Biology subjects

Vidalain, P.-O.

Publications and source records attributed to Vidalain, P.-O..

11 recordsLinked to original sources

bioRxiv2026

Ritonavir-Induced Cellular Stress Alters Viral HBs Glycoprotein Biogenesis and Production of Infectious Hepatitis D Virions

Chronic co-infections by HBV and its satellite virus HDV are associated with a high risk of progression to cirrhosis and liver cancer, and therapeutic options for achieving a cure are still unsatisfactory. HBs is the main surface glycoprotein of both viruses, and is also massively secreted by infected hepatocytes in the form of empty subviral particles which suppress the host immune responses. This makes HBs an attractive target to develop therapeutic strategies. Here, we took advantage of the known interaction between the Large form HDV antigen (HDAg-L) and the small form of HBs (S-HBs) to develop a non-infectious, minimalistic reporter assay for the assembly and secretion of HDV particles. By screening the existing pharmacopeia for drugs that could interfere with S-HBs and HDAg-L co-secretion, we found that ritonavir and other Cytochrome P450 inhibitors affect the biogenesis of HBs and impair the production of infectious HDV virions. Mechanistically, we established that these drugs induce oxidative stress which dysregulates disulfide bond formation in the endoplasmic reticulum. As a consequence, the production of HBs, which depends on a dense network of disulfide bonds, is markedly affected as evidenced by an abnormal glycosylation profile, altered antigenic properties, and a poor expression of the largest form of HBs (L-HBs) which is essential to virus entry into target cells. This is associated with induction of the unfolded protein response, with the upregulation of CHOP/DDIT3 and key enzymes involved in the synthesis of the reducing metabolite glutathione (PHGDH, SHMT2, MTHFD2). Overall, our results indicate that alterations in redox homeostasis significantly impact HBs biogenesis, and reveal a druggable pathway that could be exploited to eliminate HDV in chronically infected patients. IMPACT AND IMPLICATIONSMore effective therapies are still needed to achieve a functional cure in patients chronically co-infected by HBV and HDV. In this study, we discovered that ritonavir, along with other cytochrome P450 inhibitors, can affect the production of infectious HDV particles in human hepatocyte cultures. Mechanistically, ritonavir induces oxidative stress and the unfolded protein response in the endoplasmic reticulum, thereby altering the biogenesis of HBs, the surface glycoprotein of both viruses. This work highlights the potential benefit and mechanism of action of ritonavir and related molecules in the treatment of co-infected patients.

microbiology↗

bioRxiv2026

A rapid agarose-free protocol for preparing human organotypic lung cultures to study respiratory virus infection and evaluate antivirals ex vivo

We describe a standardized and reproducible procedure to generate human organotypic lung cultures from surgical lung resection for the study of respiratory infections. The protocol details tissue harvesting, biopsy punching, mechanical slicing, culture at the air-liquid interface. This technique enables robust ex vivo infections of human lung tissue with respiratory viruses, including Influenza A and Nipah. The described system can be used to study host-pathogen interactions, analyze innate immune responses, and evaluate antiviral candidates in physiologically relevant human lung tissue. For complete details on the use and execution of this protocol, please refer to Cezard et al1.

pathology↗

bioRxiv2025

NAD metabolism plays multiple roles in influenza A virus replication in a novel ex vivo model of mouse lung infection.

