Biology subjects

Demeret, C.

Publications and source records attributed to Demeret, C..

11 recordsLinked to original sources

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

The cellular Activating Protein 1 cFos regulates Influenza A virus replication

Previous research has demonstrated that Influenza A virus (IAV) infection activates AP-1 transcription factors as part of the antiviral response. In this study, we identified cFos as the most upregulated AP-1 transcription factor during IAV infection in A549 human lung cells. Surprisingly, the knockdown of cFos resulted in impaired IAV replication. Fluorescence microscopy and functional analyses indicated that cFos is implicated in IAV infection through its nuclear function, rather than its cytoplasmic role as an activator of lipid synthesis. The investigation into the role of cFos in IAV infection revealed increased apoptosis and elevated interferon-{beta} mRNA levels in cFos-knockdown A549 cells during IAV infection. This suggests that cFos may enhance cell survival and reduce interferon-{beta} expression during infection, thereby facilitating IAV proliferation. Furthermore, the levels of viral NA mRNA and the expression of late viral proteins NA and M2 decreased upon cFos knockdown. Overall, this study identifies cFos as a proviral factor for IAV, through the modulation of innate immunity and apoptosis during infection, and potentially by supporting the viral transcription. Author SummaryInfluenza A viruses pose a significant threat to global public health. Understanding the interaction between the virus and host is essential for the development of antiviral therapies. We identified cFos, a cellular Activating Protein-1, as a pro-viral factor for influenza A viruses. cFos was found to enhance cell survival and regulate the expression of certain cytokines during viral infection. Our findings also suggested a potential role for cFos in the viral transcription, especially of the viral proteins expressed late in the viral cycle.

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↗

bioRxiv2023

Role of two modules controlling the interaction between SKAP1 and SRC kinasesComparison with SKAP2 architecture and consequences for evolution

AbstractSRC kinase associated phosphoprotein 1 (SKAP1), an adaptor for protein assembly, plays an important role in the immune system such as stabilizing immune synapses. Understanding how these functions are controlled at the level of the protein-protein interactions is necessary to describe these processes and to develop therapeutics. Here, we dissected the SKAP1 modular organization to recognize SRC kinases and compared it to that of its paralog SRC kinase associated phosphoprotein 2 (SKAP2). Different conserved motifs common to either both proteins or specific to SKAP2 were found using this comparison. Two modules harboring different binding properties between SKAP1 and SKAP2 were identified: one composed of two conserved motifs located in the second interdomain interacting at least with the SH2 domain of SRC kinases and a second one composed of the DIM domain modulated by the SH3 domain and the activation of SRC kinases. This work suggests a convergent evolution of the binding properties of some SRC kinases interacting specifically with either SKAP1 or SKAP2.

molecular biology↗

bioRxiv2023

Identification and characterization of interactions between Influenza A Virus NS1 protein and the human ubiquitin proteasome system

As a key player involved in various cellular pathways, including innate immune response activation, the human ubiquitin-proteasome system (UPS) is particularly targeted by viral proteins upon infection. Indeed, most viruses have evolved to counteract and hijack this system, as it is the case for the influenza A virus (IAV). The non-structural protein 1 (NS1) is described as the main IAV virulence factor, which is known to interact with several cellular proteins, including some UPS factors that are important for the viral escape of the immune cell response. In this study, we profiled the overall interplay between the NS1 proteins of multiple IAV strains and the human UPS. We identified 98 UPS factors directly interacting with NS1 of all or a subset of the studied strains, and we functionally studied 18 of them. We highlighted the involvement of these UPS factors in the IAV life cycle by performing viral titrations, minigenome replicon assays and an ISRE-luc (IFN pathway) assays. Furthermore, we analyzed the expression and subcellular localizations of FZR1, MKRN3, RC3H2 and SHKBP1 upon IAV infection. This interactomics approach allows for an improved understanding of the interplay between NS1 and UPS pathway in the context of an IAV-mediated inhibition of cellular anti-viral responses. ImportanceInfluenza A viruses (IAV) are pathogens responsible for annual flu epidemics causing up to 650,000 deaths each year, resulting in a significant impact in public health and global economy. IAV are also responsible of occasionally pandemic outbreaks in human population, such as in 1918 that caused the death of 50-100 million people. Non-structural protein 1 (NS1) is the main IAV virulence factor; it acts by direct interactions with several cellular proteins, leading to the host shut-off and to the inhibition of the host cell innate immune response. Since the ubiquitin-proteasome system (UPS) plays a crucial role in the innate immune response activation, it is a designated target for NS1 upon infection. Our research thus focused on the identification of interactions between NS1 of 6 different IAV strains and the UPS, to better understand the interplay between this viral protein and the UPS upon viral infection.

molecular biology↗

bioRxiv2021

A screening pipeline identifies a broad-spectrum inhibitor of bacterial AB toxins with cross protection against influenza A virus H1N1 and SARS-CoV-2

