Biology subjects

Goujon, C.

Publications and source records attributed to Goujon, C..

10 recordsLinked to original sources

bioRxiv2025

SNX-BAR proteins 5 and 6 are required for NCOA7-AS antiviral activity against influenza A virus

Interferon-induced antiviral proteins act on the first line of defence against viruses, including influenza A virus (IAV). Among those, the short isoform of Nuclear Receptor Coactivator 7 (NCOA7-AS) has been shown to inhibit IAV entry and more especially the fusion between endosomal and viral membranes. Ectopic expression of NCOA7-AS leads to the increased acidification of the endolysosomal pathway, putatively through NCOA7-AS interaction with the vacuolar ATPase (V-ATPase). However, the precise mechanism of action of NCOA7-AS is not fully understood. Here, we identified the cellular partners of NCOA7-AS by mass spectrometry, including the V-ATPase subunits known to interact with NCOA7-AS. A point mutation disrupting the interaction with the V-ATPase led to loss of antiviral activity against IAV, demonstrating that V-ATPase engagement is required for the antiviral phenotype. Moreover, sorting nexin (SNX) 1/2/5/6 were identified as novel partners of NCOA7-AS. These proteins are involved in retrograde transport of cellular cargoes from endosomes to the trans-Golgi network. We revealed that SNX5/6 interacted directly with NCOA7-AS and were essential for NCOA7-AS antiviral activity against IAV. Interestingly, crystal structures of NCOA7-AS/SNX5 complexes showed that the SNX5-interaction motif in NCOA7-AS was similar to those found in known cargoes of SNX5/6. In addition, we could pinpoint several critical residues that were important for binding and antiviral activity. Collectively, our study identifies novel essential partners for NCOA7-AS antiviral activity and the structural basis for their interaction.

microbiology↗

bioRxiv2024

SARS-CoV-2 predation of Golgi-bound PI4P primes the massive activation of the DNA Damage Response kinase ATM in the cytoplasm

Like all viruses, SARS-CoV-2, the causative agent of COVID-19, relies on host cell resources to replicate. Our study reveals that, among these resources, SARS-CoV-2 hijacks the oxysterol-binding protein 1 (OSBP1) transporter to exploit the Golgi-bound phosphatidylinositol-4-phosphate (PI4P) pool. This leads to a depletion of Golgi-resident PI4P, triggering the activation of the ATM DNA Damage Response (DDR) kinase in the cytoplasm. As such, ATM, typically anchored to PI4P at the Golgi in an inactive state, undergoes auto-phosphorylation and cytoplasmic release upon SARS-CoV-2-induced PI4P depletion. Conversely, pharmacological inhibition of ATM auto-phosphorylation, which stabilizes its interaction with PI4P, significantly impairs SARS-CoV-2 replication. The requirement for PI4P and impact of ATM inhibition might be conserved across coronaviruses, as similar effects were observed with HCoV-229E. Finally, SARS-CoV-2-induced, cytoplasmic ATM pre-activation primes cells for an accelerated response to DNA damage, which might contribute to the severe outcomes of COVID-19 observed in cancer patients undergoing chemo- or radiotherapy. Therefore, this study uncovers a DNA damage-independent mode of ATM activation and highlights the potential of ATM inhibitors as therapeutic agents against COVID-19.

microbiology↗

bioRxiv2024

Human MX1 induces the cytoplasmic sequestration of neo-synthesized influenza A virus vRNPs

