Regulation of the Ebola Virus VP24 Protein by SUMO

Abstract

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.

The Minor Matrix Protein VP24 from Ebola Virus Lacks Direct Lipid-Binding Properties

“…Next, we hypothesized that VP24 lipid-binding may be more dependent on mammalian cell expression due to potential post-translational modifications (PTMs) that may alter VP24–lipid interactions. For instance, VP24 is known to undergo SUMOylation that covalently attaches the small ubiquitin-like modifier (SUMO) to the target protein [ 39 ]. Thus, HEK293 cells were transfected with plasmids of HA-tagged VP24 (HA-VP24) or enhanced green fluorescent protein (EGFP)-tagged lactadherin C2 domain (LactC2) (LactC2-EGFP) which is a well-established PS binding protein [ 40 ].…”

The Ebola virus interferon antagonist VP24 undergoes active nucleocytoplasmic trafficking

“…Interestingly, a recent study reported that sumoylation of residue K14 of VP24 enhances K binding and IFN antagonistic function (54). In contrast, ubiquitination, including at residue K206 within CL3 (Figure 2A), appears to negatively regulate IFN antagonist activity (54). Intriguingly, K14 is distal to CL1-3 but is within a predicted NES motif (Figure 2A).…”

The Role of Deubiquitinating Enzymes in Acute Lung Injury and Acute Respiratory Distress Syndrome

“…NLRP3 [38] Regulates NLRP3 inflammasome activation [38] NF-κB [39], NEMO [33] Regulates NF-κB signaling [33,39] VP24 [49] Involves in virus replication [49] Tat [50] Involves in virus production [50] TRAF3/TRAF6 [51] Modulates antiviral signaling [51] TRAF6/IKKγ [34] Regulates TLR signaling [34] USP-10 CFTR [37,52] Epithelial mucosal clearance [37,52] NICD1 [53] Regulates Notch signaling [53] USP-11 E2F1 [54] Regulates lung epithelia proliferation and wound healing [54] LPA1 [36] Enhances inflammation [36] USP-13 IL-1R8/Sigirr [40] Suppresses lung inflammation [40] PTEN [41] Regulates cell apoptosis [41] MCL1 [42] Regulates transformation of fibroblasts [42] STAT1 [55] Regulates IFN Signaling [55] STING [56] Negatively regulates antiviral responses [56] USP-14 I-kB [31] Increases cytokine release [31] CBP [32] Lung inflammation [32] USP-15 IκBα [57] NF-κB activation [57] USP-17 HDAC2 [58] Reverses glucocorticoid resistance [58] TRAF2/TRAF3 [59] Lung inflammation [59] [92] Inhibits type I IFN signaling and antiviral response [92] POH1 pro-IL-1β [93] Negatively regulates the immune response [93] BRCC3 NLRP3…”

“…USP7 (HAUSP)is originally identified as a viral binding protein that preferentially cleaves K11-, K63-and K48-linked ubiquitin chains [119,120]. USP7 is involved in viral infection by targeting virus related protein to modulate virus replication and production [49][50][51]. USP7 is reported to deubiquitinate and stabilize NF-κB to increase its transcriptional activity in TLR-induced inflammatory gene expression [39].…”