Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling

Uwase, G.; Luthra, P.; Vogel, O. A.; Batra, J.; LaRosa, B. A.; Sheehan, K. C. F.; Khatavkar, O.; Payton, J.; Davey, R. A.; Krogan, N. J.; Basler, C. F.; Leung, D. W.; Amarasinghe, G. K.

doi:10.1101/2025.07.27.667045

bioRxiv · 10.1101/2025.07.27.667045

Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling

Uwase, G.·Luthra, P.·Vogel, O. A.·Batra, J.·LaRosa, B. A.·Sheehan, K. C. F.·Khatavkar, O.·Payton, J.·Davey, R. A.·Krogan, N. J.·Basler, C. F.·Leung, D. W.·Amarasinghe, G. K.

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Abstract

Ebola virus (EBOV) is a non-segmented, negative-sense virus (NNSV) with a single-stranded RNA genome. EBOV encodes for a limited number of proteins and thus depends on host factors to facilitate viral replication and pathogenesis. Of the virus-encoded proteins, multifunctional EBOV VP35 (eVP35) is necessary for host immune evasion and viral RNA synthesis. Previous proteomics studies identified an interaction between eVP35 and the host E3 ubiquitin ligase Mindbomb 2 (MIB2). Here, we show how a previously uncharacterized NNLNS motif (residues 201-205) within eVP35 serves as a binding site for MIB2. This motif is critical for eVP35-dependent inhibition of MIB2-mediated IFN induction. It is also important for EBOV RNA synthesis as MIB2 binding to eVP35 inhibited EBOV minigenome activity. Altogether, these findings highlight the importance of the eVP35 protein and the role of host factors in EBOV infection. SIGNIFICANCE STATEMENTThe Ebola virus (EBOV) genome encodes for a limited number of proteins and depends on host factors to facilitate viral replication. Identification and characterization of host-viral interactions are needed to define infection, resolution, and to develop new therapeutics. EBOV VP35 (eVP35) is necessary for mediating host immune evasion and a cofactor for viral RNA synthesis. Here we characterized an interaction between eVP35 and MIB2. We show that the 201NNLNS205 motif in eVP35 is necessary and sufficient for MIB2 binding and inhibition of MIB2-mediated IFN production. Our results also reveal how the eVP35-MIB2 interaction impacts virus infection. These results support the importance of the multifunctional eVP35 to EBOV infection and highlight the significance of host proteins, including E3 ligases, during viral infection.

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Uwase, G., Luthra, P., Vogel, O. A., Batra, J., LaRosa, B. A., Sheehan, K. C. F., Khatavkar, O., Payton, J., Davey, R. A., Krogan, N. J., Basler, C. F., Leung, D. W., Amarasinghe, G. K.. 2025-07-27. Ebola virus VP35 NNLNS motif modulates viral RNA synthesis and MIB2-mediated signaling. https://doi.org/10.1101/2025.07.27.667045

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