<p>  </p> <p>Infection by Marburgvirus (MARV) or the closely related Ebolavirus (EBOV) results in potentially lethal hemorrhagic fever in humans characterized by uncontrolled viremia, a systemic pro-inflammatory response, and multi-organ failure. Currently, there are no approved countermeasures to treat or prevent MARV infection, which leaves a critical need for development of antiviral therapies. One approach to develop antiviral therapies is exploit a virus’s dependency on the host cell and disrupt critical human-viral interactions. While multiple studies identified host-viral interactions involved in EBOV infection, we are currently limited in our knowledge of host-viral interactions that occur during MARV infection and how these interactions influence the viral replication cycle. Thus, the purpose of this research was to identify and further characterize the biological significance of human-MARV protein-protein interactions that occur during infection.</p> <p>Here, we used genome-wide yeast two-hybrid (Y2H) screens to identify directly interacting human-viral proteins. We identified 431 putative interactions with MARV and used a combination of a novel NanoLuc Y2H assay and confidence criteria to prioritize a final set of 396 interactions. Bioinformatic analysis revealed that the molecular functions of the interacting human genes were significantly enriched in RNA binding, cell adhesion, and cytoskeleton binding. </p> <p>MARV and EBOV have many similarities in their genomic organization, sequence, and protein structures that could facilitate interactions to common host factors during infection. Thus, to identify shared interactions between these related viruses, we compared the MARV interactions to EBOV interactions identified in a parallel Y2H screen. We identified 145 human proteins targeted by both MARV and EBOV. The majority (77%) of shared interactions occurred between homologous viral proteins. Additional bioinformatic analyses comparing MARV and EBOV interactions revealed that these viruses interact with different host factors with similar molecular functions (RNA binding, DNA binding, actin and microtubule binding. Together, these data support the notion that while MARV and EBOV target common host factors there are still differences in protein interactions that support functions specific to each virus. </p> <p>To investigate the biological significance of the identified interactions, we focused on host interactions with the viral matrix protein, VP40. VP40 is a multifunctional protein that facilitates viral assembly and budding from the host cell. Y2H assays using VP40 mutants revealed that the WW-containing host protein MAGI1 interacted with the late domain of VP40. This interaction was validated in mammalian cells using coimmunoprecipitation and GFP complementation assays. Based on multiple reports of WW-containing host proteins interacting with VP40, we predict that the interaction between MAGI1 and VP40 regulates viral budding.</p> <p>In conclusion, the work presented here successfully identified 396 novel human-MARV interactions, which furthers the field’s understanding of host factors involved in MARV infection. Additionally, we identified interactions shared by MARV and EBOV, which could be beneficial in the development of a broad antiviral therapy against filoviruses. Lastly, we validate the interaction between MAGI1 and VP40, which has a potential role in viral budding </p>

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.