Biology subjects

Tubiana, T.

Publications and source records attributed to Tubiana, T..

9 recordsLinked to original sources

bioRxiv2026

ZDHHC17–Mediated Palmitoylation of Hepatitis E Virus ORF3 Protein Regulates Vectorial Trafficking in Polarized Epithelial Cells

The hepatitis E virus (HEV) is a leading cause of acute hepatitis worldwide and is transmitted enterically along the gut-liver axis. Epithelial cell polarity in the gut and liver plays a critical role in HEV transmission. In intestinal epithelial cells, HEV enters through the apical membrane and is released basolaterally to access the bloodstream, whereas in hepatocytes, the virus enters basolaterally and is secreted apically into the bile duct. In this study, we sought to identify the viral and host determinants governing directional HEV secretion in both tissue types. Using polarized intestinal and hepatocyte models, we found that the small phosphoprotein ORF3, which is essential for progeny secretion, localizes predominantly to the apical membrane, in contrast to the ORF2 capsid protein. We further identified the palmitoyltransferase ZDHHC17 as a specific ORF3 interactor that mediates its apical localization and promotes HEV progeny release. Using automated cell segmentation and quantitative image analysis, we screened a panel of ORF3 mutants and found that positively charged residues within the N-terminus regulate membrane association. In addition, we identified a conserved PIFIQP motif that mediates the critical interaction with the ankyrin repeat domain (ARD) of ZDHHC17. Leveraging this interaction, we generated high-confidence structural models of ORF3 in complex with the ZDHHC17 ARD using AlphaFold. These models, further validated by molecular dynamics simulations, revealed additional ORF3 residues involved in the interaction. Collectively, our findings define key mechanisms underlying directional HEV release and provide broader insights into trafficking processes in polarized epithelial cells.

Cell Biology↗

bioRxiv2026

A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReEDs mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

microbiology↗

bioRxiv2025

Functional study of two flexible regions of the hepatitis E virus ORF1 replicase

Hepatitis E virus (HEV), like other positive-sense RNA viruses, encodes a multidomain protein essential for replication, termed ORF1. However, the number, organization, and functions of its domains remain debated. Using AlphaFold2-based structural modeling, we investigated two structurally disordered regions with potential regulatory functions: (i) a 16-residue linker between the Helicase (Hel) and RNA-dependent RNA polymerase (RdRp) domains, proposed as a cleavage site and/or a flexible hinge, and (ii) the RdRp C-terminal tail, suggested to modulate polymerase activity through conformational plasticity. We performed mutagenesis of the Hel/RdRp linker and analyzed the impact of mutations on ORF1 maturation, subcellular localization, and replication efficiency. Using an extensive antibody panel combined with precise protein sizing, we found that ORF1 is predominantly expressed as a full-length protein with an apparent molecular weight of [~]235 kDa by SDS-PAGE, together with several low-abundance truncated products, including the previously described HEV-derived SMAD activator (HDSA) fragment. These results suggest that ORF1 likely undergoes post-translational modifications and partial maturation by cellular proteases and/or spontaneous truncations in exposed regions. Importantly, Hel/RdRp linker mutations did not alter the ORF1 expression profile or subcellular localization, arguing against cleavage within this region. However, substitutions of conserved residues in the linker strongly impaired HEV replication, highlighting the functional importance of this disordered segment for viral genome replication. Similarly, deletions or substitutions within the last 20 C-terminal RdRp residues abolished or severely impaired HEV replication. This demonstrates that the conformational flexibility of the RdRp C-terminal segment is likely critical for ORF1 function, e.g. for polymerase activity. In conclusion, although ORF1 likely undergoes tightly regulated processing, cleavage is unlikely to occur within the Hel/RdRp linker. Nevertheless, this segment and the conformational dynamics of the RdRp C-terminus emerge as key regulatory elements required for efficient HEV replication, pointing to novel mechanistic layers of control in the HEV replication process.

microbiology↗

bioRxiv2025

Oral 4-fluorouridine completely suppresses epidemic Oropouche virus replication in mice and confers protection against virus-induced disease.

