Biology subjects

Sorgeloos, F.

Publications and source records attributed to Sorgeloos, F..

10 recordsLinked to original sources

bioRxiv2025

A simple workflow to identify novel Small Linear Motif (SLiM)-mediated interactions with AlphaFold

Short linear motifs (SLiMs) are highly compact interaction modules embedded within disordered protein regions and are increasingly recognized for their central role in maintaining cellular homeostasis. Due to their small size, degeneracy and transient binding, SLiMs remain difficult to detect both experimentally and computationally. Here, we show that AlphaFold, used via ColabFold, offers a practical and accessible alternative for in-silico SLiM discovery. Unlike previous studies focused on structural accuracy, we evaluated AlphaFolds capacity to reveal SLiMs independently of model quality. To this end, we benchmarked several scoring metrics and showed that AlphaFold2 combined with MiniPAE yields the best performance, outperforming AlphaFold3 in this context. Building on these findings, we also provide a streamlined and cost-effective workflow for SLiM prediction requiring no installation or local computation. To overcome challenges associated with SLiM validation, we also introduce a highly sensitive detection method based on proximity labeling in living cells. This workflow was used to predict the occurrence of SLiMs that mediate binding to ribosomal protein S6 kinase A3 (RPS6KA3 or RSK2). By leveraging Colabfold and MiniPAE available through Colab notebooks, our approach provides a scalable and widely accessible strategy for identifying functional SLiMs in proteins of interest. MiniPAE can be accessed at https://github.com/martinovein/MiniPAE Short descriptionMartin Veinstein is a PhD student in Biomedical Sciences at the de Duve Institute, UCLouvain, Belgium. He specializes in Small Linear Motifs (SLiMs) in the context of host-virus interactions and has developed strong expertise in bioinformatics, structural biology, and predictive modeling. Victor J is a unfergradiate student at the ECAM Brussels Engineering School, Haute Ecole "ICHEC-ECAM-ISFSC", Brussels, Belgium. His activities span form September to November 2023. B.I. Iorga is a CNRS Research Director at the Institut de Chimie des Substances Naturelles in Gif-sur-Yvette, France. His research focuses among others on methodological developments in molecular modeling and the in-silico prediction of antibiotic resistance using machine learning and deep learning approaches. Raphael Helaers is a Senior Investigator and leads bioinformatics infrastructure at the de Duve Institute, UCLouvain, Belgium. He has developed strong expertise in next-generation sequencing and software development, along with a deep interest in biology, genetics, and evolution. Thomas Michiels is a Full Professor and researcher at the de Duve Institute, UCLouvain, Belgium. His research focuses on virus-mediated subversion of the innate immune response. Frederic Sorgeloos is an adjunct Professor at the INRS, Laval, Canada. He currently focuses on the subversion of cellular homeostasis through small linear peptides encoded by viral and bacterial pathogens. Short abstractVarious AlphaFold2/3 scoring metrics were systematically benchmarked for their ability to detect Small Linear Motifs (SLiMs) Based on this evaluation, a user-friendly and cost-effective in-silico workflow is proposed to identify novel SLiMs-targeting proteins The utility of this workflow is demonstrated through the prediction of previously uncharacterized SLiMs interacting with RSK kinases. A sensitive in-vitro assay is proposed to streamline the validation of low-affinity SLiM-target interactions. Together, our workflow and associated validation assay offer an integrated pipeline for the discovery and validation of SLiM-mediated protein-protein interactions.

bioinformatics↗

bioRxiv2025

The leader proteins of Theiler's virus and Boone cardiovirus use a combination of Short Linear Motifs (SLiMs) to target RSK kinases to the nuclear pore complex.

