An Epstein-Barr virus protein interaction map reveals NLRP3 inflammasome evasion via MAVS UFMylation

Abstract

Epstein-Barr virus (EBV) causes infectious mononucleosis, triggers multiple sclerosis, and is associated with 200,000 cancers/year. EBV colonizes the human B cell compartment and periodically reactivates, inducing expression of 80 viral proteins. However, much remains unknown about how EBV remodels host cells and dismantles key antiviral responses. We therefore created a map of EBV-host and EBV-EBV interactions in B cells undergoing EBV replication, uncovering conserved herpesvirus versus EBV-specific host cell targets. The EBV-encoded G-protein-coupled receptor BILF1 associated with MAVS and the UFM1 E3 ligase UFL1. Although UFMylation of 14-3-3 proteins drives RIG-I/MAVS signaling, BILF1-directed MAVS UFMylation instead triggered MAVS packaging into mitochondrial-derived vesicles and lysosomal proteolysis. In the absence of BILF1, EBV replication activated the NLRP3 inflammasome, which impaired viral replication and triggered pyroptosis. Our results provide a viral protein interaction network resource, reveal a UFM1-dependent pathway for selective degradation of mitochondrial cargo, and highlight BILF1 as a novel therapeutic target.

Keywords: MAVS; NLRP3 inflammasome; UFMylation; antiviral defense; gamma-herpesvirus; herpesvirus; interactome; mitochondrial-derived vesicles; viral evasion; virus/host interaction.

Figure 1.. EBV lytic cycle protein interaction map construction

(A) EBV protein interaction map workflow. Very high-confidence interacting proteins (VHCIPs) were defined as prey meeting peptide spectral match (PSM) and entropy criteria with either an NWD ≥ 1.0 or Z score ≥ 4.0. High-confidence interacting proteins (HCIPs) were defined as prey meeting PSM and entropy criteria with either an NWD R 1.0 or Z score R 3.0 (Table S2). (B and C) DAVID with default setting was applied to determine pathways enriched among all HCIPs, in comparison to all human proteins as background (full data, Table S3). Benjamini-Hochberg adjusted p values are shown in red with gradient scale to the top 16 unique pathway enriched (p < 0.01). Viral baits were assigned to their top unique enriched pathway with p < 0.05. Viral baits (blue squares), interacting proteins (circles, with viral proteins in light blue), enriched pathway members (red or green), and other host interactors not associated with the pathway (gray) are shown. Black solid lines indicate interactions identified in this study. See also Figure S1.

Figure 2.. Systematic analysis of interactome data from three herpesviruses highlights common host targets

(A) Overlap between EBV, HCMV, and KSHV HCIPs (see also Table S4). DAVID pathway enrichment analysis among HCIPs interacting with EBV, HCMV, and KSHV baits (default settings, against all human proteins as background) are shown at bottom, including a list of all commonly interacting CCR4-NOT complex components. Shown at upper right are representative terms from DAVID analysis of HCIPs interacting with EBV proteins but not HCMV or KSHV proteins, and a list of interacting components from the term “activation of innate immune response.” See Table S4 for full Venn diagram details. (B) Network propagation identification of CORUM database human protein complexes that are closely associated with proteins from each herpesvirus. Large colored nodes represent each virus (HCMV, blue; EBV, red; KSHV, yellow). Edges connect to CORUM complexes closely associated with proteins from each virus. Edge thickness is proportional to the Z score observed, whereas CORUM nodes sizes are colored pale yellow and scaled according to the numbers of proteins in each complex. (C and D) Interaction network depicting the CORUM CCR4-NOT and viral protein neighbors. Nodes represent proteins, whereas edges represent protein-protein interactions. Human proteins belonging to the CORUM CCR4-NOT complex (C) or RNA Pol II core complex (D) and edges among them are colored yellow. Viral proteins and their interactions are colored as in (B). Node size is scaled according to each node’s eigenvector centrality within the displayed subnetwork. Graph layouts were determined via gravity embedding. (E) Immunoblot of 1% input and anti-HA complexes from P3HR-1 expressing BRRF1 or GFP cDNAs, representative of n = 3 experiments. (F) Interaction of EBV proteins with components of the cellular degradation machinery. HCIP were searched against a database of E1, E2, E3 enzymes, cullins, deubiquitinases (DUBs), proteasome components (PSM), and ubiquitin-binding proteins (UBPs). Percentage of the total number of interactors for each protein from each of these categories is displayed. Values were clustered hierarchically (see also Table S5).