Oropouche virus (OROV) is an orthobunyavirus that causes increasingly frequent and severe outbreaks in Central and South America. We report that 4'-fluorouridine (4'-FlU) inhibits the in vitro replication of epidemic and pre-epidemic OROV strains in multiple cell lines. In vitro polymerase assays demonstrate that 4'-FlU (as its triphosphate) targets the Peribunyaviridae L protein, is incorporated during RNA synthesis and causes premature chain termination. Following 69 consecutive days of in vitro passages of OROV in the presence of suboptimal concentrations of 4'-FlU, no drug-resistant variants were identified in the viral polymerase. In stringent mouse (AG129) or Syrian hamster OROV-infection models, oral administration of 4'-FlU completely blocked viral replication and virus-induced disease, even when administration was delayed until 72 hours after infection. Our findings support exploring the potential of 4'-FlU for the management of OROV infections in humans.

microbiology↗

bioRxiv2025

The hepatitis E virus capsid protein ORF2 counteracts cell-intrinsic antiviral responses to enable persistence in hepatocytes

Hepatitis E virus (HEV) is a significant human pathogen causing both acute and chronic infections worldwide. The cell-intrinsic antiviral response serves as the initial defense against viruses and has been shown to be activated upon HEV infection. HEV can replicate in the presence of this response, but the underlying mechanisms remain poorly understood. Here, we investigated the role of the HEV structural proteins ORF2 and ORF3 in immunocompetent cells. Mechanistically, we validated that ectopic ORF2, but not ORF3, interfered with antiviral and inflammatory signaling downstream of pattern recognition receptors, in part through interaction with the central adaptor protein TANK binding kinase 1. In the full-length viral context, ORF2 contributed to a reduced antiviral response and consequently, more efficient viral replication. In addition, we discovered a protective mechanism mediated by ORF2 that shielded viral replication from antiviral effectors. Using single-cell RNA-sequencing, we confirmed that the presence of ORF2 in infected cells dampened antiviral responses in both actively infected cells and bystanders. As a consequence, we found that early in the infection process, the progression of authentic HEV infection relied on the presence of ORF2, facilitating a balance between viral replication and the antiviral response within immunocompetent cells. Altogether, our findings shed new light on the multifaceted role of ORF2 in the HEV life cycle and improve our understanding of the determinants that may contribute to HEV persistence. Significance statementHepatitis E virus (HEV) is an important yet often underestimated pathogen. Depending on the genotype, HEV infections can progress to chronicity, but the underlying mechanisms remain poorly understood. To gain insight into potential determinants, we investigated how HEV evades the bodys first line of defense, the cell-intrinsic antiviral response. We discovered that the HEV capsid protein ORF2 is crucial in limiting this response by interfering with antiviral signaling pathways and shielding viral replication from immune effectors. This balance between viral replication and the antiviral response contributes to persistent HEV infection in immunocompetent cells. Our findings reveal a new role for the HEV capsid protein in the viral life cycle and highlight it as an important target for novel therapeutic approaches.

microbiology↗

bioRxiv2024

Structural characterization of the ACDC domain from ApiAP2 proteins of the malaria parasite

The Apicomplexan AP2 (ApiAP2) proteins are the best characterized family of DNA-binding proteins in the malaria parasite. Apart from the AP2 DNA-binding domain, there is little sequence similarity between ApiAP2 proteins and no other functional domains have been extensively characterized. One protein domain, which is present in a subset of the ApiAP2 proteins, is the conserved AP2-coincident domain mostly at the C-terminus (ACDC domain). Here we solved for the first time the crystal structure of the ACDC domain from two distinct Plasmodium falciparum ApiAP2 proteins and one orthologue from P. vivax, revealing a non-canonical four-helix bundle. Despite little sequence conservation between the ACDC domains from the two proteins, the structures are remarkably similar and do not resemble that of any other known protein domains. Due to their unique protein architecture and lack of homologues in the human genome, we performed in silico docking calculations against a library of known antimalarial compounds and we identified a small molecule that can potentially bind to any Apicomplexan ACDC domain within a pocket highly conserved amongst ApiAP2 proteins. Inhibitors based on this compound would disrupt the function of the ACDC domain and thus of the ApiAP2 proteins containing it, providing a new therapeutic window for targeting the malaria parasite and other Apicomplexans.