Unrelated pathogens, including viruses and bacteria, use a common DDVF-like short linear motif (SLiM) to interact with cellular kinases of the RSK (p90 S6 ribosomal kinase) family. Such a "DDVF" SLiM occurs in the leader (L) protein encoded by picornaviruses of the genus Cardiovirus, including Theilers murine encephalomyelitis virus (TMEV), Boone cardiovirus (BCV), and Encephalomyocarditis virus (EMCV). The L-RSK complex is targeted to the nuclear pore, where RSK triggers FG-nucleoporins hyperphosphorylation, thereby causing nucleocytoplasmic trafficking disruption. In this work, we identified a second SLiM in the L proteins of TMEV and BCV, which enables the L-RSK complex to interact with RAE1 at the level of the nuclear pore complex. AlphaFold predictions suggest that the RAE1-interacting SLiM of L proteins is analogous to that found in unrelated viral proteins such as ORF6 of SARS-CoV-1/2, ORF10 of Kaposi sarcoma-associated herpes virus (KSHV), and the matrix (M) protein of vesicular stomatitis virus (VSV). Co-immunoprecipitations confirmed the interaction between BCV L and RAE1 and competition experiments revealed that L can compete with ORF6 for RAE1 binding, suggesting that BCV and TMEV L proteins interact with RAE1 via the same docking site as M, ORF6, or ORF10. This RAE1 binding SLiM tentatively named "M-acidic", is predicted to occur in other viral proteins such as Rift valley fever virus NSs as well as in cell proteins such as NXF1. BCV and TMEV L proteins use a combination of two independent SLiMs to hijack cellular kinases and retarget those kinases toward the nuclear pore complex. ImportanceProtein-protein interactions are critical to regulate cell physiology. Short linear motifs (SLiMs) are unstructured protein sequences, which usually bind to structured domains of partner proteins. They typically mediate low affinity, transient interactions, which are particularly suitable for fine tuning cell physiology or helping cells to react promptly to stress situations. Owing to their fast replication and to the high error rate of their polymerases, viruses, particularly RNA viruses are prone to acquire SLiMs that mimic cellular SLiMs and thereby interfere with host cell signaling. In this work, we show that the leader ("L") protein expressed by some cardioviruses (Picornaviridae family) uses two SLiMs in combination, which are individually shared by other pathogens: the first one, described previously, enables the L protein to hijack cellular kinases named RSKs, and the second one described in this work enables the L-RSK complex to target proteins RAE1 and NUP98 in the nuclear pore complex.

microbiology↗

bioRxiv2024

The "DDVF" motif used by viral and bacterial proteins to hijack RSK kinases evolved as a mimic of a short linear motif (SLiM) found in proteins related to the RAS-ERK MAP kinase pathway.

Proteins of pathogens such as cardioviruses, kaposi sarcoma-associated herpes virus, varicella zoster virus and bacteria of the genus Yersinia were previously shown to use a common "DDVF" (D/E-D/E-V-F) short linear motif (SLiM) to hijack cellular kinases of the RSK (p90 ribosomal S6 kinases) family. Remarkable conservation of the SLiM docking site in RSKs suggested a physiological role for this site. Using SLiM prediction tools and AlphaFold docking, we screened the human proteome for proteins that would interact with RSKs through a DDVF-like SLiM. Using co-immunoprecipitation experiments, we show that two candidates previously known as RSK partners, FGFR1 and SPRED2, as well as two candidates identified as novel RSK partners, GAB3 and CNKSR2 do interact with RSKs through a similar interface as the one used by pathogens, as was recently documented for SPRED2. Moreover, we show that FGFR1 employs a DSVF motif to bind RSKs and that phosphorylation of the serine in this motif increases RSK binding. FGFR1, SPRED2, GAB3 and CNKSR2 as well as other candidate RSK binders act upstream of RSK in the RAS-ERK MAP kinase pathway. Analysis of ERK activation in cells expressing a mutated form of RSK lacking the DDVF-docking site suggests that RSK might interact with the DDVF-like SLiM of several partners to provide a negative feed-back to the ERK MAPK pathway. Thus, through SLiM mimicry, pathogens not only retarget RSKs toward unconventional substrates but also likely compete with human proteins to alter the regulation of the RAS-ERK MAP kinase pathway. Author SummaryShort linear motif (SLiM) are 3 to 10 amino acid-long protein sequences that can mediate the interaction with other proteins. We previously observed that highly unrelated pathogens, including viruses and bacteria, convergently evolved to hijack cellular enzymes of their host, through a common SLiM. In this work, we tested the hypothesis that the SLiM found in proteins of pathogens evolved to mimic a SLiM found in human proteins that regulate the cellular enzymes through the same interface. Protein-protein interactions mediated by SLiMs are often, low-affinity, transient interactions that are difficult to detect by conventional biochemical methods but that can nowadays be predicted with increasing confidence by artificial intelligence-based methods such as AlphaFold. Using such predictions, we identified several candidate human proteins and we confirmed experimentally that these proteins interact with the cellular enzymes the same way as pathogens proteins do. Identified proteins belong to the well-known RAS-ERK MAPK pathway which regulates important functions of the cell, suggesting that pathogens evolved to hijack this MAPK pathway by SLiM mimicry. By doing so, they can both dysregulate cellular physiology and hijack cellular enzymes to their own benefit.

