Biology subjects

Jouvenet, N.

Publications and source records attributed to Jouvenet, N..

13 recordsLinked to original sources

bioRxiv2026

Powassan Virus NS5 Antagonizes TYK2-Mediated Immune Signaling Pathways

Powassan virus (POWV) is an emerging neurotropic tick-borne flavivirus, yet the mechanisms by which POWV evades host antiviral immunity remain poorly defined. Here, we identify multiple mechanisms of POWV innate immune antagonism with the viral polymerase NS5 protein as a central inhibitor of cytokine signaling. Both POWV lineages potently inhibited type I interferon (IFN) signaling, and NS5 expression suppressed signaling and downstream interferon-stimulated gene expression. Affinity purification-mass spectrometry identified the host kinase TYK2 as a conserved NS5 interactor. POWV NS5 binds the TYK2 kinase domain through a discrete interface within the RNA-dependent RNA polymerase (RdRp) region between catalytic motifs B and C and inhibits TYK2 phosphorylation. Disruption of this interface abrogated TYK2 binding and reduced NS5-mediated IFN antagonism, while revealing additional TYK2-independent mechanisms of immune suppression. POWV NS5 also inhibited TYK2-dependent IFN-{lambda} and IL-12 signaling, demonstrating that its immune antagonism extends beyond type I IFN. Together, these findings identify TYK2 as a central target of POWV immune evasion and implicate the variable RdRp B-C region as an interface for flavivirus-host interactions. More broadly, our results reveal how POWV can coordinately suppress multiple antiviral cytokine pathways and provide insight into mechanisms that may shape tick-borne flavivirus host adaptation and pathogenesis.

microbiology↗

bioRxiv2026

Phylogenetic analysis of enteroviruses from non-human primates reveals two new species within the genus Enterovirus and inter-species recombination

To date, 15 species have been described within the genus Enterovirus. Previous studies suggested the existence of another species comprising strains isolated from the stool specimens of non-human primates (NHPs) in Central Africa. Moreover, numerous full-length or partial genomic sequences of NHP enteroviruses (EVs) can be found in GenBank without being properly classified. To our knowledge, no comprehensive synthesis of NHP EV data exists, leaving genetic relationships between strains across independent studies unclear. To address these gaps, we sequenced the complete genome of four NHP EVs from our stool collection and conducted an extensive search of NHP EV sequences in GenBank to perform a comprehensive phylogenetic analysis. Our analyses revealed two new species tentatively named Enterovirus mbel and Enterovirus noa, which contain at least 6 and 2 virus types, respectively. We also identified new virus types within the known species EV-J. Phylogenetic analyses strongly suggest interspecies recombination events between NHP EVs in the non-structural region of the genome, challenging the long-held view that recombination is confined within narrowly defined subsets of EVs belonging to the same species. We also performed the first comprehensive comparative analysis of full length human and NHP EV genomes, focusing on GC content, dinucleotide frequencies and codon-usage bias. GC content emerged as the most robust host-associated marker: all NHP-associated virus types within species E. alphacoxsackie and E. betacoxsackie displayed GC % below 47 %, whereas human-derived virus types exhibited GC % above 47 %. Dinucleotide frequency, Effective Number of Codons (ENC) and Relative Synonymous Codon Usage highlighted distinct codon-bias clusters that mirror the phylogenetic relationships between EVs but only partially correlate with their respective hosts of origin. This work enhances our understanding of EVs circulating in NHPs and paves the way for future research aiming at understanding the mechanisms underlying host-adaptation among EVs.

genomics↗

bioRxiv2025

Compositional analysis of yellow fever virus induced stress granules reveals a functional connection to mitochondrial homeostasis

