Biology subjects

Chazal, M.

Publications and source records attributed to Chazal, M..

10 recordsLinked to original sources

bioRxiv2025

Nuclear activities and interactome of the NS5 protein of Tick-Borne Encephalitis Virus

Orthoflaviviruses are RNA viruses responsible for significant diseases in humans, domesticated animals and wildlife. Their NS5 protein is central in viral replication, functioning both as an RNA-dependent RNA polymerase and a methyltransferase, while also modulating cellular processes, including the interferon response. Although viral replication is cytoplasmic, the NS5 protein of several mosquito-borne orthoflaviviruses cycles between the cytoplasm and the nucleus of infected human cells. However, the nuclear localization and function of NS5 of tick-borne orthoflaviviruses, such as tick-borne encephalitis virus (TBEV), remained poorly understood. Microscopy analysis and cell fractionation revealed that the NS5 protein of TBEV localized to both the cytoplasm and nucleoplasm of infected cells. Mutagenesis studies identified critical residues required for its nuclear targeting. Mutating these residues in a TBEV replicon abolished viral replication. Immunoprecipitation-mass spectrometry analyses performed in two human cell lines infected with TBEV recovered 352 NS5 partners. Among them, 187 were nuclear or partially nuclear. By integrating our interactome data with that of Powassan virus (POWV), another tick-borne orthoflavivirus, we refined a list of 20 high-confidence NS5 partners, including splicing factors and chromatin modulators. Functional analysis revealed that seven of these nuclear partners significantly modulated viral replication, further underscoring the importance of nuclear NS5 in the viral life cycle. Our work advances our understanding of the nuclear function of the NS5 proteins of tick-borne orthoflaviviruses. ImportanceTick-borne orthoflaviviruses are emerging globally, spreading across Europe, Asia, and North America, where they infect humans, domesticated animals, and wildlife. These viruses produce a protein called NS5, which drives viral replication and helps evade the innate immune response. We observed that the NS5 protein of tick-borne encephalitis virus (TBEV) localized both in the cytoplasm and nucleoplasm of infected human cells. We identified the specific residues responsible for its nuclear addressing and showed that it interacts with numerous nuclear proteins, including some involved in regulating gene expression. Seven of these nuclear partners significantly influenced viral replication, highlighting the importance of NS5s nuclear activity. This work sheds light on how tick-borne orthoflaviviruses manipulate host cells, deepening our understanding of their replication strategies.

microbiology↗

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↗

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

Discovery of genes that modulate flavivirus replication in an interferon-dependent manner

Establishment of the interferon (IFN)-mediated antiviral state provides a crucial initial line of defense against viral infection. Numerous genes that contribute to this antiviral state remain to be identified. Using a loss-of-function strategy, we screened an original library of 1156 siRNAs targeting 386 individual curated human genes in stimulated microglial cells infected with Zika virus (ZIKV), an emerging RNA virus that belongs to the flavivirus genus. The screen recovered twenty-one potential host proteins that modulate ZIKV replication in an IFN-dependent manner, including the previously known IFITM3 and LY6E. Further characterization contributed to delineate the spectrum of action of these genes towards other pathogenic RNA viruses, including Hepatitis C virus and SARS-CoV-2. Our data revealed that APOL3 acts as a proviral factor for ZIKV and several other related and unrelated RNA viruses. In addition, we showed that MTA2, a chromatin remodeling factor, possesses potent flavivirus-specific antiviral functions. Our work identified previously unrecognized genes that modulate the replication of RNA viruses in an IFN-dependent way, opening new perspectives to target weakness points in the life cycle of these viruses.

microbiology↗

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↗

bioRxiv2020

The inflammasome components NLRP3 and ASC act in concert with IRGM to rearrange the Golgi during viral infections

Hepatitis C virus (HCV) infection triggers Golgi fragmentation through the Golgi-resident protein immunity-related GTPase M (IRGM). Here, we report the role of NLRP3 and ASC, two inflammasome components, in the initial events leading to this fragmentation. We show that ASC resides at the Golgi with IRGM at homeostasis. Upon infection, ASC dissociates from both IRGM and Golgi and associates with HCV-induced NLRP3. NLRP3 silencing inhibits Golgi fragmentation. ASC silencing disrupts the Golgi structure in both control and infected cells and reduces the localization of IRGM at the Golgi. Silencing IRGM cannot totally restore the Golgi structure. These data highlight a role for ASC, upstream of the formation of the inflammasome, in regulating IRGM through its control on the Golgi. A similar mechanism occurs in response to Nigericin or infection with Zika virus (ZIKV). We propose a model for a newly ascribed function of the inflammasome components in Golgi structural remodeling.

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↗