microbiology↗

bioRxiv2023

Porcine sapovirus protease controls the innate immune response and targets TBK1

Human sapoviruses (HuSaVs) and noroviruses are considered the leading cause of acute gastroenteritis worldwide. While extensive research has focused on noroviruses, our understanding of sapoviruses (SaVs) and their interactions with the hosts immune response remains limited. HuSaVs have been challenging to propagate in vitro, making the porcine sapovirus (PSaV) Cowden strain a valuable model for studying SaV pathogenesis. In this study we show, for the first time, that PSaV Cowden strain has mechanisms to evade the hosts innate immune response. The virus 3C-like protease (NS6) inhibits type I IFN production by targeting TBK1. Catalytically active NS6, both during ectopic expression and during PSaV infection, targets TBK1 which is then led for rapid degradation by the proteasome. Moreover, deletion of TBK1 from porcine cells led to a significant increase in PSaV titres, emphasizing its role in regulating PSaV infection. Additionally, we successfully established PSaV infection in IPEC-J2 cells, an enterocytic cell line originating from the jejunum of a neonatal piglet. Overall, this study provides novel insights into PSaV evasion strategies, opening the way for future investigations for SaV-host interactions, and enabling the use of a new cell line model for PSaV research.

microbiology↗

bioRxiv2022

Cardiovirus leader proteins retarget RSK kinases toward alternative substrates to perturb nucleocytoplasmic traffic

Proteins from some unrelated pathogens, including viruses and bacteria can recruit and activate cellular p90-ribosomal protein S6 kinases (RSKs) through a common linear motif. Our data suggested that such pathogens proteins might act as adapters to dock the kinase toward specific substrates. We explored this hypothesis using the Cardiovirus leader protein (L) as a paradigm. L is known to trigger phenylalanine-glycine nucleoporins (FG-NUPs) hyperphosphorylation and nucleocytoplasmic trafficking perturbation. Using a biotin ligase fused to either RSK or to L, we identified FG-NUPs as primary partners of the L-RSK complex in infected cells. Mutant analysis shows that L uses distinct motifs to recruit RSK and to dock the L-RSK complex toward the FG-NUPs. Using an analog-sensitive RSK2 mutant kinase, we show that, in infected cells, L can trigger RSK to use NUP98 and NUP214 as direct substrates. Our data illustrate a novel virulence mechanism where pathogens proteins retarget cellular protein kinases toward specific substrates.

microbiology↗

bioRxiv2021

Leaked genomic and mitochondrial DNA contribute to the host response to noroviruses in a STING-dependent manner

The cGAS-STING pathway is central to the IFN response against DNA viruses. However, recent studies are increasingly demonstrating its role in the restriction of some RNA viruses. Here we show that the cGAS-STING pathway also contributes to the IFN response against noroviruses, positive-sense single-stranded RNA viruses that are now one of the most common causes of infectious gastroenteritis world-wide. We show a significant reduction in IFN-{beta} induction and a corresponding increase in viral replication in norovirus-infected cells following STING inhibition, knockdown or deletion. Upstream of STING, we show that cells lacking either cGAS or IFI16 also have severely impaired IFN responses. Further, we demonstrate that immunostimulatory host genome-derived DNA, and to a lesser extent mitochondrial DNA, accumulate in the cytosol of norovirus-infected cells. And lastly, overexpression of the viral NS4 protein was sufficient to drive the accumulation of cytosolic DNA. Together, our data elucidate a role for cGAS, IFI16 and STING in the restriction of noroviruses, and demonstrate for the first time the utility of host genomic DNA as a damage-associated molecular pattern in cells infected with an RNA virus. HighlightsO_LIcGAS, IFI16 and STING are required for a robust IFN response against noroviruses C_LIO_LINuclear and mitochondrial DNA accumulate in the cytosols of infected cells C_LIO_LIViral NS4 mediates accumulation of cytosolic DNA C_LI

