Biology subjects

Wavreil, F.

Publications and source records attributed to Wavreil, F..

3 recordsLinked to original sources

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

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↗

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↗