Biology subjects

Roingeard, P.

Publications and source records attributed to Roingeard, P..

10 recordsLinked to original sources

bioRxiv2025

Cholesterol Remodeling by CH25h Rewires IFITM3 Trafficking and Secretion Without Enhancing Antiviral Restriction

Type I interferon induces multiple protein effectors to inhibit viral replication. Among these, the interferon-induced transmembrane protein (IFITM3) and the cholesterol-25-hydroxylase (CH25h) converge on the inhibition of viral entry by altering the behavior of membranes. Here, we dissect the functional and mechanistic relationship between these two membrane-acting effectors using HIV-1 and vesicular stomatitis virus (VSV) as models. We show that IFITM3 and CH25h restrict viral entry with similar efficiency, but act in a largely redundant manner during infection. Their redundancy is consistent across infection systems, cell types, and entry assays, indicating that both factors converge on a shared biophysical block to membrane fusion. Unexpectedly, we uncover a second layer of interplay in which the CH25h-25HC axis remodels IFITM3 trafficking. Exposure to 25-hydroxycholesterol drives IFITM3 from endolysosomal compartments to the plasma membrane, impairs its internalization into early endosomes, and increases its secretion in exosomal vesicles. These effects occur independently of direct antiviral activity and reveal that CH25h regulates IFITM3 cellular dynamics. Together, our study identifies a previously unrecognized cross-regulatory circuit between two IFN-induced antiviral pathways, highlighting how lipid remodeling by CH25h, and potentially by other cellular factors, can contribute to control the behavior of IFITM3. ImportanceType I interferons induce numerous antiviral factors that frequently target the same vulnerable steps of viral infection, but how these factors influence one another is not well understood. IFITM3 and the CH25h are two potent, broad-acting inhibitors of viral membrane fusion. Here, we show that although their antiviral activities are largely redundant against HIV-1 and VSV, CH25h profoundly alters the biology of IFITM3. The product of the enzymatic activity of CH25h, the oxysterol 25HC, redistributes IFITM3 from endosomal compartments to the plasma membrane and enhances its release in exosomal vesicles-- effects independent of direct antiviral activity. These findings reveal an unexpected layer of cross-regulation between lipid-modifying enzymes and membrane-embedded restriction factors. More broadly, they highlight how interferon-driven lipid remodeling can reshape the trafficking and secretion of antiviral proteins, expanding the functional landscape of innate immunity beyond viral entry inhibition.

cell biology↗

bioRxiv2025

Entry, replication and innate immunity evasion of BANAL-236, a SARS-CoV-2-related bat virus, in Rhinolophus and human cells

