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Abstract Retroviruses are enveloped viruses that assemble on the inner leaflet of cellular membranes. Improving biophysical techniques has recently unveiled many molecular aspects of the interaction between the retroviral structural protein Gag and the cellular membrane lipids. This interaction is driven by the N-terminal matrix domain of the protein, which probably undergoes important structural modifications during this process, and could induce membrane lipid distribution changes as well. This review aims at describing the molecular events occurring during MA-membrane interaction, and pointing out their consequences in terms of viral assembly. The striking conservation of the matrix membrane binding mode among retroviruses indicates that this particular step is most probably a relevant target for antiviral research.

Abstract Background HIV-1 uses cellular co-factors for virion formation and release. The virus is able to incorporate into the viral particles host cellular proteins, such as tetraspanins which could serve to facilitate HIV-1 egress. Here, we investigated the implication of several tetraspanins on HIV-1 formation and release in chronically infected T-lymphoblastic cells, a model that permits the study of the late steps of HIV-1 replication. Results Our data revealed that HIV-1 Gag and Env structural proteins co-localized with tetraspanins in the form of clusters. Co-immunoprecipitation experiments showed that Gag proteins interact, directly or indirectly, with CD81, and less with CD82, in tetraspanin-enriched microdomains composed of CD81/CD82/CD63. In addition, when HIV-1 producing cells were treated with anti-CD81 antibodies, or upon CD81 silencing by RNA interference, HIV-1 release was significantly impaired, and its infectivity was modulated. Finally, CD81 downregulation resulted in Gag redistribution at the cell surface. Conclusion Our findings not only extend the notion that HIV-1 assembly can occur on tetraspanin-enriched microdomains in T cells, but also highlight a critical role for the tetraspanin CD81 on the late steps of HIV replication.

Abstract Background The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three α helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the α-helices could perturb the leucine zipper like motifs. The ΔQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. Conclusion We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three α helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.

Abstract Background The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly. Results We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. Conclusion These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

Abstract In HIV-1 infected cells transcription of the integrated provirus generates the single full length 9 kb viral RNA, a major fraction of which is spliced to produce the single-spliced 4 kb RNAs and the multiple-spliced 2 kb RNAs. These spliced RNAs are the messengers for the Env glycoproteins and the viral regulatory factors. The cellular SR and hnRNP proteins were shown in vitro to control alternative splicing by binding cis-regulatory elements on the viral RNA. To better understand in vivo the role of the SR proteins on HIV-1 genomic RNA splicing and virion production, we used a human cell line expressing high levels of complete HIV-1 and either one of the ASF/SF2, SC35, and 9G8 SR proteins. Results show that over-expressing SR proteins caused a large reduction of genomic RNA and that each SR protein modified the viral 9 kb RNA splicing pattern in a specific mode. In fact, ASF/SF2 increased the level of Vpr RNA while SC35 and 9G8 caused a large increase in Tat RNA. As expected, overexpressing SR proteins caused a strong reduction of total Gag made. However, we observed by immuno-confocal microscopy an accumulation of Gag at the plasma membrane and in intracellular compartments while there is a dramatic reduction of Env protein made in most cells. Due to the negative impact of the SR proteins on the levels of genomic RNA and HIV-1 structural proteins much less virions were produced which retained part of their infectivity. In conclusion, SR proteins can down-regulate the late steps of HIV-1 replication.

Membrane targeting of the human immunodeficiency virus Gag proteins is dependent on phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2] located in the plasma membrane. In order to determine if evolutionarily distant retroviral Gag proteins are targeted by a similar mechanism, we generated mutants of the matrix (MA) domain of murine leukemia virus (MuLV) Gag, examined their binding to membrane models in vitro, and analyzed their phenotypes in cell culture. In vitro, we showed that MA bound all the phosphatidylinositol phosphates with significant affinity but displayed a strong specificity for PI(4,5)P2 only if enhanced by phosphatidylserine. Mutations in the polybasic region in MA dramatically reduced this affinity. In cells, virus production was strongly impaired by PI(4,5)P2 depletion under conditions of 5ptaseIV overexpression, and mutations in the MA polybasic region altered Gag localization, membrane binding, and virion production. Our results suggest that the N-terminal polybasic cluster of MA is essential for Gag targeting to the plasma membrane. The binding of the MA domain to PI(4,5)P2 appears to be a conserved feature among retroviruses despite the fact that the MuLV-MA domain is structurally different from that of human immunodeficiency virus types 1 and 2 and lacks a readily identifiable PI(4,5)P2 binding cleft. [ABSTRACT FROM AUTHOR]