Interferon-inducible Myxovirus resistance 1 (MX1) proteins are well-known to restrict influenza A virus (IAV) at early stages during viral replication, impairing the viral transcription/replication process. Herein, we show that this early restriction was only partial against human IAVs, whereas a strong inhibition of viral production was observed. Indeed, relatively high levels of IAV mRNAs and proteins were observed in the presence of human (Hs) and mouse (Mm) MX1 proteins but additional inhibition processes occurred at later stages of IAV life cycle. Hence, MmMx1 induced an abnormal nuclear accumulation of the viral nucleoprotein (NP) at late time points post-infection. This block was also observed, albeit to a much lower extent, with HsMX1. In most HsMX1-expressing cells, vRNPs could be exported from the nucleus to the cytoplasm however a potent defect in subsequent vRNP cytoplasmic trafficking was observed. Indeed, vRNPs were found sequestrated together with cellular co-factors YBX1 and Rab11a in large clusters in the vicinity of the microtubule organization center (MTOC). Live imaging experiments revealed that the transient association of HsMX1 with Rab11a-associated vRNPs favoured their dynein-dependant retrograde transport along microtubules towards the MTOC. Importantly, dynein inhibition prevented the vRNP sequestration and significantly rescued infectious viral production in the presence of HsMX1, showing a significant contribution of these abnormal vRNP clusters in HsMX1 antiviral activity. In conclusion, this study provides the first evidence of IAV vRNPs being re-routed and accumulated away from the plasma membrane, through the coordinated action of HsMX1 restriction factor, dynein and the microtubule network.

microbiology↗

bioRxiv2022

The N-terminal domain of MX1 proteins is essential for their antiviral activity against different families of RNA viruses

Myxovirus resistance protein 1 (MX1) and MX2, are homologous, dynamin-like large GTPases, induced upon interferon (IFN) exposure. Human MX1 (HsMX1) is known to inhibit many viruses, including influenza A virus (IAV), by likely acting at various steps of their life cycles. Despite decades of studies, the mechanism(s) of action with which MX1 proteins manage to inhibit target viruses is not fully understood. MX1 proteins are mechano-enzymes and share a similar organization to dynamin, with an amino-terminal GTPase domain and a carboxy-terminal stalk domain, connected by a Bundle Signalling Element (BSE). These three elements are known to be essential for antiviral activity. HsMX1 has two unstructured regions, the L4 loop, also essential for antiviral activity, and a short amino (N)-terminal region, which greatly varies between MX1 proteins of different species. The role of this N-terminal domain in antiviral activity is not known. Herein, using mutagenesis, imaging and biochemical approaches, we demonstrate that the N-terminal domain of HsMX1 is essential for antiviral activity against IAV and Vesicular Stomatitis Virus (VSV), and for the ability to aggregate Orthobunyavirus nucleoproteins. Furthermore, we pinpoint a highly conserved leucine within this region, which is absolutely crucial for human, mouse and bat MX1 protein antiviral activity. Importantly, mutation of this leucine does not compromise GTPase activity or oligomerization capabilities, but does modify MX1 protein subcellular localisation. The discovery of this essential and highly conserved residue defines this region as key and may reveal insights as to the mechanism of action of MX1 proteins.

microbiology↗

bioRxiv2022

Extensive uORF translation from HIV-1 transcripts conditions DDX3 dependency for expression of main ORFs and elicits specific T cell immune responses in infected individuals

Human immunodeficiency virus type-1 (HIV-1) is a complex retrovirus which relies on alternative splicing, translational and post-translational mechanisms to produce more than 15 functional proteins from its single [~]10kb transcriptional unit. Here, we have applied ribosome profiling and nascent protein labeling at different time points during infection of CD4+ T lymphocytes to characterize the translational landscape of cellular and viral transcripts during the course of infection. Our results indicate a strong impact of viral infection on host cellular transcript levels but a modest impact on global translation rates. Analysis of ribosome profiling reads from viral transcripts reveals extensive and productive non-AUG translation of small peptides from multiple upstream open reading-frames (uORFs) located in the 5 long terminal repeat. Remarkably, these uORFs derived peptides elicit specific T cell responses in HIV-infected individuals. uORFs are conserved among other retroviruses and, together with the TAR sequence, condition the dependency on DDX3 for efficient translation of the main viral open-reading frames.

microbiology↗

bioRxiv2021

Pharmacological perturbation of intracellular dynamics as a SARS-CoV-2 antiviral strategy