Figure 3.. BILF1 inhibits NLRP3 inflammasome activation

(A) Cytoscape version 3.8.1 BILF1 interaction network. DAVID pathways enriched among BILF1 interactors are annotated in black. Sub-pathways are annotated either red or green with italicized characters. Host proteins associated with the main or sub-pathways are colored in red or green, remaining proteins are in gray. (B) Immunoblot of WCL from P3HR-1 expressing the indicated sgRNA and uninduced or induced for lytic reactivation by 4HT (400 nM) for 24 h. Representative of n = 3 replicates. (C) Mean ± SEM of plasma membrane (PM) gp350 levels in P3HR-1 expressing the indicated sgRNA, uninduced or 4HT-induced for reactivation for 24 h. (D) Quantitative real-time PCR (qRT-PCR) of EBV intracellular genome copy number from P3HR-1 with the indicated sgRNA and 4HT-induced for 24 h. Mean ± SEM from n = 3 replicates. (E) Immunoblot of 2.5% input and anti-HA immunopurified mitochondria from P3HR-1 stably expressing HA-OMP25, uninduced or 4HT-induced for 24 h. Representative of n = 2 replicates. (F) Immunofluorescence analysis of NLRP3 and ASC in P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. Right: mean ± SEM percentage of cells with NLRP3/ASC specks from n = 3 replicates, as in (E), using data from 20 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin. (G) Immunoblot of ASC oligomerization from P3HR-1 expressing the indicated sgRNA, uninduced or 4HT-induced for 24 h. Representative of n = 2 replicates. (H) Mean ± SEM from n = 3 replicates of caspase-1 activity normalized by live cell number from P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h. (I) Mean ± SEM from n = 3 replicates of caspase-1 activity normalized by live cell number from P3HR-1 cells expressing the indicated sgRNA, 4HT-induced ± the NLRP3 inhibitor MCC950 (10 μM) for 24 h. (J) Mean ± SEM live cell percentages from n = 3 replicates of trypan blue staining of P3HR-1 expressing the indicated sgRNA and treated with 4HT, MCC950 (10 μM) or the caspase/pyroptosis inhibitor VX795 (10 μM) for 24 h. (K) Mean ± SEM from n = 3 replicates of caspase-1 activity normalized by live cell number from P3HR-1 expressing the indicated cDNA and nigericin treated for 24 h. Student’s t test was performed, with ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. See also Figure S2.

Figure 4.. BILF1 mediates MAVS dislocation from the mitochondria to inhibit NLRP3 inflammasome activation

(A) Immunofluorescence analysis of NLRP3 and TOMM20 in P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. Right: mean ± SEM percentage of cells with NLRP3-TOMM20 co-localization from n = 3 replicates, using data from 10 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin. (B) Immunofluorescence analysis of NLRP3 and ASC speck formation in P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h. Right: mean ± SEM percentage of cells with NLRP3/ASC specks from n = 3 replicates, using data from 20 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin. (C) Immunoblot of ASC oligomerization from P3HR-1 expressing the indicated sgRNA, uninduced, or 4HT-induced for 24 h. Representative of n = 2 replicates. (D) Immunoblot of WCL from P3HR-1 expressing the indicated sgRNA, uninduced, or 4HT-induced for 24 h. # indicates low molecular weight bands immunoreactive with anti-MAVS antibody. (E) Mean ± SEM from n = 3 replicates of caspase-1 activity normalized by live cell number from P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. (F) Mean ± SEM from n = 3 replicates of trypan blue analysis of P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. (G) Immunoblot of 293T transiently expressing the indicated cDNA. Representative of n = 2. (H) Immunofluorescence analysis of MAVS and TOMM20 in P3HR-1 expressing the indicated sgRNA, 4HT-induced for 24 h. Right: mean ±SEM percentage of cells with delocalized MAVS from n = 3 replicates, using data from 20 randomly selected panels of 400 nuclei, analyzed by ImageJ ComDet plugin. (I) Immunofluorescence analysis of MAVS and TOMM20 in P3HR-1, ± BILF1 cDNA induced by 5 mM doxycycline for 24 h. Right: mean ± SEM percentage of cells with delocalized MAVS from n = 3 replicates, using data from 30 randomly selected panels of 600 nuclei, analyzed by ImageJ ComDet plugin. Student’s t test was performed, with ****p < 0.0001. ***p < 0.001. **p < 0.01. *p < 0.05. See also Figure S3.

Figure 5.. BILF1 triggers MAVS mitochondrial dislocation through UFMylation

(A) Immunoblot of ASC oligomerization from P3HR-1 expressing the indicated sgRNA, uninduced or 4HT-induced for 24 h. (B) Immunoblot analysis of WCL from P3HR-1 expressing the indicated sgRNA and 4HT-induced, as indicated. (C) Mean ± SEM caspase-1 activity normalized by live cell number from n = 3 replicates of P3HR-1 expressing the indicated sgRNA and 4HT-treated for 24 h, as indicated. (D) Immunoblot of 1% input and anti-FLAG-MAVS complexes from 293T co-transfected with FLAG-MAVS, BILF1, or BXLF1 cDNAs for 24 h, as indicated. (E) PEAKS software identification of potential MAVS post-translational modification sites. Putative UFMylation sites were identified at lysines 362, 371, and 461. MAVS residues 321–500 are depicted. Black vertical lines represent individual peptide sequencing events. (F) Immunoblot of anti-EGFP-MAVS immunopurified from 293T co-transfected with the indicated MAVS, BILF1, and UFM1 cDNAs for 24 h. (G) Immunofluorescence analysis of wild type or K461R MAVS subcellular localization in 293T co-transfected with MAVS and BILF1 cDNAs for 24 h. (H) Immunoblot of WCL from 293T expressing MAVS and BILF1 cDNA, nigericin stimulated for 24 h, as indicated. (I) Immunoblot of 1% input and anti-HA immunopurified EGFP-MAVS and UFL1 from 293T transfected with BILF1 and EGFP-MAVS cDNAs for 24 h, as indicated. (J) AlphaFold multimer model highlighting the predicted BILF1 and MAVS interaction domain and residues. (K) Immunoblot of WCL from 293T expressing wild type, N- (ΔN) or C- (ΔC) terminal tail deletion mutant BILF1, nigericin stimulated for 24 h, as indicated. (L) Schematic of BILF1 NLRP3 inflammasome inhibition. Student’s t test was performed, with ***p < 0.001. *p < 0.05. See also Figures S4 and S5. Immunoblots are representative of n = 2.