Mechanical properties of the nucleus are remodeled not only by extracellular forces transmitted to the nucleus but also by internal modifications, such as those induced by viral infections. During herpes simplex virus type 1 infection, the viral regulation of essential nuclear functions and growth of the nuclear viral replication compartments are known to reorganize nuclear structures. However, little is known about how this infection-induced nuclear deformation changes nuclear mechanobiology. Our analyses showed that the nucleus softens during the infection. To understand why this happens, we used microscopy and computational methods to study how the mechanical components of the nucleus are modified during the infection. We discovered that the viral replication compartment occupying the nuclear center has a low biomolecular density compared to the centrally located euchromatin in noninfected cells. The nuclear lamina was also modified such that in the infection the amount of lamin proteins increased and the nuclear envelope had more outward curved regions and moved less in infection compared to noninfected cells. The computational modeling of virus-induced changes in cellular forces showed that the most probable cause for the decreasing nuclear stiffness is the removal of lamina-associated domains or the decrease in outward forces, such as reduced intranuclear osmotic pressure and cytoskeletal pull. The simulations showed that an increase in the nuclear envelope tension can occur with a decrease in nuclear stiffness. Based on these findings, we propose a mechanical model that explains mechanistic coordination between the nuclear deformation in infection and decreased nuclear stiffness.

The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus that predominantly spreads through cell-to-cell contact due to the limited infectivity of cell-free viruses. Among various modes of intercellular transmission, HTLV-1 biofilms emerge as adhesive structures, polarized at the cell surface, which encapsulate virions within a protective matrix. This biofilm is supposed to facilitate simultaneous virion delivery during infection. Yet, the molecular and functional intricacies of viral biofilms remain largely unexplored, despite their pivotal role in understanding retroviral pathogenesis. In this study, we optimized a protocol to isolate HTLV-1 biofilms from chronically infected T cells, facilitating their structural and molecular characterization using proteomic and super-resolution microscopy analyses. This protocol involves cultivating HTLV-1 chronically infected T cells at high density to facilitate the natural detachment of viral biofilms into the supernatant. Then, employing successive centrifugations, the cells are separated from the detached biofilms, and these structures are pelleted at medium speed (10,000× g). This method circumvents the need for mechanical, chemical, or enzymatic biofilm detachment, bypasses the use of ultracentrifugation, and enables us to resuspend the biofilms in the appropriate buffer for subsequent analyses such as western blotting or super-resolution microscopy imaging as presented. Key features • Isolation of viral biofilms from HTLV-1 chronically infected T cells after 4 days of culture at high cellular density. • Structural analysis of viral biofilms using super-resolution microscopy techniques. • Experiments performed in vitro within a confined biosafety level 3 (BSL3) environment. • This protocol requires at least five days to complete., Competing Interests: Competing interestsThe authors declare no competing interests., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