SARS-CoV-2 (CoV2) is the viral agent responsible for the pandemic of the coronavirus disease 2019 (COVID-19). Vaccines are being deployed all over the world with good efficacy, but there is no approved antiviral treatment to date. This is particularly needed since the emergence of variants and the potential immune escape may prolong pandemic spreading of the infection for much longer than anticipated. Here, we developed a series of small molecules and identified RG10 as a potent antiviral compound against SARS-CoV-2 in cell lines and human airway epithelia (HAE). RG10 localizes to endoplasmic reticulum (ER) membranes, perturbing ER morphology and inducing ER stress. Yet, RG10 does not associate with SARS-CoV-2 replication sites although preventing virus replication. To further investigate the antiviral properties of our compound, we developed fluorescent SARS-CoV-2 viral particles allowing us to track virus arrival to ER membranes. Live cell imaging of replication-competent virus infection revealed that RG10 stalls the intracellular virus-ER dynamics. Finally, we synthesized RG10b, a stable version of RG10, that showed increased potency in vitro and in HAE with a pharmacokinetic half-life greater than 2 h. Together, our work reports on a novel fluorescent virus model and innovative antiviral strategy consisting of the perturbation of ER/virus dynamics, highlighting the promising antiviral properties of RG10 and RG10b.

microbiology↗

bioRxiv2021

TMPRSS2 promotes SARS-CoV-2 evasion from NCOA7-mediated restriction

Interferons play a critical role in regulating host immune responses to SARS-CoV-2, but the interferon (IFN)-stimulated gene (ISG) effectors that inhibit SARS-CoV-2 are not well characterized. The IFN-inducible short isoform of human nuclear receptor coactivator 7 (NCOA7) inhibits endocytic virus entry, interacts with the vacuolar ATPase, and promotes endo-lysosomal vesicle acidification and lysosomal protease activity. Here, we used ectopic expression and gene knockout to demonstrate that NCOA7 inhibits infection by SARS-CoV-2 as well as by lentivirus particles pseudotyped with SARS-CoV-2 Spike in lung epithelial cells. Infection with the highly pathogenic, SARS-CoV-1 and MERS-CoV, or seasonal, HCoV-229E and HCoV-NL63, coronavirus Spike-pseudotyped viruses was also inhibited by NCOA7. Importantly, either overexpression of TMPRSS2, which promotes plasma membrane fusion versus endosomal fusion of SARS-CoV-2, or removal of Spikes polybasic furin cleavage site rendered SARS-CoV-2 less sensitive to NCOA7 restriction. Collectively, our data indicate that furin cleavage sensitizes SARS-CoV-2 Spike to the antiviral consequences of endosomal acidification by NCOA7, and suggest that the acquisition of furin cleavage may have favoured the co-option of cell surface TMPRSS proteases as a strategy to evade the suppressive effects of IFN-induced endo-lysosomal dysregulation on virus infection.

microbiology↗

bioRxiv2021

Bidirectional genome-wide CRISPR screens reveal host factors regulating SARS-CoV-2, MERS-CoV and seasonal coronaviruses