Figure 6.. MAVS sequestration and lysosomal fusion events captured in real time

(A) Left: immunofluorescence analysis of MAVS and LAMP1 in P3HR-1, uninduced, or 4HT-induced for 24 h. Right: fluorescence intensity line scanning of LAMP1 (red) and MAVS (green) in the white rectangle. * and # mark co-localization and non-co-localization, respectively. (B) Mean ± SEM percentage of cells with MAVS-LAMP1 co-localization from n = 3 replicates, as in (A), using data from 12 randomly selected panels of 240 nuclei, analyzed by ImageJ ComDet plugin. (C) Immunofluorescence analysis of MAVS and LAMP1 in EBV+ AGSiZ gastric carcinoma, lytic induced by doxycycline for 24 h. Right: mean ± SEM percentage of cells with MAVS-LAMP1 co-localization from n = 3 replicates, using data from 12 randomly selected panels of 240 nuclei, analyzed by ImageJ ComDet plugin. (D) Immunoblot of 2.5% input and anti-HA immunopurified lysosomes from P3HR-1 HA-TMEM192+ cells, 4HT-induced for 24 h, as indicated. High and low molecular weight bands reactive with anti-MAVS antibodies are denoted by #. UFL1 is denoted by *. Representative of n = 2. (E) Consecutive frames captured at 10 s intervals at 3.5 h post AGSiZ lytic induction by doxycycline. White arrows highlight a GFP-MAVS puncta (green) superimposed on BFP-labeled mitochondria (blue) and SIRylo-stained lysosomes (red). Consecutive frames show partial co-localization of the GFP-MAVS puncta and lysosome (red) signals at +10 and +20 s, and then loss of MAVS puncta signal at the site of lysosome overlap at +40 s. See also corresponding Video S1. (F) Fluorescence intensity line scanning of lysosome SIRylo (red) and GFP-MAVS (green) signals at the white arrow marked puncta in (F) at +10, +20, and +40 s. (G) Schematic illustration of UFMylated MAVS trafficking from mitochondria to lysosome via MDVs. Student’s t test was performed, with ****p < 0.0001. See also Figure S6 and Video S1.

Figure 7.. BILF1 dislocates MAVS to mitochondria derived vesicles.

(A) Immunofluorescence analysis of MAVS and TOMM20 in P3HR-1 expressing the indicated PARK2 or Drp1 sgRNAs and 4HT-induced for 24 h. (B) Mean ± SEM percentage of cells with dislocated MAVS from n = 3 replicates, as judged by appearance of MAVS puncta that did not overlap with TOMM20 signal as in (A), using data from 25 randomly selected panels of 500 nuclei, analyzed using ImageJ ComDet plugin. (C) Mean ± SEM percentage of P3HR-1 with NLRP3/ASC specks from n = 3 replicates, using data from 20 randomly selected panels of 200 nuclei, analyzed by ImageJ ComDet plugin. (D) Mean ± SEM caspase-1 activity normalized by live cell number from n = 3 replicates of P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h, as indicated. (E) Immunoblot of WCL from P3HR-1 expressing the indicated sgRNA and 4HT-induced for 24 h. # denotes low molecular weight bands immunoreactive with anti-MAVS antibody. Representative of n = 3. (F) PAKR2 and TOMM20 immunofluorescence analysis in 293T transfected with BILF1 or BXLF1 cDNA for 24 h. (G) Immunoblot of 1% input vs. anti-HA-MAVS immunopurified from wild type or UFL1-KO 293T transfected with MAVS and BILF1 cDNA for 24 h, as indicated. Representative of n = 2. (H) Schematic of NLRP3 inflammasome subversion by BILF1. BILF1 recruits UFL1 to mediate MAVS UFMylation, which together with PARK2 triggers selective MAVS removal from the mitochondrial outer membrane, MDV packaging and delivery to lysosomes, preventing NLRP3 inflammasome activation and pyroptosis. Student’s t test was performed, with ****p < 0.0001. ***p < 0.001. ns > 0.05. See also Figure S6.