Asian Rhinolophus bats are considered the natural reservoirs of an ancestral SARS-CoV-2. However, the biology of SARS-CoV-2-related viruses in bat cells is not well understood. Here, we investigated the replication of BANAL-236, the only bat-derived SARS-CoV-2 relative isolated to date, in Rhinolophus cells. BANAL-236 did not replicate in wild-type Rhinolophus cell lines. Entry assays using pseudoviruses expressing the spike proteins (S) of SARS-CoV-2, BANAL-236, and BANAL-52 revealed that efficient S-mediated entry depends on the expression of human ACE2 (hACE2) and human TMPRSS2 (hTMPRSS2) in human and Rhinolophus cells. Expression of Rhinolophus entry factors, either alone or in combination, did not facilitate SARS-CoV-2 or BANAL-236 entry in human cells, suggesting that the S protein of BANAL-236 interacts more efficiently with hACE2 than with its Rhinolophus counterpart (rACE2). Through biochemical, virological, and electron microscopy analyses, we showed that BANAL-236 and SARS-CoV-2 completed their replication cycles in a Rhinolophus cell line engineered to express high levels of hACE2 and hTMPRSS2. Despite efficient viral replication in modified Rhinolophus and human cells, no induction of interferon (IFN)-stimulated genes was detected. Using a screening approach, we identified several BANAL-236 proteins that antagonize IFN production and signaling in human cells. Our findings thus show that BANAL-236 possesses critical features that enabled zoonotic spillover: hACE2 usage and potent evasion of human IFN responses. The Rhinolophus cellular model we established offers a platform for further investigating the interactions between bat coronaviruses and their reservoir hosts. Author summaryBats are known reservoirs for viruses that cause severe diseases in humans, such as coronaviruses and filoviruses. Bat species naturally or experimentally infected with these viruses rarely exhibit clinical symptoms, suggesting an evolved tolerance to viral infections. To elucidate the mechanisms underlying viral tolerance and to identify factors that could facilitate zoonotic spillover, it is essential to study the replication of bat-borne viruses in relevant bat cellular models. Here, we investigated the replication of BANAL-236, a SARS-CoV-2 related virus isolated from fecal samples of Rhinolophus bats in Northen Laos, in a novel cell line derived from Rhinolophus ferrumequinum lung fibroblasts. Our findings reveal that BANAL-236 can efficiently use human entry factors and potently evade the human innate immune response, two traits that may have contributed to its zoonotic transmission. Furthermore, the R. ferrumequinum cell lines we developed is a valuable model for investigating the molecular interactions between sarbecoviruses and their natural hosts.

microbiology↗

bioRxiv2025

Microtubule remodeling by the innate immune factor Trim69 compromises dynein-dependent migration of HIV virion cores towards the nucleus

Like many viruses, HIV relies on the microtubule (MT) cytoskeleton for successful infection. MT-associated proteins (MAPs) regulate MT functions and thus bear the potential to modulate viral infection. However, while several MAPs are known to exert pro-viral effects on HIV, little is known about antiviral ones. We previously described the Tripartite motif protein 69 (Trim69) as an innate immune factor that remodels MTs, leading to inhibition of a broad spectrum of viruses, including HIV. Through in silico modeling, TIRF microscopy and cell-based assays, we determine that Trim69 binding to MTs is determined by a basic surface in its SPRY domain that interacts with the C-terminal tails of tubulins. This surface is conserved among mammalian Trim69s and is critical for its functions. We demonstrate that by binding and remodeling MTs, Trim69 inhibits the docking and the migration of virion cores on MTs by promoting the stalling of the dynein/dynactin motor complexes. Altogether, these findings shed light on a novel mechanism of viral defense that involves an innate immune regulation of the MT cytoskeleton.

cell biology↗

bioRxiv2024

Proteostasis and metabolic dysfunction in a distinct subset of storage-induced senescent erythrocytes targeted for clearance

Although refrigerated storage slows the metabolism of volunteer donor RBCs, cellular aging still occurs throughout this in vitro process, which is essential in transfusion medicine. Storage-induced microerythrocytes (SMEs) are morphologically-altered senescent RBCs that accumulate during storage and which are cleared from circulation following transfusion. However, the molecular and cellular alterations that trigger clearance of this RBC subset remain to be identified. Using a staining protocol that sorts long-stored SMEs (i.e., CFSEhigh) and morphologically-normal RBCs (CFSElow), these in vitro aged cells were characterized. Metabolomics analysis identified depletion of energy, lipid-repair, and antioxidant metabolites in CFSEhigh RBCs. By redox proteomics, irreversible protein oxidation primarily affected CFSEhigh RBCs. By proteomics, 96 proteins, mostly in the proteostasis family, had relocated to CFSEhigh RBC membranes. CFSEhigh RBCs exhibited decreased proteasome activity and deformability; increased phosphatidylserine exposure, osmotic fragility, and endothelial cell adherence; and were cleared from the circulation during human spleen ex vivo perfusion. Conversely, molecular, cellular, and circulatory properties of long-stored CFSElow RBCs resembled those of short-stored RBCs. CFSEhigh RBCs are morphologically and metabolically altered, have irreversibly oxidized and membrane-relocated proteins, and exhibit decreased proteasome activity. In vitro aging during storage selectively alters metabolism and proteostasis in SMEs, targeting these senescent cells for clearance.