Cells naturally produce vesicles that bud from different lipid membranes using dedicated molecular machineries. Enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), also generate particles that bud from host cell membranes by hijacking cellular factors and signaling pathways similar to those involved in the budding of extracellular vesicles. HIV-1 buds from the host cell plasma membrane mainly via the self-assembly of Gag, a structural protein. Gag is a polyprotein that forms assembly complexes containing viral genomic RNA (gRNA), host cell lipids and proteins. HIV-1 Gag binds and segregates host cell plasma membrane lipids while self-assembling simultaneously on the gRNA and the plasma membrane. This self-assembly causes membrane bending and formation of a new viral particle with the help of host cell proteins, likely including cortical actin-associated factors. However, it is unclear whether the energy of Gag self-assembly is sufficient to generate new HIV-1 particles. In this Review, we discuss these processes in the light of the past and recent virology literature, incorporating lessons from studies on the quantitative biophysics of viral self-assembly, and explore how Gag might reorganize the plasma membrane and divert host cell membrane curving proteins and cortical actin-related factors to achieve particle assembly and budding., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii , the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella -containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche., Competing Interests: Competing interests statement:The authors declare no competing interest.

Unknown particle screening-including virus and nanoparticles-are keys in medicine, industry, and also in water pollutant determination. Here, RYtov MIcroscopy for Nanoparticles Identification (RYMINI) is introduced, a staining-free, non-invasive, and non-destructive optical approach that is merging holographic label-free 3D tracking with high-sensitivity quantitative phase imaging into a compact optical setup. Dedicated to the identification and then characterization of single nano-object in solution, it is compatible with highly demanding environments, such as level 3 biological laboratories, with high resilience to external source of mechanical and optical noise. Metrological characterization is performed at the level of each single particle on both absorbing and transparent particles as well as on immature and infectious HIV, SARS-CoV-2 and extracellular vesicles in solution. The capability of RYMINI to determine the nature, concentration, size, complex refractive index and mass of each single particle without knowledge or model of the particles' response is demonstrated. The system surpasses 90% accuracy for automatic identification between dielectric/metallic/biological nanoparticles and ≈80% for intraclass chemical determination of metallic and dielectric. It falls down to 50-70% for type determination inside the biological nanoparticle's class., (© 2023 Wiley‐VCH GmbH.)

The 20-year revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and timely acquisition, allows the visualization of nanoscaled objects in cell biology. Currently, the use of a recent generation of super-resolution fluorescence microscope coupled with improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus particle or protein level. Here, we highlight the protocol for visualizing HIV-1 Gag assembly at the host T-cell plasma membrane using super-resolution light microscopy. Total internal reflection fluorescence microscopy (TIRF-M) coupled with single-molecule localization microscopy (SMLM) enables the detection and characterization of the assembly of viral proteins at the plasma membrane of infected host cells at the single protein level. Here, we describe the TIRF equipment, the T-cell culture for HIV-1, the sample preparation for single-molecule localization microscopies such as PALM and STORM, acquisition protocols, and Gag assembling cluster analysis., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Importance: In the early stages of infection, human T-lymphotropic virus type 1 (HTLV-1) dissemination within its host is believed to rely mostly on cell-to-cell contacts. Past studies unveiled a novel mechanism of HTLV-1 intercellular transmission based on the remodeling of the host-cell extracellular matrix and the generation of cell-surface viral assemblies whose structure, composition, and function resemble bacterial biofilms. These polarized aggregates of infectious virions, identified as viral biofilms, allow the bulk delivery of viruses to target cells and may help to protect virions from immune attacks. However, viral biofilms' molecular and functional description is still in its infancy, although it is crucial to fully decipher retrovirus pathogenesis. Here, we explore the function of cellular tetraspanins (CD9, CD81, CD82) that we detect inside HTLV-1 particles within biofilms. Our results demonstrate specific roles for CD82 in the cell-surface distribution and intercellular transmission of HTLV-1 biofilms, which we document as two essential parameters for efficient viral transmission. At last, our findings indicate that N-glycosylation of cell-surface molecules, including CD82, is required for the polarization of HTLV-1 biofilms and for the efficient transmission of HTLV-1 between T-lymphocytes., Competing Interests: The authors declare no conflict of interest.

