F-actin nanostructures rearrangements and regulation are essential for SARS-CoV-2 particle production in host pulmonary cells.

Our study focused on deciphering the role of F-actin and related regulatory factors during SARS-CoV-2 particle production and transmission in human pulmonary cells. Quantitative high-resolution microscopies revealed that the late phases of SARS-CoV-2 infection induce a strong rearrangement of F-actin nanostructures dependent on the viral M, E, and N structural proteins. Intracellular vesicles containing viral components are labeled with Rab7 and Lamp1 and are surrounded by F-actin ring-shaped structures, suggesting their role in viral trafficking toward the cell membrane for virus release. Furthermore, filopodia-like nanostructures were loaded with viruses, potentially facilitating their egress and transmission between lung cells. Gene expression analysis revealed the involvement of alpha-actinins under the regulation of the protein kinase N (PKN). The use of a PKN inhibitor efficiently reduces virus particle production, restoring endoplasmic reticulum and F-actin cellular shape. Our results highlight an important role of F-actin rearrangements during the productive phases of SARS-CoV-2 particles.

The FDA-approved drug Auranofin has a dual inhibitory effect on SARS-CoV-2 entry and NF-κB signaling.

Patients with severe COVID-19 show an altered immune response that fails to control the viral spread and suffer from exacerbated inflammatory response, which eventually can lead to death. A major challenge is to develop an effective treatment for COVID-19. NF-κB is a major player in innate immunity and inflammatory process. By a high-throughput screening approach, we identified FDA-approved compounds that inhibit the NF-κB pathway and thus dampen inflammation. Among these, we show that Auranofin prevents post-translational modifications of NF-κB effectors and their recruitment into activating complexes in response to SARS-CoV-2 infection or cytokine stimulation. In addition, we demonstrate that Auranofin counteracts several steps of SARS-CoV-2 infection. First, it inhibits a raft-dependent endocytic pathway involved in SARS-CoV-2 entry into host cells; Second, Auranofin alters the ACE2 mobility at the plasma membrane. Overall, Auranofin should prevent SARS-CoV-2 infection and inflammatory damages, offering new opportunities as a repurposable drug candidate to treat COVID-19.

Specific intracellular signature of SARS-CoV-2 infection using confocal Raman microscopy.

SARS-CoV-2 infection remains spread worldwide and requires a better understanding of virus-host interactions. Here, we analyzed biochemical modifications due to SARS-CoV-2 infection in cells by confocal Raman microscopy. Obtained results were compared with the infection with another RNA virus, the measles virus. Our results have demonstrated a virus-specific Raman molecular signature, reflecting intracellular modification during each infection. Advanced data analysis has been used to distinguish non-infected versus infected cells for two RNA viruses. Further, classification between non-infected and SARS-CoV-2 and measles virus-infected cells yielded an accuracy of 98.9 and 97.2 respectively, with a significant increase of the essential amino-acid tryptophan in SARS-CoV-2-infected cells. These results present proof of concept for the application of Raman spectroscopy to study virus-host interaction and to identify factors that contribute to the efficient SARS-CoV-2 infection and may thus provide novel insights on viral pathogenesis, targets of therapeutic intervention and development of new COVID-19 biomarkers.

Optimized production and fluorescent labeling of SARS-CoV-2 virus-like particles.

SARS-CoV-2 is an RNA enveloped virus responsible for the COVID-19 pandemic 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 100 nm diameter viral particles. Based on productive assays, we propose an optimal transfected plasmid ratio mimicking the viral RNA ratio in infected cells. This allows SARS-CoV-2 Virus-Like Particle (VLPs) formation composed of the viral structural proteins M, N, E and mature S. Furthermore, fluorescent or photoconvertible VLPs were generated by adding a fluorescent protein tag on N or M mixing with unlabeled viral proteins and characterized by western blots, atomic force microscopy coupled to fluorescence and immuno-spotting. Thanks to live fluorescence and super-resolution microscopies, we quantified VLPs size and concentration. SARS-CoV-2 VLPs present a diameter of 110 and 140 nm respectively for MNE-VLPs and MNES-VLPs with a concentration of 10e12 VLP/ml. In this condition, we were able to establish the incorporation of the Spike in the fluorescent VLPs. Finally, the Spike functionality was assessed by monitoring fluorescent MNES-VLPs docking and internalization in human pulmonary cells expressing or not the receptor hACE2. Results show a preferential maturation of S on N(GFP) labeled VLPs and an hACE2-dependent VLP internalization and a potential fusion in host cells. 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.

