Regulation of the Ebola Virus VP24 Protein by SUMO

Some viruses take advantage of conjugation of ubiquitin or ubiquitin-like proteins to enhance their own replication. One example is Ebola virus, which has evolved strategies to utilize these modification pathways to regulate the viral proteins VP40 and VP35 and to counteract the host defenses. Here, we show a novel mechanism by which Ebola virus exploits the ubiquitin and SUMO pathways. Our data reveal that minor matrix protein VP24 of Ebola virus is a bona fide SUMO target. Analysis of a SUMOylation-defective VP24 mutant revealed a reduced ability to block the type I interferon (IFN) pathway and to inhibit IFN-mediated STAT1 nuclear translocation, exhibiting a weaker interaction with karyopherin 5 and significantly diminished stability. Using glutathione S-transferase (GST) pulldown assay, we found that VP24 also interacts with SUMO in a noncovalent manner through a SIM domain. Mutation of the SIM domain in VP24 resulted in a complete inability of the protein to downmodulate the IFN pathway and in the monoubiquitination of the protein. We identified SUMO deubiquitinating enzyme ubiquitin-specific-processing protease 7 (USP7) as an interactor and a negative modulator of VP24 ubiquitination. Finally, we show that mutation of one ubiquitination site in VP24 potentiates the IFN modulatory activity of the viral protein and its ability to block IFN-mediated STAT1 nuclear translocation, pointing to the ubiquitination of VP24 as a negative modulator of the VP24 activity. Altogether, these results indicate that SUMO interacts with VP24 and promotes its USP7-mediated deubiquitination, playing a key role in the interference with the innate immune response mediated by the viral protein.IMPORTANCE The Ebola virus VP24 protein plays a critical role in escape of the virus from the host innate immune response. Therefore, deciphering the molecular mechanisms modulating VP24 activity may be useful to identify potential targets amenable to therapeutics. Here, we identify the cellular proteins USP7, SUMO, and ubiquitin as novel interactors and regulators of VP24. These interactions may represent novel potential targets to design new antivirals with the ability to modulate Ebola virus replication.
Funding at the laboratory of C.R. is provided by Ministry of Science, Innovation and Universities and FEDER (BFU-2017-88880-P). C.R. also acknowledges grants GRC GI-2119 (Xunta de Galicia) and SAF2017-90900-REDT (UBIRed Program). S.V. is a predoctoral fellow funded by Xunta de Galicia. M.B.-M. is a postdoctoral fellow funded by Xunta de Galicia (Consellería de Cultura, Educación e Ordenación Universitaria). A.E.M. is the recipient of a fellowship of the Spanish FPI program. This work was partially funded by the German Center for Infection Research (DZIF TTU 01.702_00 to C.M.-F.). R.B. and J.D.S. acknowledge grants BFU2017-84653-P (MINECO/FEDER, EU), SEV-2016-0644 (Severo Ochoa Excellence Program), 765445-EU (UbiCODE Program), and SAF2017- 90900-REDT (UBIRed Program). C.S.M. acknowledges grant BFU2016-74868-P.