biochemistry↗

bioRxiv2024

Studying self-assembly of norovirus capsid by a combination of in silico methods

Understanding how macromolecular assembly occurs is a fundamental and challenging problem because spontaneous, precise assembly is at the center of most biological processes. It is an elaborate process that requires non-covalent stable interactions between partners to stabilize the desired architecture for a specific purpose. One of the advantages of virus models is that under adequate conditions capsid proteins can be efficiently assembled in vitro in the absence of any other component, providing simplified experimental models that can be rigorously characterized. The present study aims at describing the initial steps of molecular self-assembly of norovirus-like particles (NoVLPs, composed solely of the major norovirus capsid protein VP1), by combining in silico computational approaches to explore complementary physical properties. We show that this strategy allows not only recapitulating but also revising a former NoVLP assembly model. Our approach can be applied and extended to other problems in macromolecular assembly.

bioinformatics↗

bioRxiv2022

Dissecting peripheral protein-membrane interfaces

Peripheral membrane proteins (PMPs) include a wide variety of proteins that have in common to bind transiently to the chemically complex interfacial region of membranes through their interfacial binding site (IBS). In contrast to protein-protein or protein-DNA/RNA interfaces, peripheral protein-membrane interfaces are poorly characterized. We collected a dataset of PMP domains representative of the variety of PMP functions: membrane-targeting domains (Annexin, C1, C2, discoidin C2, PH, PX), enzymes (PLA, PLC/D) and lipid-transfer proteins (START). The dataset contains 1328 experimental structures and 1194 AphaFold models. We mapped the amino acid composition and structural patterns of the IBS of each protein in this dataset, and evaluated which were more likely to be found at the IBS compared to the rest of the domains accessible surface. In agreement with earlier work we find that about two thirds of the PMPs in the dataset have protruding hydrophobes (Leu, Ile, Phe, Tyr, Trp and Met) at their IBS. The three aromatic amino acids Trp, Tyr and Phe are a hallmark of PMPs IBS regardless of whether they protrude on loops or not. This is also the case for lysines but not arginines suggesting that, unlike for Arg-rich membrane-active peptides, the less membrane-disruptive lysine is preferred in PMPs. Another striking observation was the over-representation of glycines at the IBS of PMPs compared to the rest of their surface, possibly procuring IBS loops a much-needed flexibility to insert in-between membrane lipids. The analysis of the 9 superfamilies revealed amino acid distribution patterns in agreement with their known functions and membrane-binding mechanisms. Besides revealing novel amino acids patterns at protein-membrane interfaces, our work contributes a new PMP dataset and an analysis pipeline that can be further built upon for future studies of PMPs properties, or for developing PMPs prediction tools using for example, machine learning approaches.

bioinformatics↗

bioRxiv2019

Septin 9-containging filaments and Golgi assembly depend on two polybasic domains

Septins are GTP-binding proteins involved in several membrane remodeling mechanisms. They associate with membranes, presumably by using a polybasic domain (PB1) that interacts with phosphoinositides (PIs). Membrane-bound septins assemble into microscopic structures that regulate membrane shape. How septins exactly interact with PIs, assemble, and shape membranes is weakly understood. Here, we found that septin 9 has a second polybasic domain (PB2) conserved in the human septin family. Similarly to PB1, PB2 binds specifically to PIs, and both domains are critical for septin filament formation. However, septin 9 membrane association does not depend on these PB domains but on putative PB-adjacent amphipathic helices. The presence of the PB domains guarantees the protein enrichment to PI-contained membranes, which is critical for PI-enriched organelles. In particular, we found that septin 9 PB domains control the assembly and functionality of the Golgi apparatus. Our findings bring novel insights into the role of septins in organelle morphology. HighlightsO_LITwo polybasic domains mediate septin 9 interaction with PIs C_LIO_LIHuman septins have amphipathic helices suitable for binding membrane C_LIO_LISeptin 9 polybasic domains mediate septin high order structure formation C_LIO_LIMutation or depletion of septin polybasic domains induce Golgi fragmentation C_LI

cell biology↗