cell biology↗

bioRxiv2024

A type IVB secretion system contributes to the pathogenicity of Yersinia pseudotuberculosis strains responsible for the Far East scarlet-like fever

Yersinia pseudotuberculosis is a food-borne pathogen responsible for a self-limiting gastrointestinal disease in humans known as mesenteric lymphadenitis. A phylogenetically distinct Y. pseudotuberculosis cluster from lineages 1 and 8 is associated to a specific syndrome called the Far East scarlet-like fever (FESLF), characterized by skin rash, hyperemic tongue and desquamation. Genome sequencing of FESLF strains previously revealed the presence in the plasmid pVM82 of dot/icm genes, homologous to those known to encode a T4BSS in the intracellular pathogens Legionella pneumophila and Coxiella burnetii. In the present article, we characterized the genomic features and functionality of the Y. pseudotuberculosis T4BSS (yT4BSS). We found higher dot/icm gene identity between Y. pseudotuberculosis and Pseudomonas putida genes than with those of L. pneumophila or C. burnetii. We validated the presence of all essential dot/icm genes required for the structure of a T4BSS. We then evaluated the conditions required for yT4BSS gene expression in vitro and identified an influence of temperature, with higher expression at 37{degrees}C, which mimicks the mammalian host temperature. The yT4BSS is also expressed in cellulo during the Y. pseudotuberculosis intracellular life cycle and in vivo during mouse infection. Although T4BSS functions are well characterized in the intracellular life cycle of L. pneumophila and C. burnetii, the yT4BSS appears to not be required for the intracellular survival nor for the establishment of a replication niche within cells of Y. pseudotuberculosis. Interestingly, the yT4BSS is implicated in Y. pseudotuberculosis FESLF strain pathogenicity when orally inoculated to mice but not during intravenous inoculation. Despite a role in virulence during oral infection, the yT4BSS does not influence organ colonization. However, the yT4BSS appears to be implicated in induction of important necrosis lesions in mesenteric lymph nodes and caeca of mice. Cytokine profil analyses revealed an induction of production of innate immunity related cytokines and chemokines depending on the yT4BSS in cellulo using a mouse bone marrow-derived macrophages infection model. Thus, the yT4BSS modulates cytokine responses of the host innate immune system during oral infection. In conclusion, the yT4BSS is a newly characterized virulence factor implicated in pathogenicity of Y. pseudotuberculosis strains from lineage 8 responsible for FESLF.

microbiology↗

bioRxiv2023

Tetraspanin CD82 maintains HTLV-1 biofilm polarization and is required for efficient viral transmission

The human T-lymphotropic virus type-1 (HTLV-1) is an oncogenic retrovirus whose transmission relies primarily on cell-to-cell contacts as cell-free viruses are poorly infectious. Among the intercellular transmission routes described, HTLV-1 biofilms are adhesive structures polarized at the cell surface that confine virions in a protective environment, which is believed to promote their simultaneous delivery during infection. Here, we show that several tetraspanins are enriched in HTLV-1 biofilms and incorporated into the viral envelope. However, we report that only tetraspanin CD82 interacts with HTLV-1 Gag which initiates its polarization into viral biofilms. Also, we demonstrate that CD82 maintains HTLV-1 biofilm polarization and favors viral transmission, as its silencing induces a complete reorganization of viral clusters at the cell surface and reduces the ability of infected T-cells to transmit the virus. Our results highlight the crucial role of CD82 in the architectural organization of HTLV-1 biofilms and their transfer through intercellular contacts.

microbiology↗

bioRxiv2021

The Endocytic Recycling Compartment Serves as a Viral Factory for Hepatitis E Virus