molecular biology↗

bioRxiv2021

Murine Norovirus infection results in anti-inflammatory response downstream of amino acids depletion in macrophages

Murine norovirus (MNV) infection results in a late translation shut-off, that is proposed to contribute to the attenuated and delayed innate immune response observed both in vitro and in vivo. Recently, we further demonstrated the activation of the eIF2 kinase GCN2 during MNV infection, which has been previously linked to immunomodulation and resistance to inflammatory signalling during metabolic stress. While viral infection is usually associated with activation of dsRNA binding pattern recognition receptor PKR, we hypothesised that the establishment of a metabolic stress in infected cells is a proviral event, exploited by MNV to promote replication through weakening the activation of the innate immune response. In this study, we used multi-omics approaches to characterise cellular responses during MNV replication. We demonstrate the activation of pathways related to the integrated stress response, a known driver of anti-inflammatory phenotypes in macrophages. In particular, MNV infection causes an amino acid imbalance that is associated with GCN2 and ATF2 signalling. Importantly, this reprogramming lacks the features of a typical innate immune response, with the ATF/CHOP target GDF15 contributing to the lack of antiviral responses. We propose that MNV-induced metabolic stress supports the establishment of host tolerance to viral replication and propagation. ImportanceDuring viral infection, host defences are typically characterised by the secretion of pro-inflammatory autocrine and paracrine cytokines, potentiation of the IFN response and induction of the anti-viral response via activation of JAK and Stat signalling. To avoid these and propagate viruses have evolved strategies to evade or counteract host sensing. In this study, we demonstrate that murine norovirus controls the antiviral response by activating a metabolic stress response that activates the amino acid response and impairs inflammatory signalling. This highlights novel tools in the viral countermeasures tool-kit, and demonstrates the importance of the currently poorly understood metabolic reprogramming occurring during viral infections.

microbiology↗

bioRxiv2019

Norovirus replication in human intestinal epithelial cells is restricted by the interferon-induced JAK/STAT signalling pathway and RNA Polymerase II mediated transcriptional responses.

Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis worldwide and a significant cause of morbidity and mortality in all age groups. The recent finding that HuNoV can be propagated in B cells and mucosa derived intestinal epithelial organoids (IEOs), has transformed our capability to dissect the life cycle of noroviruses. Using RNA-Seq of HuNoV infected intestinal epithelial cells (IECs), we have found that replication of HuNoV in IECs results in interferon-induced transcriptional responses and that HuNoV replication in IECs is sensitive to IFN. This contrasts with previous studies that suggest that the innate immune response may play no role in the restriction of HuNoV replication in immortalised cells. We demonstrate that the inhibition of JAK1/JAK2 enhances HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular RNA polymerase II-mediated transcription was not detrimental to HuNoV replication, but enhanced replication to a greater degree compared to blocking of JAK signalling directly. Furthermore, we demonstrate for the first time that IECs generated from genetically modified intestinal organoids, engineered to be deficient in the interferon response, are more permissive to HuNoV infection. Together our work identifies the IFN-induced transcriptional responses restrict HuNoV replication in IECs and demonstrates that the inhibition of these responses by modifications to the culture conditions can greatly enhance the robustness of the norovirus culture system.\n\nImportanceNoroviruses are a major cause of gastroenteritis worldwide yet the challenges associated with their growth culture has greatly hampered the development of therapeutic approaches and has limited our understanding of cellular pathways that control infection. Here we show that human intestinal epithelial cells, the first point of entry of human noroviruses into the host, limit virus replication by the induction of the innate responses. Furthermore we show that modulating the ability of intestinal epithelial cells to induce transcriptional responses to HuNoV infection can significantly enhance human norovirus replication in culture. Collectively our findings provide new insights into the biological pathways that control norovirus infection but also identify mechanisms to enhance the robustness of norovirus culture.

