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DTSTART;TZID=Europe/Paris:20260622T133000
DTEND;TZID=Europe/Paris:20260622T153000
DTSTAMP:20260604T143528Z
CREATED:20260604T143528Z
LAST-MODIFIED:20260604T143528Z
UID:10000188-1782135000-1782142200@sfp-alpes.fr
SUMMARY:Soutenance de Thèse de Khadeeja MUBASHIRA (CEA-Irig/IBS)
DESCRIPTION:Étude de la séparation de phase de la phosphoprotéine du virus de la rage et de sa régulation par LC8\nRésumé : \nRabies virus (RABV) replication occurs in cytoplasmic\, membrane-less compartments known as Negri bodies (NBs)\, formed through liquid-liquid phase separation (LLPS) of viral components. The phosphoprotein (RABV P) is a central\, intrinsically disordered scaf fold of the viral replication machinery. This thesis investigates the structural\, biophysical\, and dynamic properties of RABV P\, with emphasis on its phase separation behavior and interactions with molecular partners. To enable this\, recombinant expression and purification protocols were optimized to produce stable\, high-quality protein samples for reproducible analyses. \nWe first characterized the intrinsic phase behavior of RABV P in vitro. The protein undergoes thermoresponsive LLPS with a lower critical solution temperature (LCST)\, forming reversible condensates within a narrow range of protein and salt concentrations. This process is driven by multivalent interactions within a heterogeneous ensemble of conformations\, where dimers assemble into higher-order oligomers prior to phase separation. The resulting phase diagram reveals a complex\, reentrant system governed by a balance between electrostatic repulsion and attractive dipole-dipole interactions. \nThe role of ionic conditions was further examined. While NaCl induced reentrant phase separation\, LLPS strongly depended on ion identity rather than ionic strength alone. Chloride salts promoted condensate formation\, whereas bromide salts did not\, indicating ion-specific (Hofmeister-type) effects. Systematic trends showed that fluoride enhances phase separation\, while cation effects are weaker. Divalent ions also promoted LLPS\, highlighting valency contributions. Chemical perturbations confirmed that condensates are stabilized by weak interactions: 1\,6-hexanediol partially disrupted droplets\, whereas ATP fully dissolved them. Notably\, RABV P intrinsically phase separates even in water\, modulated by pH\, protein concentration\, and ionic conditions. \nTime-resolved small-angle X-ray scattering (SAXS) revealed the structural evolution underlying LLPS. Following a temperature jump\, RABV P undergoes a hierarchical assembly process\, transitioning from dispersed species to larger structures. Early conformational rearrangements precede the formation of intermediate clusters\, followed by growth into larger assemblies. These structures remain disordered and liquid-like\, supporting a multistep nucleation-and-growth mechanism. \nThe host protein LC8 was investigated as a regulator of RABV P condensation. LC8 binds a conserved motif in RABV P with high affinity\, forming a defined complex and partitioning into condensates. Functionally\, LC8 enhances phase separation by increasing condensate size\, enriching RABV P in the dense phase\, and broadening the phase-separation window. It shifts phase boundaries toward lower concentrations and temperatures while preserving liquid-like properties. These results indicate that LC8 actively promotes condensation by stabilizing interaction-competent conformations and enhancing intermolecular connectivity. \nTo assess whether LC8 can compensate for intrinsic multivalency\, a truncated RABV P lacking the dimerization domain was analyzed. Although LC8 bound this construct\, the interaction was weaker and failed to restore robust phase separation. Only weak condensation was observed under crowding conditions\, demonstrating that LC8 cannot substitute for the native dimerization-driven multivalency.\nOverall\, this work establishes RABV P as a finely tuned multivalent scaffold whose phase behavior arises from the interplay of intrinsic disorder\, ion-specific effects\, and hierarchical assembly. LLPS emerges as a multistep\, non-ideal process rather than a simple binary transition. LC8 acts as a key host regulator that enhances phase separation without altering condensate dynamics\, while intrinsic multivalency remains essential. These findings provide a mechanistic framework for understanding viral condensate formation and highlight potential avenues for antiviral intervention. \n_ \nContact : alain.farchi@cea.fr
