Les virus Ebola (EBOV) et Marburg (MARV) causent des fièvres hémorragiques chez les primates, y compris l’homme. Le taux de létalité peut atteindre 90% et il n’existe ni vaccin ni traitement contre ces virus. En raison de leurs caractéristiques moléculaires communes, EBOV et MARV sont regroupés au sein de la famille des Filoviridae. Le virion est composé de 7 protéines, dont la VP24, qui joue un rôle important dans l’assemblage et la condensation des nucléocapsides, et pour EBOV, elle est également responsable de l’inhibition de la réponse à l’IFN. Des mutations dans la séquence protéique de VP24 sont impliquées dans le processus d’adaptation chez un nouvel hôte. La protéine VP24 d’EBOV est donc multifonctionnelle. Pour MARV, cette protéine ne semble pas porter les fonctions décrites pour la VP24 d’EBOV. Afin de comprendre le rôle de la VP24 de MARV, nous avons identifié ses partenaires cellulaires par un crible double-hybride en levures. Nous avons mis en évidence l’interaction entre Keap1 et la VP24 de MARV, et confirmé ce résultat en cellules mammifères. Keap1 est une protéine impliquée dans le contrôle de la réponse au stress oxydatif, car elle inhibe le facteur de transcription Nrf2, qui régule l’expression d’enzymes impliquées dans la réduction des ERO. Nos résultats montrent que le domaine de Keap1 liant la VP24 est le même que celui liant Nrf2, et que la VP24 de MARV active Nrf2 pour la synthèse de molécules anti-oxydantes. Nous avons enfin évalué l’impact de la VP24 de MARV sur ERR, une autre cible de Keap1, et mesuré l’activité Nrf2 au cours de l’infection par EBOV. Nos résultats montrent des effets opposés des VP24 d’ EBOV et de MARV sur l’activité de Nrf2. / Ebola (EBOV) and Marburgvirus (MARV) are responsible for severe hemorrhagic syndrome in primates, including humans. The lethality rate can reach 90%, and no vaccine or treatment is available to counteract these diseases. EBOV and MARV have similar genomic organization and thus are placed in a distinct family, Filoviridae. VP24 is one of the 7 structural proteins which form the virion and has been shown to play an important role in assembly and condensation of viral nucleocapsids. VP24 of EBOV is responsible for prevention of cellular response to IFN. Mutations in EBOV VP24 gene are necessary for the adaptation to a new host. EBOV VP24 thus acts as a multifunctional factor. Available data suggest that MARV VP24 is not implicated in either the counteraction of IFN response, or in the adaptation process. In order to discover new functions for VP24 of MARV, we searched for its interaction with cellular proteins, using a yeast-double hybrid approach. We discovered an interaction between MARV VP24 and Keap1 protein and further confirmed this interaction in mammalian cells. Keap1 is a cellular protein involved in intracellular detection of Reactive Oxygen Species (ROS) and in the control of oxidative stress response. It inhibits the Nrf2 transcription factor, which regulates expression of antioxidant enzymes. Our results indicate that Keap1 binding domain for VP24 is the same as the one involved in Nrf2 binding, resulting in activation of transcriptional activity of Nrf2. Impact of MARV VP24 on ERRa, another target of Keap1, was also measured, as well as Nrf2 activity during EBOV infection. Our results showed that VP24 of EBOV and MARV have opposite effect on Nrf2 activity.

<p dir="ltr">Ebola virus is an enveloped filamentous shaped RNA virus which causes severe hemorrhagic fever in humans. Multiple outbreaks of different strains of ebolavirus have been reported in the past years with limited therapeutics available for treatment. Despite some advances in treatment, there remains a lack of knowledge about the mechanisms of ebolavirus replication in host cells.</p><p dir="ltr">Ebolavirus encodes for seven structural proteins with matrix protein (VP40) being the most abundantly expressed viral protein. VP40 is essential for viral assembly and budding as expression of VP40 alone is sufficient for formation of virus-like particles (VLPs). VP40 also disassembles during viral entry to help in viral and host cell membrane fusion. Oligomerization of VP40 has been reported to decrease viral replication and transcription. VP40 can perform these diverse functions by virtue of changes in conformation and oligomerization state. VP40 predominantly exists as a dimer through hydrophobic interactions between the alpha helices of the two protomers. Furthermore, VP40 oligomerizes into a hexamer which serves as the structural unit for cylindrical matrix layer formation. VP40 also forms a ring-shaped octamer for regulation of viral transcription. The different oligomeric forms of VP40 exist in an equilibrium for successful viral infection. However, the exact mechanism of formation, stability, and energetics of conversion between these oligomeric forms is unknown.</p><p dir="ltr">In this study, we performed biophysical analysis on the dimerization interface and identified keystone interactions which when abrogated lead to complete disruption of dimer interface. In addition, peptidomimetics approach was used to design and synthesize a library of compounds to probe the dimerization interface. The compounds were screened using thermal shift assay and then compared using MST and ITC studies. We identified that a peptide mimicking the alpha helical region stabilized by a p-xylene di-cysteine staple was able to bind to VP40 dimer. We also determined that this peptide binds near the dimer interface and was able to slightly shift equilibrium of VP40 dimer towards monomer formation.</p><p dir="ltr">Overall, this report sheds light on critical interactions required for VP40 dimer formation and stability and introduces use of peptidomimetics to probe for VP40 dimerization interface to understand energetics of oligomerization equilibrium, thereby increasing our knowledge about disease mechanism and paving way for development of therapeutics.</p>