A challenge for the development of host-targeted anti-infectives against a large spectrum of AB-like toxin-producing bacteria encompasses the identification of chemical compounds corrupting toxin transport through both endolysosomal and retrograde pathways. Here, we performed a high-throughput screening of small chemical compounds blocking active Rac1 proteasomal degradation triggered by the Cytotoxic Necrotizing Factor-1 (CNF1) toxin, followed by orthogonal screens against two AB toxins hijacking defined endolysosomal (Diphtheria toxin) or retrograde (Shiga-like toxin 1) pathways to intoxicate cells. This led to the identification of the molecule N-(3,3-diphenylpropyl)-1-propyl-4-piperidinamine, referred to as C910. This compound induces the swelling of EEA1-positive early endosomes, in absence of PIKfyve kinase inhibition, and disturbs the trafficking of CNF1 and the B-subunit of Shiga toxin along the endolysosomal or retrograde pathways, respectively. Together, we show that C910 protects cells against 8 bacterial AB toxins including large clostridial glucosylating toxins from Clostridium difficile. Of interest, C910 also reduced viral infection in vitro including influenza A virus subtype H1N1 and SARS-CoV-2. Moreover, parenteral administration of C910 to the mice resulted in its accumulation in lung tissues and reduced lethal influenza infection.

microbiology↗

bioRxiv2021

A map of direct SARS-CoV-2 protein interactions implicates specific human host processes

Key steps in viral propagation, immune suppression, and pathology are mediated by direct, binary, physical interactions between viral and host proteins. To understand the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated an unbiased systematic map of binary interactions between viral and host proteins, complementing previous co-complex association maps by conveying more direct mechanistic understanding and potentially enabling targeted disruption of direct interactions. To this end, we deployed two parallel strategies, identifying 205 virus-host and 27 intraviral binary interactions amongst 171 host and 19 viral proteins, and confirming high quality of these interactions via a calibrated orthogonal assay. Host proteins interacting with SARS-CoV-2 proteins are enriched in various cellular processes, including immune signaling and inflammation, protein ubiquitination, and membrane trafficking. Specific subnetworks provide new hypotheses related to viral modulation of host protein homeostasis and T-cell regulation. The binary virus-host protein interactions we identified can now be prioritized as targets for therapeutic intervention. More generally, we provide a resource of systematic maps describing which SARS-CoV-2 and human proteins interact directly.

systems biology↗

bioRxiv2020

A SARS-CoV-2 - host proximity interactome

Viral replication is dependent on interactions between viral polypeptides and host proteins. Identifying virus-host protein interactions can thus uncover unique opportunities for interfering with the virus life cycle via novel drug compounds or drug repurposing. Importantly, many viral-host protein interactions take place at intracellular membranes and poorly soluble organelles, which are difficult to profile using classical biochemical purification approaches. Applying proximity-dependent biotinylation (BioID) with the fast-acting miniTurbo enzyme to 27 SARS-CoV-2 proteins in a lung adenocarcinoma cell line (A549), we detected 7810 proximity interactions (7382 of which are new for SARS-CoV-2) with 2242 host proteins (results available at covid19interactome.org). These results complement and dramatically expand upon recent affinity purification-based studies identifying stable host-virus protein complexes, and offer an unparalleled view of membrane-associated processes critical for viral production. Host cell organellar markers were also subjected to BioID in parallel, allowing us to propose modes of action for several viral proteins in the context of host proteome remodelling. In summary, our dataset identifies numerous high confidence proximity partners for SARS-CoV-2 viral proteins, and describes potential mechanisms for their effects on specific host cell functions.

systems biology↗

bioRxiv2020

Global BioID-based SARS-CoV-2 proteins proximal interactome unveils novel ties between viral polypeptides and host factors involved in multiple COVID19-associated mechanisms

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

systems biology↗

bioRxiv2019

OTUB1 is a key regulator of RIG-I dependent immune signalling and is targeted for proteasomal degradation by influenza A NS1

Deubiquitylases (DUBs) regulate critical signaling pathways at the intersection of host innate immunity and viral pathogenesis. Although RIG-I activation is heavily dependent on ubiquitylation, DUBs that regulate this pathway have not been identified. Using a ubiquitin C-terminal electrophile, we profiled DUBs that function during influenza A virus (IAV) infection, and isolated OTUB1 as a key regulator of RIG-I dependent antiviral responses. OTUB1 was interferon-inducible, and interacted with RIG-I, viral PB2 and NS1. Upon infection, OTUB1 relocalised from the nucleus to mitochondrial membranes, and activated the RIG-I signaling complex via hydrolysis of K48 polyubiquitin chains and by forming a repressive complex with UBCH5c. Using a reconstituted system composed of in vitro translated [35S]IRF3, purified RIG-I, mitochondrial membranes and cytosol expressing OTUB1 variants, we recapitulated the mechanism of OTUB1-dependent RIG-I activation. A wide range of IAV NS1 proteins triggered proteasomal degradation of OTUB1, thereby antagonizing the RIG-I signaling cascade and antiviral responses.\n\nHighlightsO_LIOTUB1 is induced during influenza A virus infections in an IFN-I dependent manner\nC_LIO_LIOTUB1 regulates the RIG-I complex by hydrolysing K48-linked polyubiquitin chains and by sequestering UBCH5c to prevent K48 polyubiquitylation\nC_LIO_LIOptimal K63 versus K48 polyubiquitin chain concentrations determine RIG-I activation\nC_LIO_LIInfluenza NS1 targets OTUB1 for proteasomal degradation\nC_LI

biochemistry↗

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↗