Human respiratory syncytial virus (RSV) is the leading cause of infantile bronchiolitis in the developed world and of childhood deaths in resource-poor settings. The elderly and the immunosuppressed are also affected. It is a major unmet target for vaccines and antiviral drugs. RSV assembles and buds from the host cell plasma membrane by forming infectious viral particles which are mostly filamentous. A key interaction during RSV assembly is the interaction of the matrix (M) protein with cell plasma membrane lipids forming a layer at assembly sites. Although the structure of RSV M protein dimer is known, it is unclear how the viral M proteins interact with cell membrane lipids, and with which one, to promote viral assembly. Here, we demonstrate that M proteins are able to cluster at the plasma membrane by selectively binding with phosphatidylserine (PS). Our in vitro studies suggest that M binds PS lipid as a dimer and upon M oligomerization, PS clustering is observed. In contrast, the presence of other negatively charged lipids like PI(4, 5)P2 does not enhance M binding beyond control zwitterionic lipids, while cholesterol negatively affects M interaction with membrane lipids. Moreover, we show that the initial binding of the RSV M protein with PS lipids is independent of the cytoplasmic tail of the fusion (F) glycoprotein (FCT). Here, we highlight that M binding on membranes occurs directly through PS lipids, this interaction is electrostatic in nature, and M oligomerization generates PS clusters., Competing Interests: Conflicts of interest The authors declare that they have no conflict of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Enveloped viruses assemble and bud from the host cell membranes. Any role of cortical actin in these processes have often been a source of debate. Here, we assessed if cortical actin was involved in HIV-1 assembly in infected CD4 T lymphocytes. Our results show that preventing actin branching not only increases HIV-1 particle release but also the number of individual HIV-1 Gag assembly clusters at the T cell plasma membrane. Indeed, in infected T lymphocytes and in in vitro quantitative model systems, we show that HIV-1 Gag protein prefers areas deficient in F-actin for assembling. Finally, we found that the host factor Arpin, an inhibitor of Arp2/3 branched actin, is recruited at the membrane of infected T cells and it can associate with the viral Gag protein. Altogether, our data show that, for virus assembly and particle release, HIV-1 prefers low density of cortical actin and may favor local actin debranching by subverting Arpin., (© 2023. The Author(s).)

Flaviviruses have emerged as major arthropod-transmitted pathogens and represent an increasing public health problem worldwide. High-throughput screening can be facilitated using viruses that easily express detectable marker proteins. Therefore, developing molecular tools, such as reporter-carrying versions of flaviviruses, for studying viral replication and screening antiviral compounds represents a top priority. However, the engineering of flaviviruses carrying either fluorescent or luminescent reporters remains challenging due to the genetic instability caused by marker insertion; therefore, new approaches to overcome these limitations are needed. Here, we describe reverse genetic methods that include the design and validation of infectious clones of Zika, Kunjin, and Dengue viruses harboring different reporter genes for infection, rescue, imaging, and morphology using super-resolution microscopy. It was observed that different flavivirus constructs with identical designs displayed strikingly different genetic stabilities, and corresponding virions resembled wild-type virus particles in shape and size. A successful strategy was assessed to increase the stability of rescued reporter virus and permit antiviral drug screening based on quantitative automated fluorescence microscopy and replication studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Cherkashchenko, Gros, Trausch, Neyret, Hénaut, Dubois, Villeneuve, Chable-Bessia, Lyonnais, Merits and Muriaux.)

Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem-cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models), infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using our own and publicly available bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes ( IFI44 , IFIT1 , IFIT3 , IFI35 , IRF9 , MX1 , OAS1 , OAS3 and ISG15 ) and inflammatory genes ( NFKBIA , CSF1 , FOSL1 , IL32 and CXCL10 ) by day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes ( ITGB6 , ITGB1 and GJA1 ) were also altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins was also deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to explain the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

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., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