The phase separation of biomolecules into so-called stress granules (SGs) allows the cell to tightly regulate translation activity in response to different stimuli, such as oxidative stress, starvation, or the recognition of non-cellular RNA. Recent reports suggest SGs induced during viral infection, may act as a crossroad between the cellular stress response and the activation of the innate immune response. Here, we aimed to dissect the role of SGs in the context of yellow fever virus (YFV) infection. We found that YFV infection resulted in translational shut-off from 24 hpi on followed by the formation of SGs at 48 hpi, a delay potentially associated with the ability of the YFV capsid to inhibit SG formation, through an interaction with the major SG scaffolding protein G3BP1. To elucidate the role of YFV-induced SGs during infection, we inhibited SG assembly using a small-molecule inhibitor and find that SG formation does not influence viral replication. Uncovering the first proteome of virus-induced SGs, our compositional analysis revealed a specific enrichment of proteins associated with mitochondrial processes in YFV-induced SGs. Indeed, we show that YFV infection results in mitochondrial damage and dysfunction. Together, we propose that YFV-induced SGs may be involved not only in the modulation of cellular homeostasis but also in influencing mitochondrial functions. Author SummaryViruses impose a major burden on the infected host - from structural rearrangements needed to assemble replication complexes, to exploiting cellular energy resources and genetic rewiring associated with antiviral responses. The assembly of membrane-less organelles such as stress granules (SGs) enable cells to rapidly tune cellular processes upon sensing of stresses such as viruses. Moreover, the cells innate immune response is proposed to be regulated by SGs and in turn many viruses disrupt or highjack their components. Yet, the molecular basis for SG functions during infection remain ambiguous. Here we investigated the interplay between yellow fever virus (YFV) infection and SGs. We demonstrate that infection with attenuated or pathogenic viruses result in the formation of SGs. Their compositional analysis reveal that they sequester mitochondrial proteins, correlating with altered mitochondrial functions during infection. This highlights a novel complex interplay between membrane-bound and membrane-less organelles which could present novel opportunities for antiviral therapies.

microbiology↗

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

Coronavirus protein interaction mapping in bat and human cells identifies molecular and genetic switches for immune evasion and replication

Coronaviruses, including SARS-CoV-2, can cause severe disease in humans, whereas reservoir hosts like Rhinolophus bats remain asymptomatic. To investigate how host-specific protein-protein interactions (PPIs) influence infection, we generated comparative PPI maps for SARS-CoV-2 and its bat-origin relative RaTG13 using affinity purification-mass spectrometry (AP-MS) in human and Rhinolophus ferrumequinum (RFe) bat cells. This approach identified both conserved and virus- and host-specific interactions that regulate infection dynamics. Notably, SARS-CoV-2 required a non-synonymous mutation in nucleocapsid to replicate in bat cells expressing human ACE2 and TMPRSS2. Analysis of the viral protein Orf9b revealed differential interactions with mitochondrial proteins Tom70 and MTARC2. A single residue difference in Orf9b between SARS-CoV-2 and RaTG13 functions as a molecular switch, weakening Tom70 binding and immune evasion in human cells while enhancing interaction with the bat-specific restriction factor MTARC2. These findings demonstrate how a single-residue substitution can reshape virus-host interactions and contribute to immune evasion and host adaptation.

systems biology↗

bioRxiv2025

The type I and III interferon responses restrict infection with tick-borne orthoflaviviruses through IFI6

Tick-borne orthoflaviviruses (TBOVs) are a growing global health concern. Several representatives of this viral family cause fatal disease in humans with increasing case numbers throughout the last decades. The innate immune response, especially interferon (IFN)-dependent signaling, is an essential part of the human defense system that counteracts infection with TBOVs and other viruses. Even though they activate the same signaling cascade, IFNs belonging to the type I and III families trigger differing gene expression patterns. Which genes the two IFN families induce to restrict infection with TBOVs remains poorly characterized. Here we show that type I and III IFNs are both capable of restricting TBOV infection of human cell lines in a cell type-specific manner. Infection of C57BL/6J mice with knockouts for either IFN type I or III receptors further underscored the critical role of IFN signaling in controlling TBOV replication in vivo. To assess the contribution of single genes to controlling TBOV infection in human cells, we used a CRISPR/Cas9-KO-based screening approach. This strategy identified IFI6 as a central player for IFN type I- and III-driven responses against TBOVs. We further defined IFI6 as an ER-resident protein that restricts TBOV replication at a post-entry step. Our work thus opens new perspectives for targeting weak points in the life cycle of TBOVs and other orthoflaviviruses, potentially paving the way for the development of new antiviral therapeutics. One Sentence SummaryType I and III interferons are crucial for protection against tick-borne orthoflavivirus infection in vitro and in vivo, both relying on IFI6 as a main antiviral effector.

microbiology↗

bioRxiv2024

Hepatitis E Virus-induced antiviral response by plasmacytoid dendritic cells is modulated by the ORF2 protein

