV-1 Gag polyprotein at the inner leaflet of the cell host plasma membrane is the key orchestrator of virus assembly. The binding between Gag and the plasma membrane is mediated by specific interaction of the Gag matrix domain and the PI(4,5)P2 lipid (PIP2). It is unknown whether this interaction could lead to local reorganization of the plasma membrane lipids. In this study, using model membranes, we examined the ability of Gag to segregate specific lipids upon self-assembly. We show for the first time that Gag self-assembly is responsible for the formation of PIP2 lipid nanoclusters, enriched in cholesterol but not in sphingomyelin. We also show that Gag mainly partition into liquid-disordered domains of these lipid membranes. Our work strongly suggests that, instead of targeting pre-existing plasma membrane lipid domains, Gag is more prone to generate PIP2/Cholesterol lipid nanodomains at the inner leaflet of the plasma membrane during early events of virus assembly.

biophysics↗