Despite the availability of prophylactic and therapeutic measures, Influenza A viruses (IAV) remain a major public health concern, causing an estimated 650 000 deaths annually. In this context, the identification of metabolic vulnerabilities of IAV replication could help develop complements to existing antiviral strategies. Here, we used nicotinamide phosphoribosyltransferase (NAMPT) inhibitors to demonstrate that nicotinamide adenine dinucleotide (NAD) is essential for IAV replication and infectious particles production both in vitro and ex vivo. We established an innovant ex vivo model of murine organotypic lung cultures (mOLC) that is pertinent to analyze cell metabolic alterations triggered by IAV infection. Using untargeted metabolomic profiling and spatial transcriptomic analyses, our research revealed that IAV-infection decreased NAD+ levels in mOLCs, while PARPs, a group of NAD+-consuming enzymes, were upregulated. Pharmacological inhibition of the mono-ADP-ribosyl-transferase (mono-ART) activity of PARPs restricted IAV infection ex vivo, suggesting that NAD+ sustains the proviral activity of these enzymes. Overall, our study identifies NAD metabolism as a central regulator of IAV infection, providing redox cofactor to host cell biosynthetic processes and substrate for mono-ART activity. Our results highlight NAMPT and PARP mono-ART activity as promising antiviral targets. Significance StatementViruses are intracellular parasites that rely on the host cellular metabolism for their replication. Our results demonstrate that Influenza A virus (IAV) replication is critically dependent on NAD availability, an enzyme cofactor essential for redox metabolic reactions and a substrate for NAD+-consuming enzymes. Using a novel ex vivo infection model of mouse lung tissue, we found that IAV infection results in NAD depletion and enhanced expression of NAD-consuming PARP enzymes. Inhibitors of NAD biosynthesis and mono(ADP-ribosyl) transferase activity of PARP are restricting viral replication and infectious particles production. These findings highlight the proviral role for PARP mono-ART activity and identify NAMPT and PARP inhibitors as potential host-directed antivirals against IAV.

microbiology↗

bioRxiv2025

Cis-aconitate therapy protects against influenza mortality by dual targeting of viral polymerase and ERK/AKT/NF-κB signaling

Influenza virus poses a significant global health challenge, causing approximately 500,000 deaths annually. Its ability to evade antiviral treatments and vaccine-induced immunity underscores the need for novel therapeutic approaches. Our study identifies cis-aconitate (cis-aco), a mitochondria-derived metabolite, as a potent dual-action agent against influenza, independently of its metabolic derivative, itaconate. Cis-aco impairs viral polymerase activity, suppressing viral mRNA expression and protein synthesis to inhibit replication across a range of influenza subtypes. This antiviral efficacy is confirmed in ex vivo human airway and lung organotypic models. Beyond its antiviral properties, cis-aco exhibits potent anti-inflammatory effects, disrupting key inflammatory cascades and reducing the secretion of inflammatory mediators. In a mouse model of influenza pneumonia, cis-aco mitigates viral replication, inflammation, and immune cell activation, significantly improving survival. Notably, its efficacy persists even when administered at later stages of infection, when oseltamivir/Tamiflu(R) is no longer effective. These findings position cis-aco as a promising influenza treatment, combining antiviral and anti-inflammatory benefits within a clinically relevant timeframe.

immunology↗

bioRxiv2021

Respiratory Syncytial virus NS1 protein targets the transactivator binding domain of MED25

Respiratory syncytial virus has evolved a unique strategy to evade host immune response by coding for two non-structural proteins NS1 and NS2. Recently it was shown that in infected cells, nuclear NS1 could be involved in transcription regulation of host genes linked to innate immune response, via an interaction with chromatin and the Mediator complex. Here we identified the MED25 Mediator subunit as an NS1 interactor in a yeast two-hybrid screen. We demonstrate that NS1 directly interacts with MED25 in vitro and in cellula, and that this interaction involves the C-terminal 3 helix of NS1 and the MED25 ACID domain. More specifically we showed by NMR that the NS1 3 sequence primarily binds to the MED25 ACID H2 face, which is a transactivation domain (TAD) binding site for transcription regulators such as ATF6, a master regulator of ER stress response activated upon viral infection. Moreover, we found out that the NS1 3 helix could compete with ATF6 TAD binding to MED25. This finding points to a mechanism of NS1 interfering with innate immune response by impairing recruitment by cellular TADs of the Mediator via MED25 and hence transcription of specific genes by RNA polymerase II. ImportanceHuman RSV is the leading cause of infantile bronchiolitis in the world and one of the major causes of childhood deaths in resource-poor settings. It is a major unmet target for vaccines and anti-viral drugs. RSV non-structural protein NS1 is known to antagonize the cellular immune response and was recently shown to be involved in transcription regulation of infected cells. However, the exact mechanism of this regulation is not well defined. Here we show that nuclear NS1 interacts directly with the Mediator subunit MED25 and is able to compete with a cellular transcription activator, which is activated during viral infection. We hypothesize that this interaction may underlie regulation of the expression of genes involved in the innate immune response.