Type I and III interferons (IFN-I/III) are critical to protect the host during viral infection. Previous studies have shown that IFN-mediated antiviral responses against hepatitis E virus (HEV) are suppressed and defeated by viral escape mechanisms at play in infected hepatocytes. Here, we studied the anti-HEV function of IFN secreted by plasmacytoid dendritic cells (pDCs), which are specialized producers of IFNs. We showed that pDCs co-cultured with HEV-replicating cells secreted IFN in a cell-to-cell contact-dependent manner. Pharmacological inhibitor and antibodies targeting contact proteins revealed that pDC response against HEV required the endosomal nucleic-acid sensor TLR7 and adhesion molecules, such as ICAM-I and L{beta}2-integrin. IFNs secreted by pDCs reduced viral spread. Intriguingly, ORF2, the capsid protein of HEV, can be produced in various forms by the infected cells. During infection, a fraction of the intracellular ORF2 protein localizes into the nucleus while another ORF2 fraction packages viral genomes to produce infectious virions. In parallel, glycosylated forms of ORF2 are also massively secreted by infected cells. Using viral genome expressing ORF2 mutants, we showed that glycosylated ORF2 forms contribute to better recognition of infected cells by pDCs via regulation of contacts between infected cells and pDCs. ORF2 forms may thus modulate pDC-mediated anti-HEV response. Together, our results suggest that liver-resident pDCs, which exhibit comparable IFN-producing ability as blood-derived pDCs, may be essential to control HEV replication.

immunology↗

bioRxiv2023

Tick-borne flavivirus NS5 antagonizes interferon signaling by inhibiting the catalytic activity of TYK2

The mechanisms utilized by different flaviviruses to evade antiviral functions of interferons are varied and incompletely understood. Using virological approaches, biochemical assays and mass spectrometry analysis, we report here that the NS5 protein of tick-borne encephalitis virus (TBEV) and Louping Ill virus (LIV), two related tick-borne flaviviruses, antagonize JAK-STAT signaling through interactions with tyrosine kinase 2 (TYK2). Co-immunoprecipitation (co-IP) experiments, yeast gap-repair assays, computational protein-protein docking and functional studies identified a stretch of 10 residues of the RNA dependent RNA polymerase domain of tick-borne flavivirus NS5, but not mosquito-borne NS5, that is critical for interaction with the TYK2 kinase domain. Additional co-IP assays performed with several TYK2 orthologs revealed that the interaction was conserved across mammal species. In vitro kinase assays showed that TBEV and LIV NS5 reduced the catalytic activity of TYK2. Our results thus illustrate a novel mechanism by which viruses suppress the interferon response. TeaserInhibition of the catalytic activity of a key kinase of the JAK/STAT pathway by a viral protein

microbiology↗

bioRxiv2022

Transcriptomic landscapes of SARS-CoV-2-infected and bystander lung cells reveal a selective upregulation of NF-κB-dependent coding and non-coding proviral transcripts

Investigations of cellular responses to viral infection are commonly performed on mixed populations of infected and uninfected cells or using single-cell RNA sequencing, leading to inaccurate and low-resolution gene expression interpretations. Here, we performed deep polyA+ transcriptome analyses and novel RNA profiling of SARS-CoV-2 infected lung epithelial cells, sorted based on the expression of the viral spike (S) protein. Infection caused a massive reduction in mRNAs and lncRNAs, including transcripts coding for antiviral factors, such as interferons (IFN). This absence of IFN signaling probably explained the poor transcriptomic response of bystander cells co-cultured with S+ ones. NF-{kappa}B pathway and the inflammatory response escaped the global shutoff in S+ cells. Functional investigations revealed the proviral function of the NF-{kappa}B pathway and the antiviral activity of CYLD, a negative regulator of the pathway. Thus, our transcriptomic analysis on sorted cells revealed additional genes that modulate SARS-CoV-2 replication in lung cells.

microbiology↗

bioRxiv2021

A virus-encoded microRNA contributes to evade innate immune response during SARS-CoV-2 infection

SARS-CoV-2 infection results in impaired interferon response in severe COVID-19 patients. However, how SARS-CoV-2 interferes with host immune response is incompletely understood. Here, we sequenced small RNAs from SARS-CoV-2-infected human cells and identified a micro-RNA (miRNA) encoded in a recently evolved region of the viral genome. We show that the virus-encoded miRNA produces two miRNA isoforms in infected cells by the enzyme Dicer and they are loaded into Argonaute proteins. Moreover, the predominant miRNA isoform targets the 3UTR of interferon-stimulated genes and represses their expression in a miRNA-like fashion. Finally, the two viral miRNA isoforms were detected in nasopharyngeal swabs from COVID-19 patients. We propose that SARS-CoV-2 employs a virus-encoded miRNA to hijack the host miRNA machinery and evade the interferon-mediated immune response.