Several genome-wide CRISPR knockout screens have been conducted to identify host factors regulating SARS-CoV-2 replication, but the models used have often relied on overexpression of ACE2 receptor. Additionally, such screens have yet to identify the protease TMPRSS2, known to be important for viral entry at the plasma membrane. Here, we conducted a meta-analysis of these screens and showed a high level of cell-type specificity of the identified hits, arguing for the necessity of additional models to uncover the full landscape of SARS-CoV-2 host factors. We performed genome-wide knockout and activation CRISPR screens in Calu-3 lung epithelial cells, as well as knockout screens in Caco-2 intestinal cells. In addition to identifying ACE2 and TMPRSS2 as top hits, our study reveals a series of so far unidentified and critical host-dependency factors, including the Adaptins AP1G1 and AP1B1 and the flippase ATP8B1. Moreover, new anti-SARS-CoV-2 proteins with potent activity, including several membrane-associated Mucins, IL6R, and CD44 were identified. We further observed that these genes mostly acted at the critical step of viral entry, with the notable exception of ATP8B1, the knockout of which prevented late stages of viral replication. Exploring the pro- and anti-viral breadth of these genes using highly pathogenic MERS-CoV, seasonal HCoV-NL63 and -229E and influenza A orthomyxovirus, we reveal that some genes such as AP1G1 and ATP8B1 are general coronavirus cofactors. In contrast, Mucins recapitulated their known role as a general antiviral defense mechanism. These results demonstrate the value of considering multiple cell models and perturbational modalities for understanding SARS-CoV-2 replication and provide a list of potential new targets for therapeutic interventions.

microbiology↗

bioRxiv2020

SARS-CoV-2 replication triggers an MDA-5-dependent interferon production which is unable to efficiently control replication

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third highly pathogenic coronavirus to spill over to humans in less than 20 years, after SARS-CoV-1 in 2002-2003 and Middle East respiratory syndrome (MERS)-CoV in 2012. SARS-CoV-2 is the etiologic agent of coronavirus disease 19 (COVID-19), which ranges from mild respiratory symptoms to severe lung injury and death in the most severe cases. The COVID-19 pandemic is currently a major health issue worldwide. Immune dysregulation characterized by altered innate cytokine responses is thought to contribute to the pathology of COVID-19 patients, which is a testimony of the fundamental role of the innate immune response against SARS-CoV-2. Here, we further characterized the host cell antiviral response against SARS-CoV-2 by using primary human airway epithelia and immortalized model cell lines. We mainly focused on the type I and III interferon (IFN) responses, which lead to the establishment of an antiviral state through the expression of IFN-stimulated genes (ISGs). Our results demonstrate that both primary airway epithelial cells and model cell lines elicit a robust immune response characterized by a strong induction of type I and III IFN through the detection of viral pathogen molecular patterns (PAMPs) by melanoma differentiation associated gene (MDA)-5. However, despite the high levels of type I and III IFNs produced in response to SARS-CoV-2 infection, the IFN response was unable to control viral replication, whereas IFN pre-treatment strongly inhibited viral replication and de novo production of infectious virions. Taken together, these results highlight the complex and ambiguous interplay between viral replication and the timing of IFN responses.

microbiology↗

bioRxiv2020

A genome-wide CRISPR/Cas9 knock-out screen identifies the DEAD box RNA helicase DDX42 as a broad antiviral inhibitor

Genome-wide screens are powerful approaches to unravel new regulators of viral infections. Here, we used a CRISPR/Cas9 screen to reveal new HIV-1 inhibitors. This approach led us to identify the RNA helicase DDX42 as an intrinsic antiviral inhibitor. DDX42 was previously described as a non-processive helicase, able to bind RNA secondary structures such as G-quadruplexes, with no clearly defined function ascribed. Our data show that depletion of endogenous DDX42 significantly increased HIV-1 DNA accumulation and infection in cell lines and primary cells. DDX42 overexpression inhibited HIV-1, whereas a dominant-negative mutant increased infection. Importantly, DDX42 also restricted retrotransposition of LINE-1, infection with other retroviruses and positive-strand RNA viruses, including CHIKV and SARS-CoV-2. However, DDX42 did not inhibit infection with three negative-strand RNA viruses, arguing against a general, unspecific effect on target cells, which was confirmed by RNA-seq analysis. DDX42 was found in the vicinity of viral elements by proximity ligation assays, and cross-linking RNA immunoprecipitation confirmed a specific interaction of DDX42 with RNAs from sensitive viruses. This strongly suggested a direct mode of action of DDX42 on viral ribonucleoprotein complexes. Taken together, our results show for the first time a new and important role of DDX42 in intrinsic antiviral immunity.

microbiology↗