microbiology↗

bioRxiv2021

RACK1 associates with RNA-binding proteins Vigilin and SERBP1 to control dengue virus replication

Dengue virus (DENV), a re-emerging virus transmitted by Aedes mosquitoes, causes severe pathogenesis in humans. No effective treatment is available against this virus. We recently identified the scaffold protein RACK1 as a component of the DENV replication complex, a macromolecular complex essential for viral genome amplification. Here, we show that RACK1 is important for DENV infection. RACK1 mediates DENV replication through binding to the 40S ribosomal subunit. Mass spectrometry analysis of RACK1 partners coupled to a loss-of-function screen identified the RNA binding proteins Vigilin and SERBP1 as DENV host dependency factors. Vigilin and SERBP1 interact with DENV viral RNA (vRNA), forming a ternary complex with RACK1 to mediate viral replication. Overall, our results indicate that RACK1 recruits Vigilin and SERBP1, linking the DENV vRNA to the translation machinery for optimal translation and replication.

microbiology↗

bioRxiv2021

Depletion of TAX1BP1 amplifies innate immune responses during respiratory syncytial virus infection

Respiratory syncytial virus (RSV) is the main cause of acute respiratory infections in young children, and also has a major impact in the elderly and immunocompromised people. In the absence of vaccine or efficient treatment, a better understanding of RSV interactions with the host antiviral response during infection is needed. Previous studies revealed that cytoplasmic inclusion bodies (IBs) where viral replication and transcription occur could play a major role in the control of innate immunity during infection by recruiting cellular proteins involved in the host antiviral response. We recently showed that the morphogenesis of IBs relies on a liquid-liquid phase separation mechanism depending on the interaction between viral nucleoprotein (N) and phosphoprotein (P). These scaffold proteins are expected to play a central role in the recruitment of cellular proteins to IBs. Here, we performed a yeast two-hybrid screen using RSV N protein as a bait, and identified the cellular protein TAX1BP1 as a potential partner of N. This interaction was validated by pulldown and immunoprecipitation assays. We showed that TAX1BP1 suppression has only a limited impact on RSV infection in cell cultures. On the contrary, in vivo experiments showed that RSV replication is decreased in TAX1BP1KO mice, whereas the production of inflammatory and antiviral cytokines is enhanced. In vitro infection of either wild-type or TAX1BP1KO alveolar macrophages confirmed that the innate immune response to RSV infection is enhanced in the absence of TAX1BP1. Altogether, our results suggest that RSV could hijack TAX1BP1 to restrain the host immune response during infection. ImportanceRespiratory syncytial virus (RSV), which is the leading cause of lower respiratory tract illness in infants, still remains a medical problem in the absence of vaccine or efficient treatment. This virus is also recognized as a main pathogen in the elderly and immunocompromised people, and the occurrence of co-infections (with other respiratory viruses and bacteria) amplifies the risks of developing respiratory distress. In this context, a better understanding of the pathogenesis associated to viral respiratory infections, which depends on both viral replication and the host immune response, is needed. The present study reveals that the cellular protein TAX1BP1, which interacts with the RSV nucleoprotein N, participates in the control of the innate immune response during RSV infection, suggesting that N-TAX1BP1 interaction represents a new target for the development of antivirals.