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 non-invasive 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 K D , the diffusion coefficient on the membrane D 2D , and the diffusion coefficient in the cytosol, D 3D . 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 K D and D 2D 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 (K D and D 2D ) for peripheral membrane molecules., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The increasing frequency and magnitude of viral outbreaks in recent decades, epitomized by the COVID-19 pandemic, has resulted in an urgent need for rapid and sensitive diagnostic methods. Here, we present a methodology for virus detection and identification that uses a convolutional neural network to distinguish between microscopy images of fluorescently labeled intact particles of different viruses. Our assay achieves labeling, imaging, and virus identification in less than 5 min and does not require any lysis, purification, or amplification steps. The trained neural network was able to differentiate SARS-CoV-2 from negative clinical samples, as well as from other common respiratory pathogens such as influenza and seasonal human coronaviruses. We were also able to differentiate closely related strains of influenza, as well as SARS-CoV-2 variants. Additional and novel pathogens can easily be incorporated into the test through software updates, offering the potential to rapidly utilize the technology in future infectious disease outbreaks or pandemics. Single-particle imaging combined with deep learning therefore offers a promising alternative to traditional viral diagnostic and genomic sequencing methods and has the potential for significant impact.

RNA interference (RNAi) offers an efficient way to repress genes of interest, and it is widely used in research settings. Clinical applications emerged more recently, with 5 approved siRNAs (the RNA guides of the RNAi effector complex) against human diseases. The development of siRNAs against the SARS-CoV-2 virus could therefore provide the basis of novel COVID-19 treatments, while being easily adaptable to future variants or to other, unrelated viruses. Because the biochemistry of RNAi is very precisely described, it is now possible to design siRNAs with high predicted activity and specificity using only computational tools. While previous siRNA design algorithms tended to rely on simplistic strategies (raising fully complementary siRNAs against targets of interest), our approach uses the most up-to-date mechanistic description of RNAi to allow mismatches at tolerable positions and to force them at beneficial positions, while optimizing siRNA duplex asymmetry. Our pipeline proposes 8 siRNAs against SARS-CoV-2, and ex vivo assessment confirms the high antiviral activity of 6 out of 8 siRNAs, also achieving excellent variant coverage (with several 3-siRNA combinations recognizing each correctly-sequenced variant as of September2022). Our approach is easily generalizable to other viruses as long as avariant genome database is available. With siRNA delivery procedures being currently improved, RNAi could therefore become an efficient and versatile antiviral therapeutic strategy.

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., Competing Interests: The authors have no competing interests., (© 2022 The Authors.)

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., (© 2022 The Authors. Biology of the Cell published by Wiley-VCH GmbH on behalf of Société Française des Microscopies and Société de Biologie Cellulaire de France.)

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., (© 2022. The Author(s).)

We explored antibody response after first and second BNT162b2 vaccinations, to predict the need for subsequent injections in nursing home (NH) residents. 369 NH residents were tested for IgG against SARS-CoV-2 Receptor-Binding Domain (RBD-IgG) and nucleoprotein-IgG (SARS-CoV-2 IgG II Quant and SARS-CoV-2 IgG Alinity assays, Abbott Diagnostics). In NH residents with prior SARS-CoV-2 infection, the first dose elicited high RBD-IgG levels (≥ 4160 AU/mL) in 99/129 cases (76.9%), with no additional antibody gain after the second dose in 74 cases (74.7%). However, a low RBD-IgG level (< 1050 AU/mL) was observed in 28 (21.7%) residents. The persistence of nucleoprotein-IgG and a longer interval between infection and the first dose were associated with a higher RBD-IgG response (p < 0.0001 and p = 0.0013, respectively). RBD-IgG below 50 AU/mL after the first dose predicted failure to reach the antibody concentration associated with a neutralizing effect after the second dose (≥ 1050 AU/mL). The BNT162b2 vaccine elicited a strong humoral response after the first dose in a majority of NH residents with prior SARS-CoV-2 infection. However, about one quarter of these residents require a second injection. Consideration should be given to immunological monitoring in NH residents to optimize the vaccine response in this vulnerable population., (© 2022. The Author(s).)