Background & AimsAlthough Hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet and it is currently unknown whether HEV infection leads to cellular membrane modelling as many positive-strand RNA viruses. HEV genome encodes three proteins, the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we aimed to probe infectious particles and viral factories in HEV-producing cells, using antibodies directed against the different ORF2 isoforms. MethodsWe generated monoclonal antibodies that specifically recognize the particle-associated ORF2i form, and antibodies that recognize the different ORF2 isoforms. We used them in confocal and electron microscopy approaches to probe viral factories in HEV-producing cells. We performed an extensive colocalization study of viral proteins with subcellular markers. We analyzed the impact of silencing Rab11, a central player of the endocytic recycling compartment (ERC). ResultsOne of the antibodies, named P1H1 and targeting the N-terminus of ORF2i, recognized delipidated HEV particles. Confocal and ultrastructural microscopy analyses of HEV-producing cells revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. These subcellular structures were enriched in ORF2 and ORF3 proteins, and were dependent on the ORF3 expression and ORF2i capsid protein assembly. Colocalization and silencing analyses revealed that these structures are derived from the ERC. ConclusionsOur study reveals that HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection. Lay summaryHepatitis E virus (HEV) is the leading cause of acute hepatitis worldwide but many steps of its lifecycle are still elusive. Thanks to the development of new antibodies that recognize the different forms of the HEV capsid protein, we were able to visualize vesicular and tubular structures that were established by the virus in the host cell. In addition, extensive efforts to identify these structures led us to conclude that HEV hijacks the endocytic recycling compartment of the cell to form this network of vesicles and tubules, which might be the hallmark of HEV infection.

microbiology↗

bioRxiv2021

A novel domain within the CIL regulates egress of IFITM3 from the Golgi and prevents its deleterious accumulation in this apparatus

The InterFeron-Induced TransMembrane proteins (IFITMs) are broad viral inhibitors that protect cells by preventing viral-to-cellular membrane fusion and they belong to the dispanin/CD225 family that includes vesicle trafficking regulators and proteins of unknown functions into four subfamilies (A-D). In this study, we uncover a novel domain that regulates the egress of IFITM3 from the Golgi and that is required to prevent IFITM3-driven v- to t-SNAREs membrane fusion inhibition and Golgi dysfunctions. The S-x-K-x-R-D domain is conserved among vertebrate members of the dispanin/CD225 A subfamily that regroups all IFITMs and through the study of mutations identified in patients affected by paroxysmal kinesigenic dyskinesia (PKD), we determine that it is functionally conserved also in PRRT2, member of the B subfamily. Overall, our study defines a novel domain that regulates the egress of dispanin/CD225 members from the Golgi and stresses the importance that regulation of this process bears to preserve the functions of this apparatus.

cell 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

A comparative study of the capacity of mesenchymal stromal cell lines to form spheroids

Mesenchymal stem cells (MSCs)-derived spheroid models favor maintenance of stemness, ex vivo expansion and transplantation efficacy. Spheroids may also be considered as useful surrogate models of the hematopoietic niche. However, accessibility to primary cells, from bone marrow (BM) or adipose tissues, may limit their experimental use and the lack of consistency in methods to form spheroids may affect data interpretation. In this study, we aimed to create a simple model by examining the ability of cell lines, from human (HS-27a and HS-5) and murine (MS-5) BM origins, to form spheroids, compared to primary human MSCs (hMSCs). Our protocol efficiently allowed the spheroid formation from all cell types within 24 hours. Whilst hMSCs-derived spheroids began to shrink after twenty-four hours, the size of spheroids derived from cell lines remained constant during three weeks. The difference was partially explained by the balance between proliferation and cell death, which could be triggered by hypoxia and induced oxidative stress. Our results demonstrate that, unlike hMSCs, MSC cell lines make reproductible spheroids that are easily handled. Thus, this model could help in understanding mechanisms involved in MSC functions and may provide a simple model by which to study cell interactions in the BM niche.

bioengineering↗