Extracellular vesicles containing the I-BAR protein IRSp53 are released from the cell plasma membrane in an Arp2/3 dependent manner.

BACKGROUD: Extracellular vesicles (EVs) are nanometric membrane vesicles produced by cells and involved in cell-cell communication. EV formation can occur in endosomal compartments whose budding depends on the ESCRT machinery (i.e., exosomes), or at the cell plasma membrane (i.e., EVs or microvesicles). How these EVs bud from the cell plasma membrane is not completely understood. Membrane curvatures of the plasma membrane toward the exterior are often generated by I-BAR domain proteins. I-BAR proteins are cytosolic proteins that when activated bind to the cell plasma membrane and are involved in protrusion formation including filopodia and lamellipodia. These proteins contain a conserved I-BAR domain that senses curvature and induces negative membrane curvatures at the plasma membrane. I-BAR proteins, such as IRSp53, also interact with actin co-factors to favor membrane protrusions. RESULTS: Here, we explore whether the I-BAR protein IRSp53 is sorting with EVs and if ectopic GFP-tagged I-BAR proteins, such as IRSp53-GFP, as well as related IRTKS-GFP or Pinkbar proteins, can be found in these EVs originated from the cell plasma membrane. We found that a subpopulation of these I-BAR EVs, which are negative for the CD81 exosomal biomarker, are produced from the cell plasma membrane in a TSG101-independent manner but in an Arp2/3-dependent manner. CONCLUSIONS: Our results thus reveal that IRSp53 containing EVs represent a subset of plasma membrane EVs whose production depends on branched actin. SIGNIFICANCE: IRSp53 belongs to the I-BAR family proteins involved in curving cell membranes through a link with cortical actin. In that perspective, IRSp53 was shown to help membrane curvature of HIV-1 particles and, here, to be part of the budding process of a sub-population of EVs through its link with Arp2/3. IRSp53 is consequently a biomarker of these EVs of the cell plasma membrane.

SARS-CoV-2 Poorly Replicates in Cells of the Human Blood-Brain Barrier Without Associated Deleterious Effects.

Various neurological symptoms have been associated to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection including headache, fever, anosmia, ageusia, but also, encephalitis, Guillain-Barre syndrome and ischemic stroke. Responsible for the current coronavirus disease (COVID-19) pandemic, SARS-CoV-2 may access and affect the central nervous system (CNS) by several pathways such as axonal retrograde transport or through interaction with the blood-brain barrier (BBB) or blood-cerebrospinal fluid (CSF) barrier. Here, we explored the molecular and cellular effects of direct SARS-CoV-2 infection of human BBB cells. We observed low replication of SARS-CoV-2 that was accompanied by very moderate inflammatory response. Using a human in vitro BBB model, we also described low replication levels without strong inflammatory response or modulation of endothelium integrity. Finally, using serum samples from COVID-19 patients, we highlighted strong concentrations of pro-inflammatory factors that did not perturb BBB integrity after short term exposure. Altogether, our results show that the main mechanism of brain access following SARS-CoV-2 infection does not seem to be directed by brain infection through endothelial cells.

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. siRNA-mediated knockdown of IRSp53 gene expression 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 preferentially localizes to curved membranes induced by IRSp53 I-BAR domain 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.

Atomic force microscopy analysis of native infectious and inactivated SARS-CoV-2 virions.

SARS-CoV-2 is an enveloped virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. Here, single viruses were analyzed by atomic force microscopy (AFM) operating directly in a level 3 biosafety (BSL3) facility, which appeared as a fast and powerful method to assess at the nanoscale level and in 3D infectious virus morphology in its native conformation, or upon inactivation treatments. AFM imaging reveals structurally intact infectious and inactivated SARS-CoV-2 upon low concentration of formaldehyde treatment. This protocol combining AFM and plaque assays allows the preparation of intact inactivated SARS-CoV-2 particles for safe use of samples out of level 3 laboratory to accelerate researches against the COVID-19 pandemic. Overall, we illustrate how adapted BSL3-AFM is a remarkable toolbox for rapid and direct virus analysis based on nanoscale morphology.

Author Correction: Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Single molecule localisation microscopy reveals how HIV-1 Gag proteins sense membrane virus assembly sites in living host CD4 T cells.