microbiology↗

bioRxiv2019

Murine norovirus virulence factor 1 (VF1) protein contributes to viral fitness during persistent infection

BackgroundMurine norovirus (MNV) is widely used as a model for studying norovirus biology. While MNV isolates vary in their pathogenesis, infection of immunocompetent mice mostly results in persistent infection. The ability of a virus to establish a persistent infection is dependent on its ability to subvert or avoid the host immune response. Previously, we described the identification and characterization of virulence factor 1 (VF1) in MNV, and established that it acts as an innate immune antagonist. Here, we explore the role of VF1 during persistent MNV infection in an immunocompetent host.\n\nMethodsUsing reverse genetics, we generated VF1-knockout MNV-3 that contained a single or a triple termination codon inserted in the VF1 open reading frame.\n\nResultsVF1-knockout MNV-3 replicated to comparable levels to the wildtype virus in tissue culture. Comparative studies between MNV-3 and an acute MNV-1 strain show that MNV-3 VF1 exerts the same functions as MNV-1 VF1, but with reduced potency. Mice infected with VF1-knockout MNV-3 showed significantly reduced replication kinetics during the acute phase of the infection, but viral loads rapidly reached the levels seen in mice infected with wildtype virus after phenotypic restoration of VF1 expression. Infection with an MNV-3 mutant that had three termination codons inserted into VF1, in which reversion was suppressed, resulted in consistently lower replication throughout a three-month persistent infection in mice, suggesting a role for VF1 in viral fitness in vivo.\n\nConclusionOur results indicate that VF1 expressed by a persistent strain of MNV also functions to antagonise the innate response to infection. We found that VF1 is not essential for viral persistence, but instead contributes to viral fitness in mice. These data fit with the hypothesis that noroviruses utilise multiple mechanisms to avoid and/or control the host response to infection and that VF1 is just one component of this.

microbiology↗

bioRxiv2019

Epigenetic suppression of interferon lambda receptor expression leads to an enhanced HuNoV replication in vitro

Human norovirus (HuNoV) is the main cause of acute gastroenteritis worldwide yet no therapeutics are currently available. Here, we utilize a human norovirus replicon in epithelial human gastric tumor (HGT) cells to identify host factors involved in promoting or inhibiting HuNoV replication. We observed that an IFN-cured population of replicon-harboring HGT cells (HGT-cured) was enhanced in their ability to replicate transfected HuNoV RNA compared to parental HGT cells, suggesting that differential gene expression in HGT-cured cells created an environment favouring the replication of viral RNA. Microarray analysis was used to identify genes differentially regulated in HGT-NV and HGT-cured compared to parental HGT cells. We found that the IFN lambda receptor alpha (IFNLR1) expression was highly reduced in HGT-NV and HGT-cured cells. All three cell lines responded to exogenous IFN-{beta} by inducing interferon stimulated genes (ISGs), however, HGT-NV and HGT-cured failed to respond to exogenous IFN-{lambda}. Inhibition of DNA methyltransferase activity with 5-aza-2-deoxycytidine partially reactivated IFNLR1 expression in HGT-NV and IFN-cured cells suggesting that host adaptation occurred via epigenetic reprogramming. In line with this observation, ectopic expression of the IFN-{lambda} receptor alpha rescued HGT-NV and HGT-cured cells response to IFN-{lambda}. We conclude that type III IFN is important in inhibiting HuNoV replication in vitro and that the loss of IFNLR1 enhances replication of HuNoV. To the best of our knowledge, this study unravels for the first time epigenetic reprograming of the interferon lambda receptor as a new mechanism of cellular adaptation during long-term RNA virus replication and shows that an endogenous level of interferon lambda signalling is able to control human norovirus replication.

microbiology↗