molecular biology↗

bioRxiv2021

Species-specific molecular barriers to SARS-CoV-2 replication in bat cells

Bats are natural reservoirs of numerous coronaviruses, including the potential ancestor of SARS-CoV-2. Knowledge concerning the interaction between coronaviruses and bat cells is sparse. We investigated the susceptibility of primary cells from Rhinolophus ferrumequinum and Myotis species, as well as of established and novel cell lines from Myotis myotis, Eptesicus serotinus, Tadarida brasiliensis and Nyctalus noctula, to SARS-CoV-2 infection. None of these cells were sensitive to infection, not even the ones expressing detectable levels of angiotensin-converting enzyme 2 (ACE2), which serves as the viral receptor in many mammalian species. The resistance to infection was overcome by expression of human ACE2 (hACE2) in three cell lines, suggesting that restriction to viral replication was due to a low expression of bat ACE2 (bACE2) or absence of bACE2 binding in these cells. Infectious virions were produced but not released from hACE2-transduced M. myotis brain cells. E. serotinus brain cells and M. myotis nasal epithelial cells expressing hACE2 efficiently controlled viral replication. This ability to control viral replication correlated with a potent interferon response. Our data highlight the existence of species-specific molecular barriers to viral replication in bat cells. These novel chiropteran cellular models are valuable tools to investigate the evolutionary relationships between bats and coronaviruses. Author summaryBats host ancestors of several viruses that cause serious disease in humans, as illustrated by the on-going SARS-CoV-2 pandemic. Progress in investigating bat-virus interactions have been hampered by a limited number of bat cell lines. We have generated primary cells and cell lines from several bat species that are relevant for coronavirus research. The varying susceptibilities of the cells to SARS-CoV-2 infection offered the opportunity to uncover some species-specific molecular restrictions to viral replication. All bat cells exhibited a potent entry-dependent restriction. Once this block was overcome by over-expression of human ACE2, which serves at the viral receptor, two bat cell lines controlled well viral replication, which correlated with the inability of the virus to counteract antiviral responses. Other cells potently inhibited viral release. Our novel bat cellular models contribute to a better understanding of the molecular interplays between bats and viruses.

microbiology↗

bioRxiv2021

Identification of DAXX As A Restriction Factor Of SARS-CoV-2 Through A CRISPR/Cas9 Screen

Interferon restricts SARS-CoV-2 replication in cell culture, but only a handful of Interferon Stimulated Genes with antiviral activity against SARS-CoV-2 have been identified. Here, we describe a functional CRISPR/Cas9 screen aiming at identifying SARS-CoV-2 restriction factors. We identified DAXX, a scaffold protein residing in PML nuclear bodies known to limit the replication of DNA viruses and retroviruses, as a potent inhibitor of SARS-CoV-2 and SARS-CoV replication in human cells. Basal expression of DAXX was sufficient to limit the replication of SARS-CoV-2, and DAXX over-expression further restricted infection. In contrast with most of its previously described antiviral activities, DAXX-mediated restriction of SARS-CoV-2 was independent of the SUMOylation pathway. SARS-CoV-2 infection triggered the re-localization of DAXX to cytoplasmic sites and promoted its degradation. Mechanistically, this process was mediated by the viral papain-like protease (PLpro) and the proteasome. Together, these results demonstrate that DAXX restricts SARS-CoV-2, which in turn has evolved a mechanism to counteract its action.

microbiology↗

bioRxiv2019

Y-RNAs Lead an Endogenous Program of RIG-I Agonism Mobilized upon RNA Virus Infection and Targeted by HIV

Pattern recognition receptors (PRRs) protect against host invasion by detecting specific molecular patterns found in pathogens and initiating an immune response. While microbial-derived PRR ligands have been extensively characterized, the contribution and relevance of endogenous ligands to PRR activation during viral infection remain overlooked. In this work, we characterize the landscape of endogenous ligands that engage RIG-I-like receptors (RLRs) upon infection by a positive-sense RNA virus, a negative-sense RNA virus or a retrovirus. We found that several endogenous RNAs transcribed by RNA polymerase 3 (Pol3) specifically engage RLRs, and in particular the family of small non-coding repeats Y-RNAs, which presents the highest affinity as RIG-I ligands. We show that this recognition is dependent on Y-RNA mimicking viral secondary structure and its 5-triphosphate extremity. Further, we found that HIV-1 infection triggers a VPR-dependent downregulation of RNA triphosphatase DUSP11 in vitro and in vivo, leading to an increase of Y-RNA 5-triphosphorylation that enables their immunogenicity. Importantly, we show that altering DUSP11 expression is sufficient to induce a type-I interferon and T cell activation transcriptional program associated with HIV-1 infection. Overall, our work uncovers the critical contribution of endogenous repeat RNAs ligands to antiviral immunity and demonstrates the role of this pathway in HIV-1 infection.

immunology↗