Biology subjects

Michiels, T.

Publications and source records attributed to Michiels, T..

6 recordsLinked to original sources

bioRxiv2025

Conserved phosphorylatable residues in motif G of positive-stranded virus RdRps regulate polymerase activity and suggest targets for drug design.

RNA viruses rely on an RNA-dependent RNA polymerase (RdRp) to replicate their genome. RdRps share a conserved core replicase structure described as a right hand within which RNA replication occurs. RNA polymerases contain a series of conserved motifs (A-G) that are essential for catalysis. Motif G, located at the RNA entry channel, guides the incoming RNA into the catalytic centre and and holds it in place during catalysis. Although RdRp phosphorylation has been reported, it has been scarcely studied. In most studied cases, phosphomimetic mutations reduced viral replication. In this study, we identified Theilers murine encephalomyelitis virus (TMEV) polymerase (3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 located in motif G are highly conserved in the sequences of picornavirus polymerases and in the structure of many positive-stranded virus polymerases, including nsp12 of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Using mutagenesis and reporter viruses, we show that phosphomimetic mutation of either residue abrogates viral replication, for both TMEV and SARS-CoV-2. Mutations of 3Dpol residues 109 and 110 into all other possible residues shows that, besides negatively charged phosphomimetic residues, bulky residues strongly inhibit replication, suggesting that phosphorylation inhibits polymerase activity by steric hindrance and/or through charge repulsion with RNA entering the catalytic core. Because these phosphorylatable residues are surface-exposed and conserved among viral polymerases, they represent promising targets for the rational design of broad-spectrum antiviral agents. ImportanceRNA viruses require an RNA-dependent RNA polymerase to replicate their genome. We identified in Theilers murine encephalomyelitis virus polymerase (TMEV 3Dpol) residues that undergo some extent of phosphorylation in infected cells. Among these residues, Thr109 and Ser110 are located in the entry channel of the polymerase, a region important for directing the RNA into the polymerase and locking it in place during catalysis. Incidentally, Thr109 and Ser110 are highly conserved in the sequences of picornavirus polymerases and in the structures of many positive-stranded virus polymerases including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nsp12. Our study revealed that, in both TMEV 3Dpol and SARS-CoV-2 nsp12, mutation of either residue into a negatively charged amino acid that mimics phosphorylation abrogates viral replication, suggesting phosphorylation would block polymerase activity. As these phosphorylated residues are accessible and conserved, they provide important candidate targets for the design of antiviral molecules.

microbiology↗

bioRxiv2025

Cardiovirus-Mediated PKR Inhibition Results from Nucleocytoplasmic Trafficking Disruption

Eukaryotic translation initiation factor 2 alpha kinase 2 (EIF2AK2), know as PKR, is a key antiviral kinase activated by double-stranded RNA (dsRNA) typically produced during viral replication. Upon activation, PKR phosphorylates eIF2, leading to the inhibition of translation and viral replication. However, many viruses have evolved mechanisms to counteract PKR activity. In Cardioviruses, the Leader protein (L), a short peptide cleaved from the N-terminus of the viral polyprotein, not only inhibits PKR but also blocks interferon production and disrupts nucleocytoplasmic trafficking (NCT). L disrupts NCT by recruiting host RSK kinases to the nuclear pore complex (NPC), where RSK phosphorylates FG-nucleoporins, thereby impairing NCT. L mutations that affect NCT disruption also impact its ability to inhibit PKR, suggesting a mechanistic link. Recombinant TMEV and EMCV viruses designed to disrupt NCT through different mechanisms exhibited some extent of PKR inhibition, supporting the link between NCT disruption and PKR inhibition. Immunostaining and live-cell imaging revealed that L-induced NCT disruption redistributes a fraction of PKR to the nucleoli, where PKR remains inactive. This suggests that nucleolar sequestration contributes to PKR inhibition. Additionally, L-mediated NCT disruption leads to the release of nuclear RNA-binding proteins (nRBPs) into the cytosol, which may bind or modify viral dsRNA, further preventing PKR activation. Collectively, these results highlight nucleocytoplasmic trafficking as a critical regulatory mechanism governing PKR activation. Thus, beyond the specific action of cardiovirus L protein, our study reveals that interference with host nucleocytoplasmic transport can significantly impact the subcellular localization and functional regulation of immune effectors such as PKR. Author SummaryProtein kinase R (PKR) is a crucial component of the host innate immune response. It is activated by double-stranded RNA (dsRNA) typically produced during viral replication and triggers a shutdown of mRNA translation. This antiviral mechanism limits viral propagation by inhibiting both host and viral protein synthesis. However, many viruses have developed mechanisms to inhibit PKR, allowing them to escape immune detection. PKR downregulation facilitates viral replication whereas uncontrolled PKR activation can lead to autoimmune disorders. Therefore, PKR activity must be tightly regulated to maintain immune homeostasis. Using recombinant viruses which target the nuclear pore complex, we show that nucleocytoplasmic trafficking of cellular components is critical for regulation of PKR activity. Infection of cells with Theilers murine encephalomyelitis virus triggers an efflux of nuclear RNA binding proteins which likely compete with PKR for dsRNA binding and thereby block PKR activity. Moreover, upon TMEV infection as well as during mitosis, PKR is detected in the nucleoli where it is thought to interact with structured RNAs without being activated. Our data highlight an important link between nucleocytoplasmic trafficking and PKR activity.

immunology↗

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↗

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↗