Monitoring virus assembly at the nanoscale 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 100 nm in diameter. Interestingly, HIV-1 Gag protein expression alone is sufficient to produce virus-like particles (VLPs) that resemble the immature virus. Here, we monitored VLP formation at the plasma membrane of host CD4+ T cells using a newly developed workflow allowing the analysis of long duration recordings of single-molecule Gag protein localisation and movement. Comparison of Gag assembling platforms in CD4+ T cells expressing wild type or assembly-defective Gag mutant proteins showed that VLP formation lasts roughly 15 minutes with an assembly time of 5 minutes. Trapping energy maps, built from membrane associated Gag protein movements, showed that one third of the assembling energy is due to direct Gag capsid-capsid interaction while the remaining two thirds require the nucleocapsid-RNA interactions. Finally, we show that the viral RNA genome does not increase the attraction of Gag at the membrane towards the assembling site but rather acts as a spatiotemporal coordinator of the membrane assembly process.

Glucose and glutamine metabolism regulate human hematopoietic stem cell lineage specification.

The metabolic state of quiescent hematopoietic stem cells (HSCs) is an important regulator of self-renewal, but it is unclear whether or how metabolic parameters contribute to HSC lineage specification and commitment. Here, we show that the commitment of human and murine HSCs to the erythroid lineage is dependent upon glutamine metabolism. HSCs require the ASCT2 glutamine transporter and active glutamine metabolism for erythroid specification. Blocking this pathway diverts EPO-stimulated HSCs to differentiate into myelomonocytic fates, altering in vivo HSC responses and erythroid commitment under stress conditions such as hemolytic anemia. Mechanistically, erythroid specification of HSCs requires glutamine-dependent de novo nucleotide biosynthesis. Exogenous nucleosides rescue erythroid commitment of human HSCs under conditions of limited glutamine catabolism, and glucose-stimulated nucleotide biosynthesis further enhances erythroid specification. Thus, the availability of glutamine and glucose to provide fuel for nucleotide biosynthesis regulates HSC lineage commitment under conditions of metabolic stress.

Peptide-vector strategy bypasses P-glycoprotein efflux, and enhances brain transport and solubility of paclitaxel.

We present the results obtained with paclitaxel coupled to a peptide-vector SynB3 (PAX-OSUC-SynB3), showing that this peptide-vector enhances the solubility of paclitaxel and its brain uptake in mice using the in situ brain perfusion model. We also show by the in situ brain perfusion in P-glycoprotein (P-gp)-deficient and wild-type mice that vectorized paclitaxel bypasses the P-gp present at the luminal side of the blood-brain barrier. The effect of the vectorized paclitaxel on various cancer cells was not significantly different from that of free paclitaxel. These results indicate that vectorization of paclitaxel may have significant potential for the treatment of brain tumors.

2-Pyrrolinodoxorubicin and its peptide-vectorized form bypass multidrug resistance.

A well-known mechanism leading to the emergence of multidrug-resistant tumor cells is the overexpression of P-glycoprotein, which is capable of lowering intracellular drug concentrations. In the present study, we tested the capability of 2-pyrrolinodoxorubicin (p-DOX), a highly potent derivative of DOX, to bypass multidrug resistance. The accumulation, intracellular distribution and cytotoxicity of p-DOX were tested in two cell lines (K562 and A2780) and their DOX-resistant counterparts (K562/ADR and A2780/ADR). Cellular accumulation and cytotoxicity were dramatically lowered for DOX in resistant cell lines, in comparison with non-resistant cells. In contrast, cellular accumulation, intracellular distribution and cytotoxicity of p-DOX were independent of the nature of the cell lines. The p-DOX showed potent dose-dependent inhibition of cell growth against resistant cells as compared with DOX. After treatment of resistant cells with verapamil, the intracellular levels of DOX were markedly increased and consequent cytotoxicity improved. In contrast, treatment of resistant cells with verapamil did not cause any further enhancement of cell uptake or an increase in the cytotoxic effect of the derivative p-DOX, indicating that the compound bypasses the P-glycoprotein. Finally, we show that vectorization of p-DOX by a peptide vector (SynB3) which has been shown to enhance the brain uptake of DOX and to decrease its heart accumulation does not affect this property. These results indicate that p-DOX and its vectorized form are potent and effective in overcoming multidrug resistance.

T-cell receptor-induced phosphorylation of the zeta chain is efficiently promoted by ZAP-70 but not Syk.