URL:https://sfp-alpes.fr/event/soutenance-de-these-de-khadeeja-mubashira-cea-irig-ibs/
LOCATION:Amphi A de Biologie\, Rue de la Piscine\, Saint-Martin-d'Hères\, 38400\, France
CATEGORIES:Soutenance,Soutenance de Thèse
ORGANIZER;CN="IRIG - CEA":MAILTO:odile.rossignol@cea.fr
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DTSTART;TZID=Europe/Paris:20260626T140000
DTEND;TZID=Europe/Paris:20260626T160000
DTSTAMP:20260529T140307Z
CREATED:20260529T140204Z
LAST-MODIFIED:20260529T140307Z
UID:10000170-1782482400-1782489600@sfp-alpes.fr
SUMMARY:Soutenance de Thèse de Henri GRÖGER (Irig/IBS)
DESCRIPTION:Structural and functional characterisation of the vaccinia virus PLD- fold endonuclease K4\, telomere-binding protein i1 and the DNA polymerase complex E9A20D4\nRésumé : \n\nPoxviruses\, such as the vaccinia virus (VACV) and the monkeypox virus\, are large\, enveloped dsDNA viruses from the orthopoxvirus genus that replicate entirely within the host cytoplasm. The 2022 and 2024 outbreaks of mpox\, caused by clade IIb and Ib\, respectively\, have revealed the lack of efficient antivirals and underlined the urgency of understanding poxvirus biology. The poxvirus genome is flanked by short\, inverted complementary hairpin telomeres that feature mismatched bases and insertions essential for viral replication. ​\n​\nThis thesis presents the structural and functional characterisation of three proteins central to poxvirus DNA metabolism : the E9A20D4 DNA polymerase holoenzyme\, and two telomere-interacting proteins\, the PLD-fold nuclease K4 and I1. The project initially focused on the VACV polymerase holoenzyme\, but was reoriented towards the telomere-interacting proteins following the publication of numerous competing mpox polymerase structures. ​\n​\nHaving established that VACV polymerase activity requires K+ and is inhibited by Na+\, I undertook a structure determination of the E9A20D4 polymerase holoenzyme bound to template DNA\, primer and incoming nucleotide in the presence of K+\, using single-particle cryogenic electron microscopy (cryo-EM). I obtained both the structure of the complex E9exo−A20D4 as well as the structure of E9exo− alone bound to the primer-template DNA. The structures in the presence of K+ appear identical to published structures in the presence of Na+. However\, I identified an ion binding site in the exonuclease domain of E9. The thumb domain is disordered in the DNA-free structure\, partially disordered in DNA-bound E9 and ordered in the holoenzyme-DNA complex. SAXS data indicate conformational flexibility\, with more open conformations of E9A20D4 lacking an E9-D4 interface\, while mass photometry reveals partial dissociation of E9A20D4 at low concentrations\, even in the presence of substrate. ​\n​\nUsing cryo-EM\, I report the first structures of K4 in both apo and DNA-bound states\, revealing that the active site is occluded by an orthopoxvirus-specific C-terminal extension of the PLD fold that is displaced upon DNA binding. Biochemical characterisation demonstrates that K4 functions as a DNA-specific endonuclease with a preference for single-stranded DNA and hairpin loops. ​\n​\nI also report the first cryo-EM structure of I1 bound to DNA. I1 is known to bind to viral telomeres and is essential for virion maturation. Cryo-EM data showed the presence of dimers where the head domains 2 and 3 of I1 interact with the DNA duplex through electrostatic interactions\, while the N-terminal domain predicted to be α-helical remains disordered. In solution\, isolated I1 or I1 bound to DNA forms higher-order assemblies\, predominantly tetramers\, but also octamers. ​\n​\n​ Altogether\, these findings substantially advance the molecular understanding of poxvirus biology\, providing a foundation for future mechanistic studies and the rational development of antiviral strategies against emerging orthopoxvirus infections.\n​​\n\n\nLes séminaires et soutenances sont ouverts à tous\, notez toutefois que l’accès au campus EPN nécessite un avis de rendez-vous. Merci de remplir ce formulaire  et de l’adresser\, plus de 48h à l’avance\, à ibs.seminaires@ibs.fr. Pensez à vous munir d’une pièce d’identité le jour de votre visite.\n\n  \n  \n  \n  \n  \n 
URL:https://sfp-alpes.fr/event/soutenance-de-these-de-henri-groger-irig-ibs/
LOCATION:Salle des séminaires du CIBB\, EPN Campus - 71 avenue des Martyrs\, Grenoble\, 38000\, France
CATEGORIES:Soutenance,Soutenance de Thèse
ORGANIZER;CN="IRIG - CEA":MAILTO:odile.rossignol@cea.fr
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