Background: Limited information exists on nursing home (NH) residents regarding BNT162b2 vaccine efficacy in preventing SARS-CoV-2 and severe COVID-19, and its association with post-vaccine humoral response., Methods: 396 residents from seven NHs suffering a SARS-CoV-2 B.1.1.7 (VOC-α) outbreak at least 14 days after a vaccine campaign were repeatedly tested using SARS-CoV-2 real-time reverse-transcriptase polymerase chain reaction on nasopharyngeal swab test (RT-qPCR). SARS-CoV-2 receptor-binding domain (RBD) of the S1 subunit (RBD-IgG) was measured in all residents. Nucleocapsid antigenemia (N-Ag) was measured in RT-qPCR-positive residents and serum neutralizing antibodies in vaccinated residents from one NH., Results: The incidence of positive RT-qPCR was lower in residents vaccinated by two doses (72/317; 22.7%) vs one dose (10/31; 32.3%) or non-vaccinated residents (21/48; 43.7%; p < .01). COVID-19-induced deaths were observed in 5 of the 48 non-vaccinated residents (10.4%), in 2 of the 31 who had received one dose (6.4%), and in 3 of the 317 (0.9%) who had received two doses (p = .0007). Severe symptoms were more common in infected non-vaccinated residents (10/21; 47.6%) than in infected vaccinated residents (15/72; 21.0%; p = .002). Higher levels of RBD-IgG (n = 325) were associated with a lower SARS-CoV-2 incidence. No in vitro serum neutralization activity was found for RBD-IgG levels below 1050 AU/ml. RBD-IgG levels were inversely associated with N-Ag levels, found as a risk factor of severe COVID-19., Conclusions: Two BNT162b2 doses are associated with a 48% reduction of SARS-CoV-2 incidence and a 91.3% reduction of death risk in residents from NHs facing a VOC-α outbreak. Post-vaccine RBD-IgG levels correlate with BNT162b2 protection against SARS-CoV-2 B.1.1.7., (© 2021 European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

The cell plasma membrane is mainly composed of phospholipids, cholesterol and embedded proteins, presenting a complex interface with the environment. It maintains a barrier to control matter fluxes between the cell cytosol and its outer environment. Enveloped viruses are also surrounded by a lipidic membrane derived from the host-cell membrane and acquired while exiting the host cell during the assembly and budding steps of their viral cycle. Thus, model membranes composed of selected lipid mixtures mimicking plasma membrane properties are the tools of choice and were used to decipher the first step in the assembly of enveloped viruses. Amongst these viruses, we choose to report the three most frequently studied viruses responsible for lethal human diseases, i.e., Human Immunodeficiency Type 1 (HIV-1), Influenza A Virus (IAV) and Ebola Virus (EBOV), which assemble at the host-cell plasma membrane. Here, we review how model membranes such as Langmuir monolayers, bicelles, large and small unilamellar vesicles (LUVs and SUVs), supported lipid bilayers (SLBs), tethered-bilayer lipid membranes (tBLM) and giant unilamellar vesicles (GUVs) contribute to the understanding of viral assembly mechanisms and dynamics using biophysical approaches.

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., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2022.)

In 2019, a new pandemic virus belonging to the betacoronavirus family emerged, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This new coronavirus appeared in Wuhan, China, and is responsible for severe respiratory pneumonia in humans, namely, coronavirus disease 2019 (COVID-19). Having infected almost 200 million people worldwide and caused more than 4.1 million deaths as of today, this new disease has raised a significant number of questions about its molecular mechanism of replication and, in particular, how infectious viral particles are produced. Although viral entry is well characterized, the full assembly steps of SARS-CoV-2 have still not been fully described. Coronaviruses, including SARS-CoV-2, have four main structural proteins, namely, the spike glycoprotein (S), the membrane glycoprotein (M), the envelope protein (E), and the nucleocapsid protein (N). All these proteins have key roles in the process of coronavirus assembly and budding. In this review, we gathered the current knowledge about betacoronavirus structural proteins involved in viral particle assembly, membrane curvature and scission, and then egress in order to suggest and question a coherent model for SARS-CoV-2 particle production and release.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Constant, Barthelemy, Bolloré, Tuaillon, Gosselet, Chable-Bessia, Merida, Muriaux, Van de Perre, Salinas and Simonin.)