Engagement of the T-cell receptor (TCR) results in the activation of Lck/Fyn and ZAP-70/Syk tyrosine kinases. Lck-mediated tyrosine phosphorylation of signaling motifs (ITAMs) in the CD3-zeta subunits of the TCR is an initial step in the transduction of signaling cascades. However, zeta phosphorylation is also promoted by ZAP-70, as TCR-induced zeta phosphorylation is defective in ZAP-70-deficient T cells. We show that this defect is corrected by stable expression of ZAP-70, but not Syk, in primary and transformed T cells. Indeed, these proteins are differentially coupled to the TCR with a 5- to 10-fold higher association of ZAP-70 with zeta as compared to Syk. Low-level Syk-zeta binding is associated with significantly less Lck coupled to the TCR. Moreover, diminished coupling of Lck to zeta correlates with a poor phosphorylation of the positive regulatory tyr352 residue of Syk. Thus, recruitment of Lck into the TCR complex with subsequent zeta chain phosphorylation is promoted by ZAP-70 but not Syk. Importantly, the presence of ZAP-70 positively regulates the TCR-induced tyrosine phosphorylation of Syk. The interplay between Syk and ZAP-70 in thymocytes, certain T cells, and B-chronic lymphocytic leukemia cells, in which they are coexpressed, will therefore modulate the amplitude of antigen-mediated receptor signaling.

Intracellular single-chain variable fragments directed to the Src homology 2 domains of Syk partially inhibit Fc epsilon RI signaling in the RBL-2H3 cell line.

Intracellular expression of Ab fragments has been efficiently used to inactivate therapeutic targets, oncogene products, and to induce viral resistance in plants. Ab fragments expressed in the appropriate cell compartment may also help to elucidate the functions of a protein of interest. We report in this study the successful targeting of the protein tyrosine kinase Syk in the RBL-2H3 rat basophilic leukemia cell line. We isolated from a phage display library human single-chain variable fragments (scFv) directed against the portion of Syk containing the Src homology 2 domains and the linker region that separates them. Among them, two scFv named G4G11 and G4E4 exhibited the best binding to Syk in vivo in a yeast two-hybrid selection system. Stable transfectants of RBL-2H3 cells expressing cytosolic G4G11 and G4E4 were established. Immunoprecipitation experiments showed that intracellular G4G11 and G4E4 bind to Syk, but do not inhibit the activation of Syk following FcepsilonRI aggregation, suggesting that the scFv do not affect the recruitment of Syk to the receptor. Nevertheless, FcepsilonRI-mediated calcium mobilization and the release of inflammatory mediators are inhibited, and are consistent with a defect in Bruton's tyrosine kinase and phospholipase C-gamma2 tyrosine phosphorylation and activation. Interestingly, FcepsilonRI-induced mitogen-activated protein kinase phosphorylation is not altered, suggesting that intracellular G4G11 and G4E4 do not prevent the coupling of Syk to the Ras pathway, but they selectively inhibit the pathway involving phospholipase C-gamma2 activation.

Reconstitution of lymphoid development and function in ZAP-70-deficient mice following gene transfer into bone marrow cells.

Mutations in the ZAP-70 protein tyrosine kinase gene result in a severe combined immunodeficiency (SCID) characterized by a selective inability to produce CD8(+) T cells and a signal transduction defect in peripheral CD4(+) cells. Transplantation of genetically modified hematopoietic progenitor cells that express the wild-type ZAP-70 gene may provide significant benefit to some of these infants. The feasibility of stem cell gene correction for human ZAP-70 deficiency was assessed using a ZAP-70 knock-out model. ZAP-70-deficient murine bone marrow progenitor cells were transduced with a retroviral vector expressing the human ZAP-70 gene. Engraftment of these cells in irradiated ZAP-70-deficient animals resulted in the development of mature CD4(+) and CD8(+) T cells. In marked contrast, both populations were absent in ZAP-70(-/-) mice undergoing transplantation with bone marrow progenitor cells transduced with a control vector. Importantly, ZAP-70-reconstituted T cells proliferated in response to T-cell receptor stimulation. Moreover, these ZAP-70-expressing T cells demonstrated a diverse T-cell receptor repertoire as monitored by the relative usage of each T-cell receptor beta chain hypervariable region subfamily. The presence of ZAP-70 in B cells did not affect either lipopolysaccharide- or lipopolysaccharide/interleukin-4-mediated immunoglobulin isotype switching. Altogether, these data indicate that retroviral-mediated gene transfer of the ZAP-70 gene may prove to have a therapeutic benefit for patients with ZAP-70-SCID.