microbiology↗

bioRxiv2021

Characterization and functional interrogation of SARS-CoV-2 RNA interactome

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19 pandemic, which has caused a devastating global health crisis. The emergence of highly transmissible novel viral strains that escape neutralizing responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2 biology and to develop additional therapeutic strategies. Using a comprehensive identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S) approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-2 viral genome during infection. By systematically knocking down their expression in a human lung epithelial cell line, we found that the majority of the RBPs identified in our study are proviral factors that regulate SARS-CoV-2 genome replication. We showed that some of these proteins represented drug targets of interest for inhibiting SARS-CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-CoV-2 RNA interactome during infection and highlights candidates for host-centered antiviral therapies.

microbiology↗

bioRxiv2020

Replication of Equine arteritis virus is efficiently suppressed by purine and pyrimidine biosynthesis inhibitors

RNA viruses are responsible for a large variety of animal infections. Equine Arteritis Virus (EAV) is a positive single-stranded RNA virus member of the family Arteriviridae from the order Nidovirales like the Coronaviridae. EAV causes respiratory and reproductive diseases in equids. Although two vaccines are available, the vaccination coverage of the equine population is largely insufficient to prevent new EAV outbreaks around the world. In this study, we present a high-throughput in vitro assay suitable for testing candidate antiviral molecules on equine dermal cells infected by EAV. Using this assay, we identified three molecules that impair EAV infection in equine cells: the broad-spectrum antiviral and nucleoside analog ribavirin, and two compounds previously described as inhibitors of dihydroorotate dehydrogenase (DHODH), the fourth enzyme of the pyrimidine biosynthesis pathway. These molecules effectively suppressed cytopathic effects associated to EAV infection, and strongly inhibited viral replication and production of infectious particles. Since ribavirin is already approved in human and small animal, and that several DHODH inhibitors are in advanced clinical trials, our results open new perspectives for the management of EAV outbreaks.

microbiology↗

bioRxiv2020

Lipogenesis and innate immunity in hepatocellular carcinoma cells reprogrammed by an isoenzyme switch of hexokinases

During the cancerous transformation of normal hepatocytes into hepatocellular carcinoma (HCC), the enzyme catalyzing the first rate-limiting step of glycolysis, namely the glucokinase (GCK), is replaced by the higher affinity isoenzyme, hexokinase 2 (HK2). The transcriptomic analysis of HCC tumors shows that highest expression level of HK2 in tumor lesions is inversely correlated to GCK expression, and is associated to poor prognosis for patient survival. To further explore functional consequences of the GCK-to-HK2 isoenzyme switch occurring during carcinogenesis, HK2 was knocked-out in the HCC cell line Huh7 and replaced by GCK, to generate the Huh7-GCK+/HK2- cell line. HK2 knockdown and GCK expression rewired central carbon metabolism, stimulated mitochondrial respiration and restored essential metabolic functions of normal hepatocytes such as lipogenesis, VLDL secretion, glycogen storage. It also reactivated innate immune responses and sensitivity to natural killer cells, showing that consequences of the HK switch extend beyond metabolic reprogramming.

cancer biology↗

bioRxiv2019

Towards an ″assayome″ for binary interactome mapping

Complementary assays are required to comprehensively map complex biological entities such as genomes, proteomes and interactome networks. However, how various assays can be optimally combined to approach completeness while maintaining high precision often remains unclear. Here, we propose the concept of an "assayome" for binary protein-protein interaction (PPI) mapping as an optimal combination of assays and/or assay versions that maximizes detection of true positive interactions, while avoiding detection of random protein pairs. We engineered a novel NanoLuc two-hybrid (N2H) system that integrates 12 different versions differing by protein expression systems and tagging configurations. The resulting N2H assayome recovers as many PPIs as 10 distinct assays combined. Thus, to further improve PPI mapping, developing alternative versions of existing assays might be as productive as designing completely new assays. Our assayome concept should be applicable to systematic mapping of other biological landscapes.

systems biology↗